Synthetic niches for differentiation of human embryonic stem cells bypassing embryoid body formation.

PubMed

Liu, Yarong; Fox, Victoria; Lei, Yuning; Hu, Biliang; Joo, Kye-Il; Wang, Pin

2014-07-01

The unique self-renewal and pluripotency features of human embryonic stem cells (hESCs) offer the potential for unlimited development of novel cell therapies. Currently, hESCs are cultured and differentiated using methods, such as monolayer culture and embryoid body (EB) formation. As such, achieving efficient differentiation into higher order structures remains a challenge, as well as maintaining cell viability during differentiation into homogeneous cell populations. Here, we describe the application of highly porous polymer scaffolds as synthetic stem cell niches. Bypassing the EB formation step, these scaffolds are capable of three-dimensional culture of undifferentiated hESCs and subsequent directed differentiation into three primary germ layers. H9 hESCs were successfully maintained and proliferated in biodegradable polymer scaffolds based on poly (lactic-co-glycolic acid) (PLGA). The results showed that cells within PLGA scaffolds retained characteristics of undifferentiated pluripotent stem cells. Moreover, the scaffolds allowed differentiation towards the lineage of interest by the addition of growth factors to the culture system. The in vivo transplantation study revealed that the scaffolds could provide a microenvironment that enabled hESCs to interact with their surroundings, thereby promoting cell differentiation. Therefore, this approach, which provides a unique culture/differentiation system for hESCs, will find its utility in various stem cell-based tissue-engineering applications. © 2013 Wiley Periodicals, Inc.

Evaluation of human embryonic stem cells and their differentiated fibroblastic progenies as cellular models for in vitro genotoxicity screening.

PubMed

Vinoth, Kumar Jayaseelan; Manikandan, Jayapal; Sethu, Swaminathan; Balakrishnan, Lakshmidevi; Heng, Alexis; Lu, Kai; Hande, Manoor Prakash; Cao, Tong

2014-08-20

This study evaluated human embryonic stem cells (hESC) and their differentiated fibroblastic progenies as cellular models for genotoxicity screening. The DNA damage response of hESCs and their differentiated fibroblastic progenies were compared to a fibroblastic cell line (HEPM, CRL1486) and primary cultures of peripheral blood lymphocytes (PBL), upon exposure to Mitomycin C, gamma irradiation and H2O2. It was demonstrated that hESC-derived fibroblastic progenies (H1F) displayed significantly higher chromosomal aberrations, micronuclei formation and double strand break (DSB) formation, as compared to undifferentiated hESC upon exposure to genotoxic stress. Nevertheless, H1F cell types displayed comparable sensitivities to genotoxic challenge as HEPM and PBL, both of which are representative of somatic cell types commonly used for genotoxicity screening. Subsequently, transcriptomic and pathways analysis identified differential expression of critical genes involved in cell death and DNA damage response upon exposure to gamma irradiation. The results thus demonstrate that hESC-derived fibroblastic progenies are as sensitive as commonly-used somatic cell types for genotoxicity screening. Moreover, hESCs have additional advantages, such as their genetic normality compared to immortalized cell lines, as well as their amenability to scale-up for producing large, standardized quantities of cells for genotoxicity screening on an industrial scale, something which can never be achieved with primary cell cultures. Copyright © 2014. Published by Elsevier B.V.

Effect of Antibiotics against Mycoplasma sp. on Human Embryonic Stem Cells Undifferentiated Status, Pluripotency, Cell Viability and Growth

PubMed Central

Romorini, Leonardo; Riva, Diego Ariel; Blüguermann, Carolina; Videla Richardson, Guillermo Agustin; Scassa, Maria Elida; Sevlever, Gustavo Emilio; Miriuka, Santiago Gabriel

2013-01-01

Human embryonic stem cells (hESCs) are self-renewing pluripotent cells that can differentiate into specialized cells and hold great promise as models for human development and disease studies, cell-replacement therapies, drug discovery and in vitro cytotoxicity tests. The culture and differentiation of these cells are both complex and expensive, so it is essential to extreme aseptic conditions. hESCs are susceptible to Mycoplasma sp. infection, which is hard to detect and alters stem cell-associated properties. The purpose of this work was to evaluate the efficacy and cytotoxic effect of PlasmocinTM and ciprofloxacin (specific antibiotics used for Mycoplasma sp. eradication) on hESCs. Mycoplasma sp. infected HUES-5 884 (H5 884, stable hESCs H5-brachyury promoter-GFP line) cells were effectively cured with a 14 days PlasmocinTM 25 µg/ml treatment (curative treatment) while maintaining stemness characteristic features. Furthermore, cured H5 884 cells exhibit the same karyotype as the parental H5 line and expressed GFP, through up-regulation of brachyury promoter, at day 4 of differentiation onset. Moreover, H5 cells treated with ciprofloxacin 10 µg/ml for 14 days (mimic of curative treatment) and H5 and WA09 (H9) hESCs treated with PlasmocinTM 5 µg/ml (prophylactic treatment) for 5 passages retained hESCs features, as judged by the expression of stemness-related genes (TRA1-60, TRA1-81, SSEA-4, Oct-4, Nanog) at mRNA and protein levels. In addition, the presence of specific markers of the three germ layers (brachyury, Nkx2.5 and cTnT: mesoderm; AFP: endoderm; nestin and Pax-6: ectoderm) was verified in in vitro differentiated antibiotic-treated hESCs. In conclusion, we found that PlasmocinTM and ciprofloxacin do not affect hESCs stemness and pluripotency nor cell viability. However, curative treatments slightly diminished cell growth rate. This cytotoxic effect was reversible as cells regained normal growth rate upon antibiotic withdrawal. PMID:23936178

A single EBV-based vector for stable episomal maintenance and expression of GFP in human embryonic stem cells.

PubMed

Thyagarajan, Bhaskar; Scheyhing, Kelly; Xue, Haipeng; Fontes, Andrew; Chesnut, Jon; Rao, Mahendra; Lakshmipathy, Uma

2009-03-01

Stable expression of transgenes in stem cells has been a challenge due to the nonavailability of efficient transfection methods and the inability of transgenes to support sustained gene expression. Several methods have been reported to stably modify both embryonic and adult stem cells. These methods rely on integration of the transgene into the genome of the host cell, which could result in an expression pattern dependent on the number of integrations and the genomic locus of integration. To overcome this issue, site-specific integration methods mediated by integrase, adeno-associated virus or via homologous recombination have been used to generate stable human embryonic stem cell (hESC) lines. In this study, we describe a vector that is maintained episomally in hESCs. The vector used in this study is based on components derived from the Epstein-Barr virus, containing the Epstein-Barr virus nuclear antigen 1 expression cassette and the OriP origin of replication. The vector also expresses the drug-resistance marker gene hygromycin, which allows for selection and long-term maintenance of cells harboring the plasmid. Using this vector system, we show sustained expression of green fluorescent protein in undifferentiated hESCs and their differentiating embryoid bodies. In addition, the stable hESC clones show comparable expression with and without drug selection. Consistent with this observation, bulk-transfected adipose tissue-derived mesenchymal stem cells showed persistent marker gene expression as they differentiate into adipocytes, osteoblasts and chondroblasts. Episomal vectors offer a fast and efficient method to create hESC reporter lines, which in turn allows one to test the effect of overexpression of various genes on stem cell growth, proliferation and differentiation.

Identification and Single-Cell Functional Characterization of an Endodermally Biased Pluripotent Substate in Human Embryonic Stem Cells.

PubMed

Allison, Thomas F; Smith, Andrew J H; Anastassiadis, Konstantinos; Sloane-Stanley, Jackie; Biga, Veronica; Stavish, Dylan; Hackland, James; Sabri, Shan; Langerman, Justin; Jones, Mark; Plath, Kathrin; Coca, Daniel; Barbaric, Ivana; Gokhale, Paul; Andrews, Peter W

2018-05-09

Human embryonic stem cells (hESCs) display substantial heterogeneity in gene expression, implying the existence of discrete substates within the stem cell compartment. To determine whether these substates impact fate decisions of hESCs we used a GFP reporter line to investigate the properties of fractions of putative undifferentiated cells defined by their differential expression of the endoderm transcription factor, GATA6, together with the hESC surface marker, SSEA3. By single-cell cloning, we confirmed that substates characterized by expression of GATA6 and SSEA3 include pluripotent stem cells capable of long-term self-renewal. When clonal stem cell colonies were formed from GATA6-positive and GATA6-negative cells, more of those derived from GATA6-positive cells contained spontaneously differentiated endoderm cells than similar colonies derived from the GATA6-negative cells. We characterized these discrete cellular states using single-cell transcriptomic analysis, identifying a potential role for SOX17 in the establishment of the endoderm-biased stem cell state. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Embryonic Stem Cells: Isolation, Characterization and Culture

NASA Astrophysics Data System (ADS)

Amit, Michal; Itskovitz-Eldor, Joseph

Embryonic stem cells are pluripotent cells isolated from the mammalian blastocyst. Traditionally, these cells have been derived and cultured with mouse embryonic fibroblast (MEF) supportive layers, which allow their continuous growth in an undifferentiated state. However, for any future industrial or clinical application hESCs should be cultured in reproducible, defined, and xeno-free culture system, where exposure to animal pathogens is prevented. From their derivation in 1998 the methods for culturing hESCs were significantly improved. This chapter wills discuss hESC characterization and the basic methods for their derivation and maintenance.

Comparison of the glycosphingolipids of human-induced pluripotent stem cells and human embryonic stem cells.

PubMed

Säljö, Karin; Barone, Angela; Vizlin-Hodzic, Dzeneta; Johansson, Bengt R; Breimer, Michael E; Funa, Keiko; Teneberg, Susann

2017-04-01

High expectations are held for human-induced pluripotent stem cells (hiPSC) since they are established from autologous tissues thus overcoming the risk of allogeneic immune rejection when used in regenerative medicine. However, little is known regarding the cell-surface carbohydrate antigen profile of hiPSC compared with human embryonic stem cells (hESC). Here, glycosphingolipids were isolated from an adipocyte-derived hiPSC line, and hiPSC and hESC glycosphingolipids were compared by concurrent characterization by binding assays with carbohydrate-recognizing ligands and mass spectrometry. A high similarity between the nonacid glycosphingolipids of hiPSC and hESC was found. The nonacid glycosphingolipids P1 pentaosylceramide, x2 pentaosylceramide and H type 1 heptaosylceramide, not previously described in human pluripotent stem cells (hPSC), were characterized in both hiPSC and hESC. The composition of acid glycosphingolipids differed, with increased levels of GM3 ganglioside, and reduced levels of GD1a/GD1b in hiPSC when compared with hESC. In addition, the hESC glycosphingolipids sulf-globopentaosylceramide and sialyl-globotetraosylceramide were lacking in hiPSC. Neural stem cells differentiating from hiPSC had a reduced expression of sialyl-lactotetra, whereas expression of the GD1a ganglioside was significantly increased. Thus, while sialyl-lactotetra is a marker of undifferentiated hPSC, GD1a is a novel marker of neural differentiation. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Epigenetic Mechanisms Regulate MHC and Antigen Processing Molecules in Human Embryonic and Induced Pluripotent Stem Cells

PubMed Central

Suárez-Álvarez, Beatriz; Rodriguez, Ramón M.; Calvanese, Vincenzo; Blanco-Gelaz, Miguel A.; Suhr, Steve T.; Ortega, Francisco; Otero, Jesus; Cibelli, Jose B.; Moore, Harry; Fraga, Mario F.; López-Larrea, Carlos

2010-01-01

Background Human embryonic stem cells (hESCs) are an attractive resource for new therapeutic approaches that involve tissue regeneration. hESCs have exhibited low immunogenicity due to low levels of Mayor Histocompatibility Complex (MHC) class-I and absence of MHC class-II expression. Nevertheless, the mechanisms regulating MHC expression in hESCs had not been explored. Methodology/Principal Findings We analyzed the expression levels of classical and non-classical MHC class-I, MHC class-II molecules, antigen-processing machinery (APM) components and NKG2D ligands (NKG2D-L) in hESCs, induced pluripotent stem cells (iPSCs) and NTera2 (NT2) teratocarcinoma cell line. Epigenetic mechanisms involved in the regulation of these genes were investigated by bisulfite sequencing and chromatin immunoprecipitation (ChIP) assays. We showed that low levels of MHC class-I molecules were associated with absent or reduced expression of the transporter associated with antigen processing 1 (TAP-1) and tapasin (TPN) components in hESCs and iPSCs, which are involved in the transport and load of peptides. Furthermore, lack of β2-microglobulin (β2m) light chain in these cells limited the expression of MHC class I trimeric molecule on the cell surface. NKG2D ligands (MICA, MICB) were observed in all pluripotent stem cells lines. Epigenetic analysis showed that H3K9me3 repressed the TPN gene in undifferentiated cells whilst HLA-B and β2m acquired the H3K4me3 modification during the differentiation to embryoid bodies (EBs). Absence of HLA-DR and HLA-G expression was regulated by DNA methylation. Conclusions/Significance Our data provide fundamental evidence for the epigenetic control of MHC in hESCs and iPSCs. Reduced MHC class I and class II expression in hESCs and iPSCs can limit their recognition by the immune response against these cells. The knowledge of these mechanisms will further allow the development of strategies to induce tolerance and improve stem cell allograft acceptance. PMID:20419139

HLA Class I Depleted hESC as a Source of Hypoimmunogenic Cells for Tissue Engineering Applications.

PubMed

Karabekian, Zaruhi; Ding, Hao; Stybayeva, Gulnaz; Ivanova, Irina; Muselimyan, Narine; Haque, Amranul; Toma, Ian; Posnack, Nikki G; Revzin, Alexander; Leitenberg, David; Laflamme, Michael A; Sarvazyan, Narine

2015-10-01

Rapidly improving protocols for the derivation of autologous cells from stem cell sources is a welcome development. However, there are many circumstances when off-the-shelf universally immunocompatible cells may be needed. Embryonic stem cells (ESCs) provide a unique opportunity to modify the original source of differentiated cells to minimize their rejection by nonautologous hosts. Immune rejection of nonautologous human embryonic stem cell (hESC) derivatives can be reduced by downregulating human leukocyte antigen (HLA) class I molecules, without affecting the ability of these cells to differentiate into specific lineages. Beta-2-microglobulin (B2M) expression was decreased by lentiviral transduction using human anti-HLA class I light-chain B2M short hairpin RNA. mRNA levels of B2M were decreased by 90% in a RUES2-modified hESC line, as determined by quantitative real time-polymerase chain reaction analysis. The transduced cells were selected under puromycin pressure and maintained in an undifferentiated state. The latter was confirmed by Oct4 and Nanog expression, and by the formation of characteristic round-shaped colonies. B2M downregulation led to diminished HLA-I expression on the cell surface, as determined by flow cytometry. When used as target cells in a mixed lymphocyte reaction assay, transduced hESCs and their differentiated derivatives did not stimulate allogeneic T-cell proliferation. Using a cardiac differentiation protocol, transduced hESCs formed a confluent layer of cardiac myocytes and maintained a low level of B2M expression. Transduced hESCs were also successfully differentiated into a hepatic lineage, validating their capacity to differentiate into multiple lineages. HLA-I depletion does not preclude hESC differentiation into cardiac or hepatic lineages. This methodology can be used to engineer tissue from nonautologous hESC sources with improved immunocompatibility.

HLA Class I Depleted hESC as a Source of Hypoimmunogenic Cells for Tissue Engineering Applications

PubMed Central

Karabekian, Zaruhi; Ding, Hao; Stybayeva, Gulnaz; Ivanova, Irina; Muselimyan, Narine; Haque, Amranul; Toma, Ian; Posnack, Nikki G.; Revzin, Alexander; Leitenberg, David; Laflamme, Michael A.

2015-01-01

Background: Rapidly improving protocols for the derivation of autologous cells from stem cell sources is a welcome development. However, there are many circumstances when off-the-shelf universally immunocompatible cells may be needed. Embryonic stem cells (ESCs) provide a unique opportunity to modify the original source of differentiated cells to minimize their rejection by nonautologous hosts. Hypothesis: Immune rejection of nonautologous human embryonic stem cell (hESC) derivatives can be reduced by downregulating human leukocyte antigen (HLA) class I molecules, without affecting the ability of these cells to differentiate into specific lineages. Methods and Results: Beta-2-microglobulin (B2M) expression was decreased by lentiviral transduction using human anti-HLA class I light-chain B2M short hairpin RNA. mRNA levels of B2M were decreased by 90% in a RUES2-modified hESC line, as determined by quantitative real time-polymerase chain reaction analysis. The transduced cells were selected under puromycin pressure and maintained in an undifferentiated state. The latter was confirmed by Oct4 and Nanog expression, and by the formation of characteristic round-shaped colonies. B2M downregulation led to diminished HLA-I expression on the cell surface, as determined by flow cytometry. When used as target cells in a mixed lymphocyte reaction assay, transduced hESCs and their differentiated derivatives did not stimulate allogeneic T-cell proliferation. Using a cardiac differentiation protocol, transduced hESCs formed a confluent layer of cardiac myocytes and maintained a low level of B2M expression. Transduced hESCs were also successfully differentiated into a hepatic lineage, validating their capacity to differentiate into multiple lineages. Conclusions: HLA-I depletion does not preclude hESC differentiation into cardiac or hepatic lineages. This methodology can be used to engineer tissue from nonautologous hESC sources with improved immunocompatibility. PMID:26218149

KEEPING AN EYE ON RETINOBLASTOMA CONTROL OF HUMAN EMBRYONIC STEM CELLS

PubMed Central

Conklin, Jamie F.; Sage, Julien

2010-01-01

Human embryonic stem cells (hESCs) hold great promise in regenerative medicine. However, before the full potential of these cells is achieved, major basic biological questions need to be addressed. In particular, there are still gaps in our knowledge of the molecular mechanisms underlying the derivation of hESCs from blastocysts, the regulation of the undifferentiated, pluripotent state, and the control of differentiation into specific lineages. Furthermore, we still do not fully understand the tumorigenic potential of hESCs, limiting their use in regenerative medicine. The RB pathway is a key signaling module that controls cellular proliferation, cell survival, chromatin structure, and cellular differentiation in mammalian cells. Members of the RB pathway are important regulators of hESC biology and manipulation of the activity of this pathway may provide novel means to control the fate of hESCs. Here we review what is known about the expression and function of members of the RB pathway in hESCs and discuss areas of interest in this field. PMID:19760644

Uncovering the Role of Hypermethylation by CTG Expansion in Myotonic Dystrophy Type 1 Using Mutant Human Embryonic Stem Cells

PubMed Central

Yanovsky-Dagan, Shira; Avitzour, Michal; Altarescu, Gheona; Renbaum, Paul; Eldar-Geva, Talia; Schonberger, Oshrat; Mitrani-Rosenbaum, Stella; Levy-Lahad, Ephrat; Birnbaum, Ramon Y.; Gepstein, Lior; Epsztejn-Litman, Silvina; Eiges, Rachel

2015-01-01

Summary CTG repeat expansion in DMPK, the cause of myotonic dystrophy type 1 (DM1), frequently results in hypermethylation and reduced SIX5 expression. The contribution of hypermethylation to disease pathogenesis and the precise mechanism by which SIX5 expression is reduced are unknown. Using 14 different DM1-affected human embryonic stem cell (hESC) lines, we characterized a differentially methylated region (DMR) near the CTGs. This DMR undergoes hypermethylation as a function of expansion size in a way that is specific to undifferentiated cells and is associated with reduced SIX5 expression. Using functional assays, we provide evidence for regulatory activity of the DMR, which is lost by hypermethylation and may contribute to DM1 pathogenesis by causing SIX5 haplo-insufficiency. This study highlights the power of hESCs in disease modeling and describes a DMR that functions both as an exon coding sequence and as a regulatory element whose activity is epigenetically hampered by a heritable mutation. PMID:26190529

Non-canonical TAF complexes regulate active promoters in human embryonic stem cells.

PubMed

Maston, Glenn A; Zhu, Lihua Julie; Chamberlain, Lynn; Lin, Ling; Fang, Minggang; Green, Michael R

2012-11-13

The general transcription factor TFIID comprises the TATA-box-binding protein (TBP) and approximately 14 TBP-associated factors (TAFs). Here we find, unexpectedly, that undifferentiated human embryonic stem cells (hESCs) contain only six TAFs (TAFs 2, 3, 5, 6, 7 and 11), whereas following differentiation all TAFs are expressed. Directed and global chromatin immunoprecipitation analyses reveal an unprecedented promoter occupancy pattern: most active genes are bound by only TAFs 3 and 5 along with TBP, whereas the remaining active genes are bound by TBP and all six hESC TAFs. Consistent with these results, hESCs contain a previously undescribed complex comprising TAFs 2, 6, 7, 11 and TBP. Altering the composition of hESC TAFs, either by depleting TAFs that are present or ectopically expressing TAFs that are absent, results in misregulated expression of pluripotency genes and induction of differentiation. Thus, the selective expression and use of TAFs underlies the ability of hESCs to self-renew.DOI:http://dx.doi.org/10.7554/eLife.00068.001.

Cell cycle regulation in human embryonic stem cells: links to adaptation to cell culture.

PubMed

Barta, Tomas; Dolezalova, Dasa; Holubcova, Zuzana; Hampl, Ales

2013-03-01

Cell cycle represents not only a tightly orchestrated mechanism of cell replication and cell division but it also plays an important role in regulation of cell fate decision. Particularly in the context of pluripotent stem cells or multipotent progenitor cells, regulation of cell fate decision is of paramount importance. It has been shown that human embryonic stem cells (hESCs) show unique cell cycle characteristics, such as short doubling time due to abbreviated G1 phase; these properties change with the onset of differentiation. This review summarizes the current understanding of cell cycle regulation in hESCs. We discuss cell cycle properties as well as regulatory machinery governing cell cycle progression of undifferentiated hESCs. Additionally, we provide evidence that long-term culture of hESCs is accompanied by changes in cell cycle properties as well as configuration of several cell cycle regulatory molecules.

Non-canonical TAF complexes regulate active promoters in human embryonic stem cells

PubMed Central

Maston, Glenn A; Zhu, Lihua Julie; Chamberlain, Lynn; Lin, Ling; Fang, Minggang; Green, Michael R

2012-01-01

The general transcription factor TFIID comprises the TATA-box-binding protein (TBP) and approximately 14 TBP-associated factors (TAFs). Here we find, unexpectedly, that undifferentiated human embryonic stem cells (hESCs) contain only six TAFs (TAFs 2, 3, 5, 6, 7 and 11), whereas following differentiation all TAFs are expressed. Directed and global chromatin immunoprecipitation analyses reveal an unprecedented promoter occupancy pattern: most active genes are bound by only TAFs 3 and 5 along with TBP, whereas the remaining active genes are bound by TBP and all six hESC TAFs. Consistent with these results, hESCs contain a previously undescribed complex comprising TAFs 2, 6, 7, 11 and TBP. Altering the composition of hESC TAFs, either by depleting TAFs that are present or ectopically expressing TAFs that are absent, results in misregulated expression of pluripotency genes and induction of differentiation. Thus, the selective expression and use of TAFs underlies the ability of hESCs to self-renew. DOI: http://dx.doi.org/10.7554/eLife.00068.001 PMID:23150797

Effect of mitomycin-C on human foreskin fibroblasts used as feeders in human embryonic stem cells: immunocytochemistry MIB1 score and DNA ploidy and apoptosis evaluated by flow cytometry.

PubMed

Nieto, A; Cabrera, C M; Catalina, P; Cobo, F; Barnie, A; Cortés, J L; Barroso del Jesus, A; Montes, R; Concha, A

2007-03-01

Mitomycin C (MMC) treatment has been used to arrest cell proliferation but not much is known about the effect of MMC on human foreskin fibroblasts (HFF) used as feeders for human embryonic stem cells (hESC). We tested the ability of MMC to stop the proliferation of HFF and to induce apoptosis. MMC inhibited the proliferation of HFF at 10 microg/ml over 2.5h of MMC treatment showing a decrease in the proliferation index measured by Ki-67 and S and G2/M phases related to active HFF. A low percentage of cells showed necrotic or apoptotic features using different lengths of incubation. Over time, the majority of cells remained in a mitotically inactive state. The percentage of apoptotic cells increased from day 2 to day 10, at the same time as the necrotic ones increased. The HS181 hESC line grew in an undifferentiated state on inactive HFF throughout the study.

Uncovering the Role of Hypermethylation by CTG Expansion in Myotonic Dystrophy Type 1 Using Mutant Human Embryonic Stem Cells.

PubMed

Yanovsky-Dagan, Shira; Avitzour, Michal; Altarescu, Gheona; Renbaum, Paul; Eldar-Geva, Talia; Schonberger, Oshrat; Mitrani-Rosenbaum, Stella; Levy-Lahad, Ephrat; Birnbaum, Ramon Y; Gepstein, Lior; Epsztejn-Litman, Silvina; Eiges, Rachel

2015-08-11

CTG repeat expansion in DMPK, the cause of myotonic dystrophy type 1 (DM1), frequently results in hypermethylation and reduced SIX5 expression. The contribution of hypermethylation to disease pathogenesis and the precise mechanism by which SIX5 expression is reduced are unknown. Using 14 different DM1-affected human embryonic stem cell (hESC) lines, we characterized a differentially methylated region (DMR) near the CTGs. This DMR undergoes hypermethylation as a function of expansion size in a way that is specific to undifferentiated cells and is associated with reduced SIX5 expression. Using functional assays, we provide evidence for regulatory activity of the DMR, which is lost by hypermethylation and may contribute to DM1 pathogenesis by causing SIX5 haplo-insufficiency. This study highlights the power of hESCs in disease modeling and describes a DMR that functions both as an exon coding sequence and as a regulatory element whose activity is epigenetically hampered by a heritable mutation. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Morphological Analysis of Human Induced Pluripotent Stem Cells During Induced Differentiation and Reverse Programming

PubMed Central

Magniez, Aurélie; Oudrhiri, Noufissa; Féraud, Olivier; Bacci, Josette; Gobbo, Emilie; Proust, Stéphanie; Turhan, Ali G.

2014-01-01

Abstract The fine analysis of cell components during the generation of pluripotent cells and their comparison to bone fide human embryonic stem cells (hESCs) are valuable tools to understand their biological behavior. In this report, human mesenchymal cells (hMSCs) generated from the human ES cell line H9, were reprogrammed back to induced pluripotent state using Oct-4, Sox2, Nanog, and Lin28 transgenes. Human induced pluripotent stem cells (hIPSCs) were analyzed using electron microscopy and compared with regard to the original hESCs and the hMSCs from which they were derived. This analysis shows that hIPSCs and the original hESCs are morphologically undistinguishable but differ from the hMSCs with respect to the presence of several morphological features of undifferentiated cells at both the cytoplasmic (ribosomes, lipid droplets, glycogen, scarce reticulum) and nuclear levels (features of nuclear plasticity, presence of euchromatin, reticulated nucleoli). We show that hIPSC colonies generated this way presented epithelial aspects with specialized junctions highlighting morphological criteria of the mesenchymal–epithelial transition in cells engaged in a successful reprogramming process. Electron microscopic analysis revealed also specific morphological aspects of partially reprogrammed cells. These results highlight the valuable use of electron microscopy for a better knowledge of the morphological aspects of IPSC and cellular reprogramming. PMID:25371857

Elasticity of human embryonic stem cells as determined by atomic force microscopy.

PubMed

Kiss, Robert; Bock, Henry; Pells, Steve; Canetta, Elisabetta; Adya, Ashok K; Moore, Andrew J; De Sousa, Paul; Willoughby, Nicholas A

2011-10-01

The expansive growth and differentiation potential of human embryonic stem cells (hESCs) make them a promising source of cells for regenerative medicine. However, this promise is off set by the propensity for spontaneous or uncontrolled differentiation to result in heterogeneous cell populations. Cell elasticity has recently been shown to characterize particular cell phenotypes, with undifferentiated and differentiated cells sometimes showing significant differences in their elasticities. In this study, we determined the Young's modulus of hESCs by atomic force microscopy using a pyramidal tip. Using this method we are able to take point measurements of elasticity at multiple locations on a single cell, allowing local variations due to cell structure to be identified. We found considerable differences in the elasticity of the analyzed hESCs, reflected by a broad range of Young's modulus (0.05-10 kPa). This surprisingly high variation suggests that elasticity could serve as the basis of a simple and efficient large scale purification/separation technique to discriminate subpopulations of hESCs.

Effects of Feeder Cells on Dopaminergic Differentiation of Human Embryonic Stem Cells

PubMed Central

Zhao, Zhenqiang; Ma, Yanlin; Chen, Zhibin; Liu, Qian; Li, Qi; Kong, Deyan; Yuan, Kunxiong; Hu, Lan; Wang, Tan; Chen, Xiaowu; Peng, Yanan; Jiang, Weimin; Yu, Yanhong; Liu, Xinfeng

2016-01-01

Mouse embryonic fibroblasts (MEFs) and human foreskin fibroblasts (HFFs) are used for the culture of human embryonic stem cells (hESCs). MEFs and HFFs differed in their capacity to support the proliferation and pluripotency of hESCs and could affect cardiac differentiation potential of hESCs. The aim of this study was to evaluate the effect of MEFs and HFFs feeders on dopaminergic differentiation of hESCs lines. To minimize the impact of culture condition variation, two hESCs lines were cultured on mixed feeder cells (MFCs, MEFs: HFFs = 1:1) and HFFs feeder, respectively, and then were differentiated into dopaminergic (DA) neurons under the identical protocol. Dopaminergic differentiation was evaluated by immunocytochemistry, quantitative fluorescent real-time PCR, transmission and scanning electron microscopy, and patch clamp. Our results demonstrated that these hESCs-derived neurons were genuine and functional DA neurons. However, compared to hESCs line on MFCs feeder, hESCs line on HFFs feeder had a higher proportion of tyrosine hydroxylase (TH) positive cells and expressed higher levels of FOXA2, PITX3, NURR1, and TH genes. In addition, the values of threshold intensity and threshold membrane potential of DA neurons from hESCs line on HFFs feeder were lower than those of DA neurons from hESCs line on the MFCs feeder. In conclusion, HFFs feeder not only facilitated the differentiation of hESCs cells into dopaminergic neurons, but also induced hESCs-derived DA neurons to express higher electrophysiological excitability. Therefore, feeder cells could affect not only dopaminergic differentiation potential of different hESCs lines, but also electrophysiological properties of hESCs-derived DA neurons. PMID:28066186

Defining the Genomic Signature of Totipotency and Pluripotency during Early Human Development

PubMed Central

Galan, Amparo; Diaz-Gimeno, Patricia; Poo, Maria Eugenia; Valbuena, Diana; Sanchez, Eva; Ruiz, Veronica; Dopazo, Joaquin; Montaner, David; Conesa, Ana; Simon, Carlos

2013-01-01

The genetic mechanisms governing human pre-implantation embryo development and the in vitro counterparts, human embryonic stem cells (hESCs), still remain incomplete. Previous global genome studies demonstrated that totipotent blastomeres from day-3 human embryos and pluripotent inner cell masses (ICMs) from blastocysts, display unique and differing transcriptomes. Nevertheless, comparative gene expression analysis has revealed that no significant differences exist between hESCs derived from blastomeres versus those obtained from ICMs, suggesting that pluripotent hESCs involve a new developmental progression. To understand early human stages evolution, we developed an undifferentiation network signature (UNS) and applied it to a differential gene expression profile between single blastomeres from day-3 embryos, ICMs and hESCs. This allowed us to establish a unique signature composed of highly interconnected genes characteristic of totipotency (61 genes), in vivo pluripotency (20 genes), and in vitro pluripotency (107 genes), and which are also proprietary according to functional analysis. This systems biology approach has led to an improved understanding of the molecular and signaling processes governing human pre-implantation embryo development, as well as enabling us to comprehend how hESCs might adapt to in vitro culture conditions. PMID:23614026

Antibody engineering of a cytotoxic monoclonal antibody 84 against human embryonic stem cells: Investigating the effects of multivalency on cytotoxicity.

PubMed

Klement, Maximilian; Zheng, Jiyun; Liu, Chengcheng; Tan, Heng-Liang; Wong, Victor Vai Tak; Choo, Andre Boon-Hwa; Lee, Dong-Yup; Ow, Dave Siak-Wei

2017-02-10

Antibody fragments have shown targeted specificity to their antigens, but only modest tissue retention times in vivo and in vitro. Multimerization has been used as a protein engineering tool to increase the number of binding units and thereby enhance the efficacy and retention time of antibody fragments. In this work, we explored the effects of valency using a series of self-assembling polypeptides based on the GCN4 leucine zipper multimerization domain fused to a single-chain variable fragment via an antibody upper hinge sequence. Four engineered antibody fragments with a valency from one to four antigen-binding units of a cytotoxic monoclonal antibody 84 against human embryonic stem cells (hESC) were constructed. We hypothesized that higher cytotoxicity would be observed for fragments with increased valency. Flow cytometry analysis revealed that the trimeric and tetrameric engineered antibody fragments resulted in the highest degree of cytotoxicity to the undifferentiated hESC, while the engineered antibody fragments were observed to have improved tissue penetration into cell clusters. Thus, a trade off was made for the trimeric versus tetrameric fragment due to improved tissue penetration. These results have direct implications for antibody-mediated removal of undifferentiated hESC during regenerative medicine and cell therapy. Copyright © 2016 The Author(s). Published by Elsevier B.V. All rights reserved.

A review of the emerging potential therapy for neurological disorders: human embryonic stem cell therapy

PubMed Central

Shroff, Geeta; Dhanda Titus, Jyoti; Shroff, Rhea

2017-01-01

The first human embryonic stem cell (hESC) line was developed in the late nineties. hESCs are capable of proliferating indefinitely and differentiate into all the three embryonic germ layers. Further, the differentiation of hESC lines into neural precursor cells and neurons, astrocytes and oligodendrocytes showed their potential in treating several incurable neurological disorders such as spinal cord injury (SCI), cerebral palsy (CP), Parkinson’s disease (PD). In this review, we will discuss the global scenario of research and therapeutic use of hESCs in the treatment of neurological disorders. Following this, we will discuss the development of a unique hESC line, how it differs from the other available hESC lines and its use in the treatment of neurological disorders. hESCs were isolated from mixture of neuronal and non-neuronal progenitor cells in their pre progenitor state in a Good Laboratory Practices, Good Tissue Practices and Good Manufacturing Practices compliant laboratory. Blastomere cells have served as a source to derive the hESCs and the xeno-free culture was demonstrated to be more safe and effective in clinical therapeutic application of hESCs. All the patients showed a remarkable improvement in their conditions and no serious adverse events were reported. This study concluded that hESC lines could be scalable and used in the treatment of various neurological disorders such as SCI, CP, and PD. PMID:28533935

Human ESCs predisposition to karyotypic instability: Is a matter of culture adaptation or differential vulnerability among hESC lines due to inherent properties?

PubMed

Catalina, Puri; Montes, Rosa; Ligero, Gertru; Sanchez, Laura; de la Cueva, Teresa; Bueno, Clara; Leone, Paola E; Menendez, Pablo

2008-10-03

The use of human embryonic stem cells (hESCs) in research is increasing and hESCs hold the promise for many biological, clinical and toxicological studies. Human ESCs are expected to be chromosomally stable since karyotypic changes represent a pitfall for potential future applications. Recently, several studies have analysed the genomic stability of several hESC lines maintained after prolonged in vitro culture but controversial data has been reported. Here, we prompted to compare the chromosomal stability of three hESC lines maintained in the same laboratory using identical culture conditions and passaging methods. Molecular cytogenetic analyses performed in three different hESC lines maintained in parallel in identical culture conditions revealed significant differences among them in regard to their chromosomal integrity. In feeders, the HS181, SHEF-1 and SHEF-3 hESC lines were chromosomally stable up to 185 passages using either mechanical or enzymatic dissection methods. Despite the three hESC lines were maintained under identical conditions, each hESC line behaved differently upon being transferred to a feeder-free culture system. The two younger hESC lines, HS181 (71 passages) and SHEF-3 (51 passages) became chromosomally unstable shortly after being cultured in feeder-free conditions. The HS181 line gained a chromosome 12 by passage 17 and a marker by passage 21, characterized as a gain of chromosome 20 by SKY. Importantly, the mosaicism for trisomy 12 gradually increased up to 89% by passage 30, suggesting that this karyotypic abnormality provides a selective advantage. Similarly, the SHEF-3 line also acquired a trisomy of chromosome 14 as early as passage 10. However, this karyotypic aberration did not confer selective advantage to the genetically abnormal cells within the bulk culture and the level of mosaicism for the trisomy 14 remained overtime between 15%-36%. Strikingly, however, a much older hESC line, SHEF-1, which was maintained for 185 passages in feeders did not undergo any numerical or structural chromosomal change after 30 passages in feeder-free culture and over 215 passages in total. These results support the concept that feeder-free conditions may partially contribute to hESC chromosomal changes but also confirm the hypothesis that regardless of the culture conditions, culture duration or splitting methods, some hESC lines are inherently more prone than others to karyotypic instability.

Unsuccessful derivation of human embryonic stem cell lines from pairs of human blastomeres.

PubMed

Fong, Chui-Yee; Richards, Mark; Bongso, Ariff

2006-08-01

Human embryonic stem cells (hESC) that differentiate into all three primordial germ layers have been established. Differentiation of these cells into desirable lineages offers hope for future transplantation therapies. Currently, hESC lines are derived from the inner cell mass (ICM) of blastocysts, leading to destruction of the embryo, and thus the process is ethically controversial. Successful attempts at deriving hESC lines from blastomeres without destruction of the ensuing embryo have not been reported. One or two blastomeres are routinely biopsied from 8-cell embryos for preimplantation genetic diagnosis. In this study it was therefore attempted to derive hESC lines from paired blastomeres. Of 66 pairs of 8-cell stage blastomeres, four pairs produced two morula and two blastocyst-like structures. When plated on mitomycin-C-treated mouse embryonic fibroblasts, one morula and one blastocyst-like structure separately produced small colonies containing hESC-like cells with prominent nucleoli and high nuclear-cytoplasmic ratios. When these colonies were detached and plated onto fresh feeders, there was no further colony formation or ensuing hESC lines. The results showed that it might not be possible to derive hESC lines directly from paired blastomeres. A minimum number of blastomeres in close contact with one another may be required to successfully generate an hESC line as blastomeres, like ICM and hESC cells, may be 'social' cells.

No Relationship between Embryo Morphology and Successful Derivation of Human Embryonic Stem Cell Lines

PubMed Central

Ström, Susanne; Rodriguez-Wallberg, Kenny; Holm, Frida; Bergström, Rosita; Eklund, Linda; Strömberg, Anne-Marie; Hovatta, Outi

2010-01-01

Background The large number (30) of permanent human embryonic stem cell (hESC) lines and additional 29 which did not continue growing, in our laboratory at Karolinska Institutet have given us a possibility to analyse the relationship between embryo morphology and the success of derivation of hESC lines. The derivation method has been improved during the period 2002–2009, towards fewer xeno-components. Embryo quality is important as regards the likelihood of pregnancy, but there is little information regarding likelihood of stem cell derivation. Methods We evaluated the relationship of pronuclear zygote stage, the score based on embryo morphology and developmental rate at cleavage state, and the morphology of the blastocyst at the time of donation to stem cell research, to see how they correlated to successful establishment of new hESC lines. Results Derivation of hESC lines succeeded from poor quality and good quality embryos in the same extent. In several blastocysts, no real inner cell mass (ICM) was seen, but permanent well growing hESC lines could be established. One tripronuclear (3PN) zygote, which developed to blastocyst stage, gave origin to a karyotypically normal hESC line. Conclusion Even very poor quality embryos with few cells in the ICM can give origin to hESC lines. PMID:21217828

Differences in lymphocyte developmental potential between human embryonic stem cell and umbilical cord blood–derived hematopoietic progenitor cells

PubMed Central

Martin, Colin H.; Woll, Petter S.; Ni, Zhenya; Zúñiga-Pflücker, Juan Carlos

2008-01-01

Hematopoietic progenitor cells derived from human embryonic stem cells (hESCs) develop into diverse mature hematopoietic lineages, including lymphocytes. Whereas functional natural killer (NK) cells can be efficiently generated in vitro from hESC-derived CD34+ cells, studies of T- and B-cell development from hESCs have been much more limited. Here, we demonstrate that despite expressing functional Notch-1, CD34+ cells from hESCs did not derive T cells when cocultured with OP9 cells expressing Delta-like 1, or in fetal thymus organ culture. hESC-derived CD34+ cells also did not produce B cells in vitro. In contrast, CD34+ cells isolated from UCB routinely generated T and B cells when cultured in the same conditions. Notably, both undifferentiated hESCs, and sorted hESC-derived populations with hematopoietic developmental potential exhibited constitutive expression of ID family genes and of transcriptional targets of stem cell factor–induced signaling. These pathways both inhibit T-cell development and promote NK-cell development. Together, these results demonstrate fundamental differences between hESC-derived hematopoietic progenitors and analogous primary human cells. Therefore, hESCs can be more readily supported to differentiate into certain cell types than others, findings that have important implications for derivation of defined lineage-committed populations from hESCs. PMID:18621931

Differences in lymphocyte developmental potential between human embryonic stem cell and umbilical cord blood-derived hematopoietic progenitor cells.

PubMed

Martin, Colin H; Woll, Petter S; Ni, Zhenya; Zúñiga-Pflücker, Juan Carlos; Kaufman, Dan S

2008-10-01

Hematopoietic progenitor cells derived from human embryonic stem cells (hESCs) develop into diverse mature hematopoietic lineages, including lymphocytes. Whereas functional natural killer (NK) cells can be efficiently generated in vitro from hESC-derived CD34(+) cells, studies of T- and B-cell development from hESCs have been much more limited. Here, we demonstrate that despite expressing functional Notch-1, CD34(+) cells from hESCs did not derive T cells when cocultured with OP9 cells expressing Delta-like 1, or in fetal thymus organ culture. hESC-derived CD34(+) cells also did not produce B cells in vitro. In contrast, CD34(+) cells isolated from UCB routinely generated T and B cells when cultured in the same conditions. Notably, both undifferentiated hESCs, and sorted hESC-derived populations with hematopoietic developmental potential exhibited constitutive expression of ID family genes and of transcriptional targets of stem cell factor-induced signaling. These pathways both inhibit T-cell development and promote NK-cell development. Together, these results demonstrate fundamental differences between hESC-derived hematopoietic progenitors and analogous primary human cells. Therefore, hESCs can be more readily supported to differentiate into certain cell types than others, findings that have important implications for derivation of defined lineage-committed populations from hESCs.

Podocalyxin as a major pluripotent marker and novel keratan sulfate proteoglycan in human embryonic and induced pluripotent stem cells.

PubMed

Toyoda, Hidenao; Nagai, Yuko; Kojima, Aya; Kinoshita-Toyoda, Akiko

2017-04-01

Podocalyxin (PC) was first identified as a heavily sialylated transmembrane protein of glomerular podocytes. Recent studies suggest that PC is a remarkable glycoconjugate that acts as a universal glyco-carrier. The glycoforms of PC are responsible for multiple functions in normal tissue, human cancer cells, human embryonic stem cells (hESCs), and human induced pluripotent stem cells (hiPSCs). PC is employed as a major pluripotent marker of hESCs and hiPSCs. Among the general antibodies for human PC, TRA-1-60 and TRA-1-81 recognize the keratan sulfate (KS)-related structures. Therefore, It is worthwhile to summarize the outstanding chemical characteristic of PC, including the KS-related structures. Here, we review the glycoforms of PC and discuss the potential of PC as a novel KS proteoglycan in undifferentiated hESCs and hiPSCs.

Brief report: ectopic expression of NUP98-HOXA10 augments erythroid differentiation of human embryonic stem cells.

PubMed

Ji, Junfeng; Risueño, Ruth M; Hong, Seokho; Allan, David; Rosten, Patty; Humphries, Keith; Bhatia, Mickie

2011-04-01

Hox genes encode highly conserved transcription factors that have been implicated in hematopoietic development and self-renewal of hematopoietic stem cells (HSCs) and hematopoietic development. The potency of NUP98-HOXA10hd (NA10) on adult murine bone marrow HSC self-renewal prompted us to examine its effect on specification and proliferation of hematopoietic cells derived from human embryonic stem cells (hESCs). Here, we demonstrate that expression of NA10 in hESCs influences the hematopoietic differentiation program. The specific effect of NA10 is dependent on the developmental stage of hematopoietic emergence from hESCs. Overexpression of NA10 in either undifferentiated hESCs or early hemogenic precursors augmented the frequency of CD45(-) GlycophorinA(+) cells and erythroid progenitors (blast-forming unit-erythrocyte). In contrast, targeted NA10 expression in primitive CD34+ cells committed to the hematopoietic lineage had no effect on erythropoietic capacity but instead increased hematopoietic progenitor proliferation. Our study reveals a novel neomorphic effect of NA10 in early human erythroid development from pluripotent stem cells. Copyright © 2011 AlphaMed Press.

Effects of ß-TCP scaffolds on neurogenic and osteogenic differentiation of human embryonic stem cells.

PubMed

Arpornmaeklong, Premjit; Pressler, Michael J

2018-01-01

Extracellular matrix (ECM) and adhesion molecules play crucial roles in regulating growth and differentiation of stem cells. The current study aimed to investigate the effects of beta-tricalcium phosphate (ß-TCP) scaffolds on differentiation and expression of ECM and adhesion molecules of human embryonic stem cells (hESCs). Undifferentiated hESCs were seeded on ß-TCP scaffolds and cell culture plates and cultured in growth and osteogenic medium for 21 days. Scanning electron microscopy (SEM) displayed adhesion and growth of hESCs on the porous ß-TCP scaffolds. Histological analysis, immunohistochemical staining and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) demonstrated that the scaffolds supported growth and differentiation of hESCs. Expression levels of neural crest related genes (AP2a, FoxD3, HNK1, P75, Sox1, Sox10) and osteoblast-related genes (Runx2, SPP1 and BGLA) on the scaffolds in osteogenic medium were significantly higher than on the scaffolds in growth and cell culture plates in osteogenic medium, respectively (p<0.05). Polymerase chain reaction array experiments demonstrated increased expression of ECM and adhesion molecule-related genes on the scaffolds. In conclusion, osteoconductive scaffolds such as ß-TCP scaffolds promoted differentiation of hESCs, particularly expression of genes related to neural crest stem cell and osteoblastic differentiations. Beta-TCP scaffolds could be an alternative cell culture substrate for neural crest and osteogenic differentiation of hESCs. Optimization of culture medium may be necessary to enhance lineage restriction of hESCs on the ß-TCP scaffolds. Copyright © 2017 Elsevier GmbH. All rights reserved.

The influence of early embryo traits on human embryonic stem cell derivation efficiency.

PubMed

O'Leary, Thomas; Heindryckx, Björn; Lierman, Sylvie; Van der Jeught, Margot; Menten, Björn; Deforce, Dieter; Cornelissen, Ria; de Sousa Lopes, Susana Chuva; De Sutter, Petra

2011-05-01

Despite its prognostic value in in vitro fertilization, early embryo morphology is not reported on in the derivation of human embryonic stem cell (hESC) lines. Standard hESC derivation does rely on blastocyst development and its efficiency is highly correlated to inner cell mass (ICM) quality. Poor-quality embryos (PQEs) donated for hESC derivation may have a range of cleavage-stage abnormalities that are known to compromise further development. This study was implemented to determine whether specific PQEs traits influence the efficiency of good-quality ICMs to derive new hESC lines. We found that although the types of PQEs investigated were all able to make blastocysts with good-quality ICMs, the ICMs were unequal in their ability to derive hESCs. Good-quality ICMs from embryos with multiple poor-quality traits were unable to generate hESC lines, in contrast to good-quality ICMs from embryos with a single poor-quality trait. In addition, our data suggest a direct correlation between the number of ICM cells present in the blastocyst and its capacity to derive new hESC lines. This study is the first to demonstrate that ICM quality alone is an incomplete indicator of hESC derivation and that application of in vitro fertilization-based early embryo scoring can help predict hESC derivation efficiency. Experiments aiming to quantify, improve upon, or compare hESC derivation efficiency should thus take into consideration early embryo morphology scoring for the comparison of groups with equal developmental competence.

Derivation of Human Skin Fibroblast Lines for Feeder Cells of Human Embryonic Stem Cells.

PubMed

Unger, Christian; Felldin, Ulrika; Rodin, Sergey; Nordenskjöld, Agneta; Dilber, Sirac; Hovatta, Outi

2016-02-03

After the first derivations of human embryonic stem cell (hESC) lines on fetal mouse feeder cell layers, the idea of using human cells instead of mouse cells as feeder cells soon arose. Mouse cells bear a risk of microbial contamination, and nonhuman immunogenic proteins are absorbed from the feeders to hESCs. Human skin fibroblasts can be effectively used as feeder cells for hESCs. The same primary cell line, which can be safely used for up to 15 passages after stock preparations, can be expanded and used for large numbers of hESC derivations and cultures. These cells are relatively easy to handle and maintain. No animal facilities or animal work is needed. Here, we describe the derivation, culture, and cryopreservation procedures for research-grade human skin fibroblast lines. We also describe how to make feeder layers for hESCs using these fibroblasts. Copyright © 2016 John Wiley & Sons, Inc.

Feeder & basic fibroblast growth factor-free culture of human embryonic stem cells: Role of conditioned medium from immortalized human feeders.

PubMed

Teotia, Pooja; Sharma, Shilpa; Airan, Balram; Mohanty, Sujata

2016-12-01

Human embryonic stem cell (hESC) lines are commonly maintained on inactivated feeder cells, in the medium supplemented with basic fibroblast growth factor (bFGF). However, limited availability of feeder cells in culture, and the high cost of growth factors limit their use in scalable expansion of hESC cultures for clinical application. Here, we describe an efficient and cost-effective feeder and bFGF-free culture of hESCs using conditioned medium (CM) from immortalized feeder cells. KIND-1 hESC cell line was cultured in CM, collected from primary mouse embryonic fibroblast, human foreskin fibroblast (HFF) and immortalized HFF (I-HFF). Pluripotency of KIND-1 hESC cell line was confirmed by expression of genes, proteins and cell surface markers. In culture, these cells retained normal morphology, expressed all cell surface markers, could differentiate to embryoid bodies upon culture in vitro. Furthermore, I-HFF feeder cells without supplementation of bFGF released ample amount of endogenous bFGF to maintain stemness of hESC cells. The study results described the use of CM from immortalized feeder cells as a consistent source and an efficient, inexpensive feeder-free culture system for the maintenance of hESCs. Moreover, it was possible to maintain hESCs without exogenous supplementation of bFGF. Thus, the study could be extended to scalable expansion of hESC cultures for therapeutic purposes.

Derivation and characterization of human embryonic stem cell lines from poor quality embryos.

PubMed

Liu, Weiqiang; Yin, Yifei; Long, Xiaolin; Luo, Yumei; Jiang, Yonghua; Zhang, Wenhong; Du, Hongzi; Li, Shaoying; Zheng, Yuhong; Li, Qing; Chen, Xinjie; Liao, Baoping; Xiao, Guohong; Wang, Weihua; Sun, Xiaofang

2009-04-01

Poor quality embryos discarded from in vitro fertilization (IVF) laboratories are good sources for deriving human embryonic stem cell (hESC) lines. In this study, 166 poor quality embryos donated from IVF centers on day 3 were cultured in a blastocyst medium for 2 days, and 32 early blastocysts were further cultured in a blastocyst optimum culture medium for additional 2 days so that the inner cell masses (ICMs) could be identified and isolated easily. The ICMs of 17 blastocysts were isolated by a mechanical method, while those of the other 15 blastocysts were isolated by immunosurgery. All isolated ICMs were inoculated onto a feeder layer for subcultivation. The rates of ICM attachment, primary ICM colony formation and the efficiency of hESC derivation were similar between the ICMs isolated by the two methods (P>0.05). As a result, four new hESC lines were established. Three cell lines had normal karyotypes and one had an unbalanced Robertsonian translocation. All cell lines showed normal hESC characteristics and had the differentiation ability. In conclusion, we established a stable and effective method for hESC isolation and culture, and it was confirmed that the mechanical isolation was an effective method to isolate ICMs from poor embryos. These results further indicate that hESC lines can be derived from poor quality embryos discarded by IVF laboratories.

Establishment of Homozygote Mutant Human Embryonic Stem Cells by Parthenogenesis.

PubMed

Epsztejn-Litman, Silvina; Cohen-Hadad, Yaara; Aharoni, Shira; Altarescu, Gheona; Renbaum, Paul; Levy-Lahad, Ephrat; Schonberger, Oshrat; Eldar-Geva, Talia; Zeligson, Sharon; Eiges, Rachel

2015-01-01

We report on the derivation of a diploid 46(XX) human embryonic stem cell (HESC) line that is homozygous for the common deletion associated with Spinal muscular atrophy type 1 (SMA) from a pathenogenetic embryo. By characterizing the methylation status of three different imprinted loci (MEST, SNRPN and H19), monitoring the expression of two parentally imprinted genes (SNRPN and H19) and carrying out genome-wide SNP analysis, we provide evidence that this cell line was established from the activation of a mutant oocyte by diploidization of the entire genome. Therefore, our SMA parthenogenetic HESC (pHESC) line provides a proof-of-principle for the establishment of diseased HESC lines without the need for gene manipulation. As mutant oocytes are easily obtained and readily available during preimplantation genetic diagnosis (PGD) cycles, this approach should provide a powerful tool for disease modelling and is especially advantageous since it can be used to induce large or complex mutations in HESCs, including gross DNA alterations and chromosomal rearrangements, which are otherwise hard to achieve.

A Scalable System for Production of Functional Pancreatic Progenitors from Human Embryonic Stem Cells

PubMed Central

Schulz, Thomas C.; Young, Holly Y.; Agulnick, Alan D.; Babin, M. Josephine; Baetge, Emmanuel E.; Bang, Anne G.; Bhoumik, Anindita; Cepa, Igor; Cesario, Rosemary M.; Haakmeester, Carl; Kadoya, Kuniko; Kelly, Jonathan R.; Kerr, Justin; Martinson, Laura A.; McLean, Amanda B.; Moorman, Mark A.; Payne, Janice K.; Richardson, Mike; Ross, Kelly G.; Sherrer, Eric S.; Song, Xuehong; Wilson, Alistair Z.; Brandon, Eugene P.; Green, Chad E.; Kroon, Evert J.; Kelly, Olivia G.; D’Amour, Kevin A.; Robins, Allan J.

2012-01-01

Development of a human embryonic stem cell (hESC)-based therapy for type 1 diabetes will require the translation of proof-of-principle concepts into a scalable, controlled, and regulated cell manufacturing process. We have previously demonstrated that hESC can be directed to differentiate into pancreatic progenitors that mature into functional glucose-responsive, insulin-secreting cells in vivo. In this study we describe hESC expansion and banking methods and a suspension-based differentiation system, which together underpin an integrated scalable manufacturing process for producing pancreatic progenitors. This system has been optimized for the CyT49 cell line. Accordingly, qualified large-scale single-cell master and working cGMP cell banks of CyT49 have been generated to provide a virtually unlimited starting resource for manufacturing. Upon thaw from these banks, we expanded CyT49 for two weeks in an adherent culture format that achieves 50–100 fold expansion per week. Undifferentiated CyT49 were then aggregated into clusters in dynamic rotational suspension culture, followed by differentiation en masse for two weeks with a four-stage protocol. Numerous scaled differentiation runs generated reproducible and defined population compositions highly enriched for pancreatic cell lineages, as shown by examining mRNA expression at each stage of differentiation and flow cytometry of the final population. Islet-like tissue containing glucose-responsive, insulin-secreting cells was generated upon implantation into mice. By four- to five-months post-engraftment, mature neo-pancreatic tissue was sufficient to protect against streptozotocin (STZ)-induced hyperglycemia. In summary, we have developed a tractable manufacturing process for the generation of functional pancreatic progenitors from hESC on a scale amenable to clinical entry. PMID:22623968

Human embryonic stem cell lines derived from single blastomeres of two 4-cell stage embryos

PubMed Central

Geens, Mieke; Mateizel, Ileana; Sermon, Karen; De Rycke, Martine; Spits, Claudia; Cauffman, Greet; Devroey, Paul; Tournaye, Herman; Liebaers, Inge; Van de Velde, Hilde

2009-01-01

BACKGROUND Recently, we demonstrated that single blastomeres of a 4-cell stage human embryo are able to develop into blastocysts with inner cell mass and trophectoderm. To further investigate potency at the 4-cell stage, we aimed to derive pluripotent human embryonic stem cells (hESC) from single blastomeres. METHODS Four 4-cell stage embryos were split on Day 2 of preimplantation development and the 16 blastomeres were individually cultured in sequential medium. On Day 3 or 4, the blastomere-derived embryos were plated on inactivated mouse embryonic fibroblasts (MEFs). RESULTS Ten out of sixteen blastomere-derived morulae attached to the MEFs, and two produced an outgrowth. They were mechanically passaged onto fresh MEFs as described for blastocyst ICM-derived hESC, and shown to express the typical stemness markers by immunocytochemistry and/or RT–PCR. In vivo pluripotency was confirmed by the presence of all three germ layers in the teratoma obtained after injection in immunodeficient mice. The first hESC line displays a mosaic normal/abnormal 46, XX, dup(7)(q33qter), del(18)(q23qter) karyotype. The second hESC line displays a normal 46, XY karyotype. CONCLUSION We report the successful derivation and characterization of two hESC lines from single blastomeres of four split 4-cell stage human embryos. These two hESC lines were derived from distinct embryos, proving that at least one of the 4-cell stage blastomeres is pluripotent. PMID:19633307

Derivation of new human embryonic stem cell lines reveals rapid epigenetic progression in vitro that can be prevented by chemical modification of chromatin

PubMed Central

Diaz Perez, Silvia V.; Kim, Rachel; Li, Ziwei; Marquez, Victor E.; Patel, Sanjeet; Plath, Kathrin; Clark, Amander T.

2012-01-01

Human embryonic stem cells (hESCs) are pluripotent cell types derived from the inner cell mass of human blastocysts. Recent data indicate that the majority of established female XX hESC lines have undergone X chromosome inactivation (XCI) prior to differentiation, and XCI of hESCs can be either XIST-dependent (class II) or XIST-independent (class III). XCI of female hESCs precludes the use of XX hESCs as a cell-based model for examining mechanisms of XCI, and will be a challenge for studying X-linked diseases unless strategies are developed to reactivate the inactive X. In order to recover nuclei with two active X chromosomes (class I), we developed a reprogramming strategy by supplementing hESC media with the small molecules sodium butyrate and 3-deazaneplanocin A (DZNep). Our data demonstrate that successful reprogramming can occur from the XIST-dependent class II nuclear state but not class III nuclear state. To determine whether these small molecules prevent XCI, we derived six new hESC lines under normoxic conditions (UCLA1–UCLA6). We show that class I nuclei are present within the first 20 passages of hESC derivation prior to cryopreservation, and that supplementation with either sodium butyrate or DZNep preserve class I nuclei in the self-renewing state. Together, our data demonstrate that self-renewal and survival of class I nuclei are compatible with normoxic hESC derivation, and that chemical supplementation after derivation provides a strategy to prevent epigenetic progression and retain nuclei with two active X chromosomes in the self-renewing state. PMID:22058289

Comparison of a teratogenic transcriptome-based predictive test based on human embryonic versus inducible pluripotent stem cells.

PubMed

Shinde, Vaibhav; Perumal Srinivasan, Sureshkumar; Henry, Margit; Rotshteyn, Tamara; Hescheler, Jürgen; Rahnenführer, Jörg; Grinberg, Marianna; Meisig, Johannes; Blüthgen, Nils; Waldmann, Tanja; Leist, Marcel; Hengstler, Jan Georg; Sachinidis, Agapios

2016-12-30

Human embryonic stem cells (hESCs) partially recapitulate early embryonic three germ layer development, allowing testing of potential teratogenic hazards. Because use of hESCs is ethically debated, we investigated the potential for human induced pluripotent stem cells (hiPSCs) to replace hESCs in such tests. Three cell lines, comprising hiPSCs (foreskin and IMR90) and hESCs (H9) were differentiated for 14 days. Their transcriptome profiles were obtained on day 0 and day 14 and analyzed by comprehensive bioinformatics tools. The transcriptomes on day 14 showed that more than 70% of the "developmental genes" (regulated genes with > 2-fold change on day 14 compared to day 0) exhibited variability among cell lines. The developmental genes belonging to all three cell lines captured biological processes and KEGG pathways related to all three germ layer embryonic development. In addition, transcriptome profiles were obtained after 14 days of exposure to teratogenic valproic acid (VPA) during differentiation. Although the differentially regulated genes between treated and untreated samples showed more than 90% variability among cell lines, VPA clearly antagonized the expression of developmental genes in all cell lines: suppressing upregulated developmental genes, while inducing downregulated ones. To quantify VPA-disturbed development based on developmental genes, we estimated the "developmental potency" (D p ) and "developmental index" (D i ). Despite differences in genes deregulated by VPA, uniform D i values were obtained for all three cell lines. Given that the D i values for VPA were similar for hESCs and hiPSCs, D i can be used for robust hazard identification, irrespective of whether hESCs or hiPSCs are used in the test systems.

Brief Report: External Beam Radiation Therapy for the Treatment of Human Pluripotent Stem Cell-Derived Teratomas.

PubMed

Lee, Andrew S; Tang, Chad; Hong, Wan Xing; Park, Sujin; Bazalova-Carter, Magdalena; Nelson, Geoff; Sanchez-Freire, Veronica; Bakerman, Isaac; Zhang, Wendy; Neofytou, Evgenios; Connolly, Andrew J; Chan, Charles K; Graves, Edward E; Weissman, Irving L; Nguyen, Patricia K; Wu, Joseph C

2017-08-01

Human pluripotent stem cells, including human embryonic stem cells (hESCs) and human induced PSCs (hiPSCs), have great potential as an unlimited donor source for cell-based therapeutics. The risk of teratoma formation from residual undifferentiated cells, however, remains a critical barrier to the clinical application of these cells. Herein, we describe external beam radiation therapy (EBRT) as an attractive option for the treatment of this iatrogenic growth. We present evidence that EBRT is effective in arresting growth of hESC-derived teratomas in vivo at day 28 post-implantation by using a microCT irradiator capable of targeted treatment in small animals. Within several days of irradiation, teratomas derived from injection of undifferentiated hESCs and hiPSCs demonstrated complete growth arrest lasting several months. In addition, EBRT reduced reseeding potential of teratoma cells during serial transplantation experiments, requiring irradiated teratomas to be seeded at 1 × 10 3 higher doses to form new teratomas. We demonstrate that irradiation induces teratoma cell apoptosis, senescence, and growth arrest, similar to established radiobiology mechanisms. Taken together, these results provide proof of concept for the use of EBRT in the treatment of existing teratomas and highlight a strategy to increase the safety of stem cell-based therapies. Stem Cells 2017;35:1994-2000. © 2017 AlphaMed Press.

Formation and hematopoietic differentiation of human embryoid bodies by suspension and hanging drop cultures.

PubMed

Cerdan, Chantal; Hong, Seok Ho; Bhatia, Mickie

2007-10-01

The in vitro aggregation of human embryonic stem cells (hESCs) into clusters termed embryoid bodies (EBs) allows for the spontaneous differentiation of cells representing endoderm, mesoderm, and ectoderm lineages. This stochastic process results however, in the generation of low numbers of differentiated cells, and can be enhanced to some extent by the addition of exogenous growth factors or overexpression of regulatory genes. In the authors' laboratory, the use of hematopoietic cytokines in combination with the mesoderm inducer bone morphogenetic protein-4 (BMP-4) was able to generate up to 90% of CD45(+) hematopoietic cells with colony-forming unit (CFU) activity. This unit describes two protocols that have been successfully applied in the authors' laboratory for the generation of EBs in (1) suspension and (2) hanging drop (HD) cultures from enzymatically digested clumps of undifferentiated hESC colonies.

Progenitor cells for regenerative medicine and consequences of ART and cloning-associated epimutations.

PubMed

Laprise, Shari L

2010-06-01

The "holy grail" of regenerative medicine is the identification of an undifferentiated progenitor cell that is pluripotent, patient specific, and ethically unambiguous. Such a progenitor cell must also be able to differentiate into functional, transplantable tissue, while avoiding the risks of immune rejection. With reports detailing aberrant genomic imprinting associated with assisted reproductive technologies (ART) and reproductive cloning, the idea that human embryonic stem cells (hESCs) derived from surplus in vitro fertilized embryos or nuclear transfer ESCs (ntESCs) harvested from cloned embryos may harbor dangerous epigenetic errors has gained attention. Various progenitor cell sources have been proposed for human therapy, from hESCs to ntESCs, and from adult stem cells to induced pluripotent stem cells (iPS and piPS cells). This review highlights the advantages and disadvantages of each of these technologies, with particular emphasis on epigenetic stability.

Derivation of Xeno-Free and GMP-Grade Human Embryonic Stem Cells – Platforms for Future Clinical Applications

PubMed Central

Aizenman, Einat; Kirshberg, Sophie; Ilouz, Nili; Gil, Yaniv; Berman-Zaken, Yael; Perlman, Temima Schnitzer; Geva, Nitshia; Levy, Ora; Arbell, Daniel; Simon, Alex; Ben-Meir, Assaf; Shufaro, Yoel; Laufer, Neri; Reubinoff, Benjamin E.

2012-01-01

Clinically compliant human embryonic stem cells (hESCs) should be developed in adherence to ethical standards, without risk of contamination by adventitious agents. Here we developed for the first time animal-component free and good manufacturing practice (GMP)-compliant hESCs. After vendor and raw material qualification, we derived xeno-free, GMP-grade feeders from umbilical cord tissue, and utilized them within a novel, xeno-free hESC culture system. We derived and characterized three hESC lines in adherence to regulations for embryo procurement, and good tissue, manufacturing and laboratory practices. To minimize freezing and thawing, we continuously expanded the lines from initial outgrowths and samples were cryopreserved as early stocks and banks. Batch release criteria included DNA-fingerprinting and HLA-typing for identity, characterization of pluripotency-associated marker expression, proliferation, karyotyping and differentiation in-vitro and in-vivo. These hESCs may be valuable for regenerative therapy. The ethical, scientific and regulatory methodology presented here may serve for development of additional clinical-grade hESCs. PMID:22745653

Targeted Disruption of the β2-Microglobulin Gene Minimizes the Immunogenicity of Human Embryonic Stem Cells.

PubMed

Wang, Dachun; Quan, Yuan; Yan, Qing; Morales, John E; Wetsel, Rick A

2015-10-01

Human embryonic stem cells (hESCs) are a promising source of cells for tissue regeneration, yet histoincompatibility remains a major challenge to their clinical application. Because the human leukocyte antigen class I (HLA-I) molecules are the primary mediators of immune rejection, we hypothesized that cells derived from a hESC line lacking HLA-I expression could be transplanted without evoking a robust immune response from allogeneic recipients. In the present study, we used the replacement targeting strategy to delete exons 2 and 3 of β2-microglobulin on both gene alleles in hESCs. Because β2-microglobulin serves as the HLA-I light chain, disruption of the β2-microglobulin gene led to complete HLA-I deficiency on the cell surface of hESCs and their derivatives. Therefore, these cells were resistant to CD8+ T-cell-mediated destruction. Although interferon-γ (IFN-γ) treatment significantly induced β2-microglobulin expression, promoting CD8+ T cell-mediated killing of control hESCs and their derivatives, CD8+ T-cell-mediated cytotoxicity was barely observed with β2-microglobulin-null hESCs and their derivatives treated with IFN-γ. This genetic manipulation to disrupt HLA-I expression did not affect the self-renewal capacity, genomic stability, or pluripotency of hESCs. Despite being relatively sensitive to natural killer (NK) cell-mediated killing due to the lack of HLA-I expression, when transplanted into NK cell-depleted immunocompetent mice, β2-microglobulin-null hESCs developed into tumors resembling those derived from control hESCs in severe combined immunodeficiency mice. These results demonstrate that β2-microglobulin-null hESCs significantly reduce immunogenicity to CD8+ T cells and might provide a renewable source of cells for tissue regeneration without the need for HLA matching in the future. This study reports the generation of a novel β2-microglobulin (B2M)-/- human embryonic stem cell (hESC) line. Differentiated mature cells from this line do not express cell surface human leukocyte antigen molecules even after interferon-γ stimulation and are resistant to alloreactive CD8+ T cells. Moreover, this B2M-/- hESC line contains no off-target integration or cleavage events, is devoid of stable B2M mRNA, exhibits a normal karyotype, and retains its self-renewal capacity, genomic stability, and pluripotency. Although B2M-/- hESC-derived cells are more susceptible to natural killer (NK) cells, murine transplantation studies have indicated that they are, overall, much less immunogenic than normal hESCs. Thus, these data show for the first time that, in vivo, the advantages provided by B2M-/- hESC-derived cells in avoiding CD8+ T-cell killing appear significantly greater than any disadvantage caused by increased susceptibility to NK cells. ©AlphaMed Press.

Targeted Disruption of the β2-Microglobulin Gene Minimizes the Immunogenicity of Human Embryonic Stem Cells

PubMed Central

Quan, Yuan; Yan, Qing; Morales, John E.

2015-01-01

Human embryonic stem cells (hESCs) are a promising source of cells for tissue regeneration, yet histoincompatibility remains a major challenge to their clinical application. Because the human leukocyte antigen class I (HLA-I) molecules are the primary mediators of immune rejection, we hypothesized that cells derived from a hESC line lacking HLA-I expression could be transplanted without evoking a robust immune response from allogeneic recipients. In the present study, we used the replacement targeting strategy to delete exons 2 and 3 of β2-microglobulin on both gene alleles in hESCs. Because β2-microglobulin serves as the HLA-I light chain, disruption of the β2-microglobulin gene led to complete HLA-I deficiency on the cell surface of hESCs and their derivatives. Therefore, these cells were resistant to CD8+ T-cell-mediated destruction. Although interferon-γ (IFN-γ) treatment significantly induced β2-microglobulin expression, promoting CD8+ T cell-mediated killing of control hESCs and their derivatives, CD8+ T-cell-mediated cytotoxicity was barely observed with β2-microglobulin-null hESCs and their derivatives treated with IFN-γ. This genetic manipulation to disrupt HLA-I expression did not affect the self-renewal capacity, genomic stability, or pluripotency of hESCs. Despite being relatively sensitive to natural killer (NK) cell-mediated killing due to the lack of HLA-I expression, when transplanted into NK cell-depleted immunocompetent mice, β2-microglobulin-null hESCs developed into tumors resembling those derived from control hESCs in severe combined immunodeficiency mice. These results demonstrate that β2-microglobulin-null hESCs significantly reduce immunogenicity to CD8+ T cells and might provide a renewable source of cells for tissue regeneration without the need for HLA matching in the future. Significance This study reports the generation of a novel β2-microglobulin (B2M)−/− human embryonic stem cell (hESC) line. Differentiated mature cells from this line do not express cell surface human leukocyte antigen molecules even after interferon-γ stimulation and are resistant to alloreactive CD8+ T cells. Moreover, this B2M−/− hESC line contains no off-target integration or cleavage events, is devoid of stable B2M mRNA, exhibits a normal karyotype, and retains its self-renewal capacity, genomic stability, and pluripotency. Although B2M−/− hESC-derived cells are more susceptible to natural killer (NK) cells, murine transplantation studies have indicated that they are, overall, much less immunogenic than normal hESCs. Thus, these data show for the first time that, in vivo, the advantages provided by B2M−/− hESC-derived cells in avoiding CD8+ T-cell killing appear significantly greater than any disadvantage caused by increased susceptibility to NK cells. PMID:26285657

[Embryonic stem cells - a scientific by-product of the assisted reproduction technology?].

PubMed

Sterthaus, Oliver; Zhang, Hong; De Geyter, Christian

2009-12-01

The differentiation potential of embryonic stem (ES) cells seems to be higher when compared to adult stem cells, which mainly differentiate into certain tissue types only. ES cells have the potential to play an important role in regenerative medicine as demonstrated with murine ES cells. However, with human embryonic stem cells (hESC) several obstacles still have to be overcome, when these are to be used in clinical applications. The expansion of hESC, safety issues as well as the immune-tolerance after transplantation are all problems that still have to be solved. Since 2005 the derivation of hESC lines from super-numerous embryos has become permitted in Switzerland, albeit under strictly restrictive guidelines. In 2008 the Basler hESC laboratory was successful in derivating the first hESC line with a normal chromosome complement in Switzerland (CHES2). Now, new applications allow the personalized establishment of immune-tolerant stem cells, which lead to the replacement of therapeutic cloning by induced pluripotent stem cells (iPS).

The quantitative proteomes of human-induced pluripotent stem cells and embryonic stem cells

PubMed Central

Munoz, Javier; Low, Teck Y; Kok, Yee J; Chin, Angela; Frese, Christian K; Ding, Vanessa; Choo, Andre; Heck, Albert J R

2011-01-01

Assessing relevant molecular differences between human-induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) is important, given that such differences may impact their potential therapeutic use. Controversy surrounds recent gene expression studies comparing hiPSCs and hESCs. Here, we present an in-depth quantitative mass spectrometry-based analysis of hESCs, two different hiPSCs and their precursor fibroblast cell lines. Our comparisons confirmed the high similarity of hESCs and hiPSCS at the proteome level as 97.8% of the proteins were found unchanged. Nevertheless, a small group of 58 proteins, mainly related to metabolism, antigen processing and cell adhesion, was found significantly differentially expressed between hiPSCs and hESCs. A comparison of the regulated proteins with previously published transcriptomic studies showed a low overlap, highlighting the emerging notion that differences between both pluripotent cell lines rather reflect experimental conditions than a recurrent molecular signature. PMID:22108792

Human amniotic epithelial cells as feeder layer to derive and maintain human embryonic stem cells from poor-quality embryos.

PubMed

Ávila-González, Daniela; Vega-Hernández, Eva; Regalado-Hernández, Juan Carlos; De la Jara-Díaz, Julio Francisco; García-Castro, Irma Lydia; Molina-Hernández, Anayansi; Moreno-Verduzco, Elsa Romelia; Razo-Aguilera, Guadalupe; Flores-Herrera, Héctor; Portillo, Wendy; Díaz-Martínez, Néstor Emmanuel; García-López, Guadalupe; Díaz, Néstor Fabián

2015-09-01

Data from the literature suggest that human embryonic stem cell (hESC) lines used in research do not genetically represent all human populations. The derivation of hESC through conventional methods involve the destruction of viable human embryos, as well the use of mouse embryonic fibroblasts as a feeder layer, which has several drawbacks. We obtained the hESC line (Amicqui-1) from poor-quality (PQ) embryos derived and maintained on human amniotic epithelial cells (hAEC). This line displays a battery of markers of pluripotency and we demonstrated the capacity of these cells to produce derivates of the three germ layers. Copyright © 2015. Published by Elsevier B.V.

Paradoxical Role of DNA Methylation in Activation of FoxA2 Gene Expression during Endoderm Development*

PubMed Central

Bahar Halpern, Keren; Vana, Tal; Walker, Michael D.

2014-01-01

The transcription factor FoxA2 is a master regulator of endoderm development and pancreatic beta cell gene expression. To elucidate the mechanisms underlying the activation of the FoxA2 gene during differentiation, we have compared the epigenetic status of undifferentiated human embryonic stem cells (hESCs), hESC-derived early endoderm stage cells (CXCR4+ cells), and pancreatic islet cells. Unexpectedly, a CpG island in the promoter region of the FoxA2 gene displayed paradoxically high levels of DNA methylation in expressing tissues (CXCR4+, islets) and low levels in nonexpressing tissues. This CpG island region was found to repress reporter gene expression and bind the Polycomb group protein SUZ12 and the DNA methyltransferase (DNMT)3b preferentially in undifferentiated hESCs as compared with CXCR4+ or islets cells. Consistent with this, activation of FoxA2 gene expression, but not CXCR4 or SOX17, was strongly inhibited by 5-aza-2′-deoxycytidine and by knockdown of DNMT3b. We hypothesize that in nonexpressing tissues, the lack of DNA methylation allows the binding of DNA methyltransferases and repressing proteins, such as Polycomb group proteins; upon differentiation, DNMT activation leads to CpG island methylation, causing loss of repressor protein binding. These results suggest a novel and unexpected role for DNA methylation in the activation of FoxA2 gene expression during differentiation. PMID:25016019

Effect of Chromatin Structure on the Extent and Distribution of DNA Double Strand Breaks Produced by Ionizing Radiation; Comparative Study of hESC and Differentiated Cells Lines.

PubMed

Venkatesh, Priyanka; Panyutin, Irina V; Remeeva, Evgenia; Neumann, Ronald D; Panyutin, Igor G

2016-01-02

Chromatin structure affects the extent of DNA damage and repair. Thus, it has been shown that heterochromatin is more protective against DNA double strand breaks (DSB) formation by ionizing radiation (IR); and that DNA DSB repair may proceed differently in hetero- and euchromatin regions. Human embryonic stem cells (hESC) have a more open chromatin structure than differentiated cells. Here, we study the effect of chromatin structure in hESC on initial DSB formation and subsequent DSB repair. DSB were scored by comet assay; and DSB repair was assessed by repair foci formation via 53BP1 antibody staining. We found that in hESC, heterochromatin is confined to distinct regions, while in differentiated cells it is distributed more evenly within the nuclei. The same dose of ionizing radiation produced considerably more DSB in hESC than in differentiated derivatives, normal human fibroblasts; and one cancer cell line. At the same time, the number of DNA repair foci were not statistically different among these cells. We showed that in hESC, DNA repair foci localized almost exclusively outside the heterochromatin regions. We also noticed that exposure to ionizing radiation resulted in an increase in heterochromatin marker H3K9me3 in cancer HT1080 cells, and to a lesser extent in IMR90 normal fibroblasts, but not in hESCs. These results demonstrate the importance of chromatin conformation for DNA protection and DNA damage repair; and indicate the difference of these processes in hESC.

The growth hormone-releasing hormone (GHRH) antagonist JV-1-36 inhibits proliferation and survival of human ectopic endometriotic stromal cells (ESCs) and the T HESC cell line.

PubMed

Annunziata, Marta; Grande, Cristina; Scarlatti, Francesca; Deltetto, Francesco; Delpiano, Elena; Camanni, Marco; Ghigo, Ezio; Granata, Riccarda

2010-08-01

To determine the effect of the GHRH antagonist JV-1-36 on proliferation and survival of primary ectopic human endometriotic stromal cells (ESCs) and the T HESC cell line. Prospective laboratory study. University hospital. 22 women with endometriosis (aged 34.8+/-5.7 years) undergoing therapeutic laparoscopy. Eutopic (n=10) and ectopic (n=22) endometrial tissues were collected from women who underwent therapeutic laparoscopic surgery for endometriosis (stage III/IV). Expression of GHRH, GHRH receptor (GHRH-R) and GHRH-R splice variant (SV) 1 mRNA was determined by reverse-transcription polymerase chain reaction (RT-PCR). The ESC proliferation was assessed by 5-bromo-2-deoxyuridine incorporation, cell survival by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and Trypan blue assay. The T HESC survival was evaluated by MTT, cyclic adenosine monophosphate (cAMP) levels by ELISA, extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation by Western blot, and insulin-like growth factor (IGF)-2 mRNA by real-time PCR. The ESCs and T HESCs, but not normal endometrial tissues, expressed GHRH-R mRNA; SV1 mRNA was determined in normal endometrial tissues, ESCs, and T HESCs; GHRH mRNAwas found in T HESCs; JV-1-36 inhibited ESC proliferation and ESC and T HESC survival. In T HESCs, JV-1-36 reduced cAMP production and ERK1/2 phosphorylation but had no effect on IGF-2 mRNA expression. The GHRH antagonist JV-1-36 inhibits endometriotic cell proliferation and survival, suggesting that GHRH antagonist may represent promising tools for treatment of endometriosis. Copyright (c) 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Derivation of Stromal (Skeletal and Mesenchymal) Stem-Like Cells from Human Embryonic Stem Cells

PubMed Central

Harkness, Linda; Abdallah, Basem M.; Elsafadi, Mona; Al-Nbaheen, May S.; Aldahmash, Abdullah; Kassem, Moustapha

2012-01-01

Derivation of bone forming cells (osteoblasts) from human embryonic stem cells (hESCs) is a prerequisite for their use in clinical applications. However, there is no standard protocol for differentiating hESCs into osteoblastic cells. The aim of this study was to identify the emergence of a human stromal (mesenchymal and skeletal) stem cell (hMSC)-like population, known to be osteoblastic cell precursors and to test their osteoblastic differentiation capacity in ex vivo cultures and in vivo. We cultured hESCs in a feeder-free environment using serum replacement and as suspension aggregates (embryoid bodies; hEBs). Over a 20 day developmental period, the hEBs demonstrated increasing enrichment for cells expressing hMSC markers: CD29, CD44, CD63, CD56, CD71, CD73, CD105, CD106, and CD166 as revealed by immunohistochemical staining and flow cytometry (fluorescence-activated cell sorting) analysis. Ex vivo differentiation of hEBs using bone morphogenic protein 2 (BMP2) combined with standard osteoblast induction medium led to weak osteoblastic induction. Conversely, subcutaneous in vivo implantation of day 20 hEBs in immune deficient mice, mixed with hydroxyapatite/tricalcium phosphate (HA/TCP) as an osteoconductive scaffold, revealed bone and cartilage, and fibrous tissue elements after 8 weeks. These tissues were of human origin and there was no evidence of differentiation to nonmesodermal tissues. hEBs implanted in the absence of HA/TCP formed vacuolated tissue containing glandular, fibrous and muscle-like tissue elements. Conversely, implantation of undifferentiated hESCs resulted in the formation of a teratoma containing a mixture of endodermal, mesodermal, and ectodermal tissues. Our study demonstrates that hMSC-like cells can be obtained from hESCs and they can be induced to form skeletal tissues in vivo when combined with HA/TCP. These findings are relevant for tissue engineering and suggest that differentiated hEBs can provide an unlimited source for functional osteogenic cells. PMID:22612317

Derivation of the King's College London human embryonic stem cell lines.

PubMed

Stephenson, Emma L; Braude, Peter R

2010-04-01

Since the derivation of the first human embryonic stem cell (hESC) line in 1998, there has been substantial interest in the potential of these cells for regenerative medicine and cell therapy and in the use of hESCs carrying clinically relevant genetic mutations as models for disease research and therapeutic target identification. There is still a need to improve derivation efficiency and further the understanding of the basic biology of these cells and to develop clinical grade culture systems with the aim of producing cell lines suitable for subsequent manipulation for therapy. The derivation of initial hESC lines at King's College London is discussed here, with focus on derivation methodology. Each of the derivations was distinctive. Although the stage and morphology of each blastocyst were generally similar in each attempt, the behaviour of the colonies was unpredictable; colony morphology and development was different with each attempt. Days 5, 6 and 7 blastocysts were used successfully, and the number of days until appearance of stem-like cells varied from 4 to 14 d. Routine characterisation analyses were performed on three lines, all of which displayed appropriate marker expression and survived cryopreservation-thaw cycles. From the lines discussed, four are at various stages of the deposition process with the UKSCB, one is pending submission and two are unsuitable for banking. Continued open and transparent reporting of results and collaborations will maximise the efficiency of derivation and facilitate the development of standardised protocols for the derivation and early culture of hESC lines.

The human embryonic stem cell proteome revealed by multidimensional fractionation followed by tandem mass spectrometry

PubMed Central

Zhao, Peng; Schulz, Thomas C.; Sherrer, Eric S.; Weatherly, D. Brent; Robins, Allan J.; Wells, Lance

2015-01-01

Human embryonic stem cells (hESCs) have received considerable attention due to their therapeutic potential and usefulness in understanding early development and cell fate commitment. In order to appreciate the unique properties of these pluripotent, self-renewing cells, we have performed an in-depth multidimensional fractionation followed by LC-MS/MS analysis of the hESCs harvested from defined media to elucidate expressed, phosphorylated, O-linked β-N-acetylglucosamine (O-GlcNAc) modified, and secreted proteins. From the triplicate analysis, we were able to assign more than 3000 proteins with less than 1% false-discovery rate. This analysis also allowed us to identify nearly 500 phosphorylation sites and 68 sites of O-GlcNAc modification with the same high confidence. Investigation of the phosphorylation sites allowed us to deduce the set of kinases that are likely active in these cells. We also identified more than 100 secreted proteins of hESCs that likely play a role in extracellular matrix formation and remodeling, as well as autocrine signaling for self-renewal and maintenance of the undifferentiated state. Finally, by performing in-depth analysis in triplicate, spectral counts were obtained for these proteins and posttranslationally modified peptides, which will allow us to perform relative quantitative analysis between these cells and any derived cell type in the future. PMID:25367160

Publishing SNP genotypes of human embryonic stem cell lines: policy statement of the International Stem Cell Forum Ethics Working Party.

PubMed

Knoppers, Bartha M; Isasi, Rosario; Benvenisty, Nissim; Kim, Ock-Joo; Lomax, Geoffrey; Morris, Clive; Murray, Thomas H; Lee, Eng Hin; Perry, Margery; Richardson, Genevra; Sipp, Douglas; Tanner, Klaus; Wahlström, Jan; de Wert, Guido; Zeng, Fanyi

2011-09-01

Novel methods and associated tools permitting individual identification in publicly accessible SNP databases have become a debatable issue. There is growing concern that current technical and ethical safeguards to protect the identities of donors could be insufficient. In the context of human embryonic stem cell research, there are no studies focusing on the probability that an hESC line donor could be identified by analyzing published SNP profiles and associated genotypic and phenotypic information. We present the International Stem Cell Forum (ISCF) Ethics Working Party's Policy Statement on "Publishing SNP Genotypes of Human Embryonic Stem Cell Lines (hESC)". The Statement prospectively addresses issues surrounding the publication of genotypic data and associated annotations of hESC lines in open access databases. It proposes a balanced approach between the goals of open science and data sharing with the respect for fundamental bioethical principles (autonomy, privacy, beneficence, justice and research merit and integrity).

Identification of Proteins Related to Epigenetic Regulation in the Malignant Transformation of Aberrant Karyotypic Human Embryonic Stem Cells by Quantitative Proteomics

PubMed Central

Sun, Yi; Yang, Yixuan; Zeng, Sicong; Tan, Yueqiu; Lu, Guangxiu; Lin, Ge

2014-01-01

Previous reports have demonstrated that human embryonic stem cells (hESCs) tend to develop genomic alterations and progress to a malignant state during long-term in vitro culture. This raises concerns of the clinical safety in using cultured hESCs. However, transformed hESCs might serve as an excellent model to determine the process of embryonic stem cell transition. In this study, ITRAQ-based tandem mass spectrometry was used to quantify normal and aberrant karyotypic hESCs proteins from simple to more complex karyotypic abnormalities. We identified and quantified 2583 proteins, and found that the expression levels of 316 proteins that represented at least 23 functional molecular groups were significantly different in both normal and abnormal hESCs. Dysregulated protein expression in epigenetic regulation was further verified in six pairs of hESC lines in early and late passage. In summary, this study is the first large-scale quantitative proteomic analysis of the malignant transformation of aberrant karyotypic hESCs. The data generated should serve as a useful reference of stem cell-derived tumor progression. Increased expression of both HDAC2 and CTNNB1 are detected as early as the pre-neoplastic stage, and might serve as prognostic markers in the malignant transformation of hESCs. PMID:24465727

Generation of Corneal Keratocytes from Human Embryonic Stem Cells.

PubMed

Hertsenberg, Andrew J; Funderburgh, James L

2016-01-01

Human Embryonic Stem Cells (hESC) offer an important resource as a limitless supply of any differentiated cell type of the human body. Keratocytes, cells from the corneal stroma, may have the potential for restoration of vision in cell therapy and biomedical engineering applications, but these specialized cells are not readily expanded in vitro. Here we describe a two-part method to produce keratocytes from the H1 hESC cell line. The hESC cells, maintained and expanded in feeder-free culture medium are first differentiated to neural crest cells using the stromal-derived inducing activity (SDIA) of the PA6 mouse embryonic fibroblast cell line. The resulting neural crest cells are selected by their expression of cell-surface CD271 and subsequently cultured as 3D pellets in a defined differentiation medium to induce a keratocyte phenotype.

Protein kinase A inhibitor, H89, significantly enhances survival rate of dissociated human embryonic stem cells following cryopreservation.

PubMed

Zhang, Liang; Xu, Yanqing; Xu, Jiandong; Wei, Yuping; Xu, Xia

2016-10-01

Human embryonic stem cells (hESCs) have huge potential for establishment of disease models and for treating degenerative diseases. However, the extremely low survival level of dissociated hESCs following cryopreservation is been a tremendous problem to allow for their rapid expansion, genetic manipulation and future medical applications. In this study, we have aimed to develop an efficient strategy to improve survival of dissociated hESCs after cryopreservation. Human embryonic stem cells (H9 line), dissociated into single cells, were cryopreserved using the slow-freezing method. Viable cells and their colony numbers in culture after cryopreservation were evaluated when treated with protein kinase A inhibitor H89. Western blotting was carried out to investigate mechanisms of low survival levels of dissociated hESCs following cryopreservation. Immunofluorescence, reverse transcription-polymerase chain reaction (RT-PCR), in vitro and in vivo differentiation were performed to testify to pluripotency and differentiation ability of hte cryopreserved cells treated with H89. H89 significantly improved survival level of dissociated hESCs after cryopreservation through ROCK inhibition. H89-treated cells still maintained their pluripotency and differentiation capacity. This new approach for cryopreservation of single hESCs, using H89, can promote potential use of hESCs in regenerative medicine in the future. © 2016 John Wiley & Sons Ltd.

Self-renewal of human embryonic stem cells requires insulin-like growth factor-1 receptor and ERBB2 receptor signaling

PubMed Central

Wang, Linlin; Schulz, Thomas C.; Sherrer, Eric S.; Dauphin, Derek S.; Shin, Soojung; Nelson, Angelique M.; Ware, Carol B.; Zhan, Mei; Song, Chao-Zhong; Chen, Xiaoji; Brimble, Sandii N.; McLean, Amanda; Galeano, Maria J.; Uhl, Elizabeth W.; D'Amour, Kevin A.; Chesnut, Jonathan D.; Rao, Mahendra S.

2007-01-01

Despite progress in developing defined conditions for human embryonic stem cell (hESC) cultures, little is known about the cell-surface receptors that are activated under conditions supportive of hESC self-renewal. A simultaneous interrogation of 42 receptor tyrosine kinases (RTKs) in hESCs following stimulation with mouse embryonic fibroblast (MEF) conditioned medium (CM) revealed rapid and prominent tyrosine phosphorylation of insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R); less prominent tyrosine phosphorylation of epidermal growth factor receptor (EGFR) family members, including ERBB2 and ERBB3; and trace phosphorylation of fibroblast growth factor receptors. Intense IGF1R and IR phosphorylation occurred in the absence of MEF conditioning (NCM) and was attributable to high concentrations of insulin in the proprietary KnockOut Serum Replacer (KSR). Inhibition of IGF1R using a blocking antibody or lentivirus-delivered shRNA reduced hESC self-renewal and promoted differentiation, while disruption of ERBB2 signaling with the selective inhibitor AG825 severely inhibited hESC proliferation and promoted apoptosis. A simple defined medium containing an IGF1 analog, heregulin-1β (a ligand for ERBB2/ERBB3), fibroblast growth factor-2 (FGF2), and activin A supported long-term growth of multiple hESC lines. These studies identify previously unappreciated RTKs that support hESC proliferation and self-renewal, and provide a rationally designed medium for the growth and maintenance of pluripotent hESCs. PMID:17761519

Derivation, characterization and differentiation of a new human embryonic stem cell line from a Chinese hatched blastocyst assisted by a non-contact laser system.

PubMed

Wu, Rongrong; Xu, Chenming; Jin, Fan; Tan, Zhou; Gu, Bin; Chen, Liangbiao; Yao, Xing; Zhang, Ming

2010-08-01

Currently worldwide attention has focused on the derivation of human embryonic stem cells (hESCs) for future therapeutic medicine. However, the majority of existing hESCs are directly or indirectly exposed to non-human materials during their derivation and/or propagation, which greatly restrict their therapeutic potential. Besides the efforts to improve culture systems, the derivation procedure, especially blastocyst manipulation, needs to be optimized. We adopted a non-contact laser-assisted hatching system in combination with sequential culture process to obtain hatched blastocysts as materials for hESC derivation, and derived a hESC line ZJUhES-1 of a Chinese population without exposure to any non-human materials during blastocyst manipulation. ZJUhES-1 satisfies the criteria of pluripotent hESCs: typically morphological characteristics; the expression of alkaline phosphatase, human telomerase reverse transcriptase and multiple hESC-specific markers including SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, OCT-4, Nanog, Rex-1, Sox-2, UTF-1, Connexins 43 and 45, TERF-1 and TERF-2, Glut-1, BCRP-1/ABCG-2, GDF3, LIN28, FGF4, Thy-1, Cripto1/TDGF1, AC133 as well as SMAD1/2/3/5; extended proliferative capacity; maintenance of a stable male karyotype after long-term cultivation; and robust multiple-lineage developmental potentials both in vivo and in vitro. Moreover, ZJUhES-1 has distinct identity revealed from DNA fingerprinting. Our xeno-free blastocyst manipulation procedure may promote the progression toward clinical-grade hESC derivation. © 2010 The Authors. Human Cell © 2010 Japan Human Cell Society.

Function of FEZF1 during early neural differentiation of human embryonic stem cells.

PubMed

Liu, Xin; Su, Pei; Lu, Lisha; Feng, Zicen; Wang, Hongtao; Zhou, Jiaxi

2018-01-01

The understanding of the mechanism underlying human neural development has been hampered due to lack of a cellular system and complicated ethical issues. Human embryonic stem cells (hESCs) provide an invaluable model for dissecting human development because of unlimited self-renewal and the capacity to differentiate into nearly all cell types in the human body. In this study, using a chemical defined neural induction protocol and molecular profiling, we identified Fez family zinc finger 1 (FEZF1) as a potential regulator of early human neural development. FEZF1 is rapidly up-regulated during neural differentiation in hESCs and expressed before PAX6, a well-established marker of early human neural induction. We generated FEZF1-knockout H1 hESC lines using CRISPR-CAS9 technology and found that depletion of FEZF1 abrogates neural differentiation of hESCs. Moreover, loss of FEZF1 impairs the pluripotency exit of hESCs during neural specification, which partially explains the neural induction defect caused by FEZF1 deletion. However, enforced expression of FEZF1 itself fails to drive neural differentiation in hESCs, suggesting that FEZF1 is necessary but not sufficient for neural differentiation from hESCs. Taken together, our findings identify one of the earliest regulators expressed upon neural induction and provide insight into early neural development in human.

2005 Donor Eligibility Requirements: Unintended Consequences for Stem Cell Development.

PubMed

Couture, Larry A; Carpenter, Melissa K

2015-10-01

Several human embryonic stem cell (hESC)-derived cell therapeutics have entered clinical testing and more are in various stages of preclinical development. The U.S. Food and Drug Administration (FDA) regulates these products under existing regulations and has stated that these products do not constitute a new class of biologic. However, as human tissue, hESCs are subject to regulations that were developed before hESCs were first described. The regulations have not been revised since 2005, well before the first hESC-derived product entered clinical studies. The current regulations require donors of hESCs to be tested in the same manner as donors of tissues intended for transplantation. However, because hESC-derived cell products are more than minimally manipulated, they are also subject to the same end-of-production release testing as most other biologic agents. In effect, this makes hESC products subject to redundant testing. No other biologic is subject to a similar testing requirement. Furthermore, the regulations that require donor testing are specifically applicable to hESC cells harvested from donors after a date in 2005. It is unclear which regulations cover hESCs harvested before 2005. Ambiguity in the guidelines and redundant testing requirements have unintentionally created a burdensome regulatory paradigm for these products and reluctance on the part of developers to invest in these promising therapeutics. We propose a simple solution that would address FDA safety concerns, eliminate regulatory uncertainty and risk, and provide flexibility for the FDA in the regulation of hESC-derived cell therapies. Regulatory ambiguity concerning donor eligibility screening and testing requirements for human embryonic stem cell lines, in particular those lines created before 2005, are causing significant concern for drug developers. Technically, most of these lines fail to meet eligibility under U.S. Food and Drug Administration (FDA) rules for product licensure, and many developers are unaware that FDA approval to begin trials under an exemption is not an assurance that the FDA will grant licensure of the product. This Perspective outlines the ambiguity and the problem it has caused and proposes a workable solution. The intent is to generate stakeholder and FDA discussion on this issue. ©AlphaMed Press.

A unique epigenetic signature is associated with active DNA replication loci in human embryonic stem cells.

PubMed

Li, Bing; Su, Trent; Ferrari, Roberto; Li, Jing-Yu; Kurdistani, Siavash K

2014-02-01

The cellular epigenetic landscape changes as pluripotent stem cells differentiate to somatic cells or when differentiated cells transform to a cancerous state. These epigenetic changes are commonly correlated with differences in gene expression. Whether active DNA replication is also associated with distinct chromatin environments in these developmentally and phenotypically diverse cell types has not been known. Here, we used BrdU-seq to map active DNA replication loci in human embryonic stem cells (hESCs), normal primary fibroblasts and a cancer cell line, and correlated these maps to the epigenome. In all cell lines, the majority of BrdU peaks were enriched in euchromatin and at DNA repetitive elements, especially at microsatellite repeats, and coincided with previously determined replication origins. The most prominent BrdU peaks were shared between all cells but a sizable fraction of the peaks were specific to each cell type and associated with cell type-specific genes. Surprisingly, the BrdU peaks that were common to all cell lines were associated with H3K18ac, H3K56ac, and H4K20me1 histone marks only in hESCs but not in normal fibroblasts or cancer cells. Depletion of the histone acetyltransferases for H3K18 and H3K56 dramatically decreased the number and intensity of BrdU peaks in hESCs. Our data reveal a unique epigenetic signature that distinguishes active replication loci in hESCs from normal somatic or malignant cells.

Physiological oxygen prevents frequent silencing of the DLK1-DIO3 cluster during human embryonic stem cells culture.

PubMed

Xie, Pingyuan; Sun, Yi; Ouyang, Qi; Hu, Liang; Tan, Yueqiu; Zhou, Xiaoying; Xiong, Bo; Zhang, Qianjun; Yuan, Ding; Pan, Yi; Liu, Tiancheng; Liang, Ping; Lu, Guangxiu; Lin, Ge

2014-02-01

Genetic and epigenetic alterations are observed in long-term culture (>30 passages) of human embryonic stem cells (hESCs); however, little information is available in early cultures. Through a large-scale gene expression analysis between initial-passage hESCs (ihESCs, <10 passages) and early-passage hESCs (ehESCs, 20-30 passages) of 12 hESC lines, we found that the DLK1-DIO3 gene cluster was normally expressed and showed normal methylation pattern in ihESC, but was frequently silenced after 20 passages. Both the DLK1-DIO3 active status in ihESCs and the inactive status in ehESCs were inheritable during differentiation. Silencing of the DLK1-DIO3 cluster did not seem to compromise the multilineage differentiation ability of hESCs, but was associated with reduced DNA damage-induced apoptosis in ehESCs and their differentiated hepatocyte-like cell derivatives, possibly through attenuation of the expression and phosphorylation of p53. Furthermore, we demonstrated that 5% oxygen, instead of the commonly used 20% oxygen, is required for preserving the expression of the DLK1-DIO3 cluster. Overall, the data suggest that active expression of the DLK1-DIO3 cluster represents a new biomarker for epigenetic stability of hESCs and indicates the importance of using a proper physiological oxygen level during the derivation and culture of hESCs. © AlphaMed Press.

Characterization and In Vivo Testing of Mesenchymal Stem Cells Derived from Human Embryonic Stem Cells

PubMed Central

Gruenloh, William; Kambal, Amal; Sondergaard, Claus; McGee, Jeannine; Nacey, Catherine; Kalomoiris, Stefanos; Pepper, Karen; Olson, Scott; Fierro, Fernando

2011-01-01

Mesenchymal stem cells (MSCs) have been shown to contribute to the recovery of tissues through homing to injured areas, especially to hypoxic, apoptotic, or inflamed areas and releasing factors that hasten endogenous repair. In some cases genetic engineering of the MSC is desired, since they are excellent delivery vehicles. We have derived MSCs from the human embryonic stem cell (hESC) line H9 (H9-MSCs). They expressed CD105, CD90, CD73, and CD146, and lacked expression of CD45, CD34, CD14, CD31, and HLA-DR, the hESC pluripotency markers SSEA-4 and Tra-1-81, and the hESC early differentiation marker SSEA-1. Marrow-derived MSCs showed a similar phenotype. H9-MSCs did not form teratoma in our initial studies, whereas the parent H9 line did so robustly. H9-MSCs differentiated into bone, cartilage, and adipocytes in vitro, and displayed increased migration under hypoxic conditions. Finally, using a hindlimb ischemia model, H9-MSCs were shown to home to the hypoxic muscle, but not the contralateral limb, by 48 h after IV injection. In summary, we have defined methods for differentiation of hESCs into MSCs and have defined their characteristics and in vivo migratory properties. PMID:21275830

The effect of a germline mutation in the APC gene on β-catenin in human embryonic stem cells.

PubMed

Yedid, Nofar; Kalma, Yael; Malcov, Mira; Amit, Ami; Kariv, Revital; Caspi, Michal; Rosin-Arbesfeld, Rina; Ben-Yosef, Dalit

2016-12-23

Most cases of colorectal cancer (CRC) are initiated by inactivation mutations in the APC gene, which is a negative regulator of the Wnt-β-catenin pathway. Patients with familial adenomatous polyposis (FAP) inherit a germline mutation in one APC allele, and loss of the second allele leads to the development of polyps that will turn malignant if not removed. It is not fully understood which molecular mechanisms are activated by APC loss and when the loss of the second APC allele occurs. Two FAP human embryonic stem cell (hESCs) lines were derived from APC mutated embryos following pre-implantation genetic diagnosis (PGD) for FAP. These FAP-hESCs were cultured in vitro and following extended culture: 1) β-catenin expression was analyzed by Western blot analysis; 2) Wnt-β-catenin/TCF-mediated transcription luciferase assay was performed; 3) cellular localization of β-catenin was evaluated by immunoflorecence confocal microscopy; and 4) DNA sequencing of the APC gene was performed. We have established a novel human in-vitro model for studying malignant transformation, using hESCs that carry a germline mutation in the APC gene following PGD for FAP. Extended culturing of FAP1 hESCs led to activation of the Wnt signaling pathway, as demonstrated by enhanced β-catenin/TCF-mediated activity. Additionally, β-catenin showed a distinct perinuclear distribution in most (91 %) of the FAP1 hESCs high passage colonies. DNA sequencing of the whole gene detected several polymorphisms in FAP1 hESCs, however, no somatic mutations were discovered in the APC gene. On the other hand, no changes in β-catenin were detected in the FAP2 hESCs, demonstrating the natural diversity of the human FAP population. Our results describe the establishment of novel hESC lines from FAP patients with a predisposition for cancer mutation. These cells can be maintained in culture for long periods of time and may serve as a platform for studying the initial molecular and cellular changes that occur during early stages of malignant transformation.

The promise of human embryonic stem cells in aging-associated diseases

PubMed Central

Yabut, Odessa; Bernstein, Harold S.

2011-01-01

Aging-associated diseases are often caused by progressive loss or dysfunction of cells that ultimately affect the overall function of tissues and organs. Successful treatment of these diseases could benefit from cell-based therapy that would regenerate lost cells or otherwise restore tissue function. Human embryonic stem cells (hESCs) promise to be an important therapeutic candidate in treating aging-associated diseases due to their unique capacity for self-renewal and pluripotency. To date, there are numerous hESC lines that have been developed and characterized. We will discuss how hESC lines are derived, their molecular and cellular properties, and how their ability to differentiate into all three embryonic germ layers is determined. We will also outline the methods currently employed to direct their differentiation into populations of tissue-specific, functional cells. Finally, we will highlight the general challenges that must be overcome and the strategies being developed to generate highly-purified hESC-derived cell populations that can safely be used for clinical applications. PMID:21566262

A targeted neuroglial reporter line generated by homologous recombination in human embryonic stem cells.

PubMed

Xue, Haipeng; Wu, Sen; Papadeas, Sophia T; Spusta, Steve; Swistowska, Anna Maria; MacArthur, Chad C; Mattson, Mark P; Maragakis, Nicholas J; Capecchi, Mario R; Rao, Mahendra S; Zeng, Xianmin; Liu, Ying

2009-08-01

In this study, we targeted Olig2, a basic helix-loop-helix transcription factor that plays an important role in motoneuron and oligodendrocyte development, in human embryonic stem cell (hESC) line BG01 by homologous recombination. One allele of Olig2 locus was replaced by a green fluorescent protein (GFP) cassette with a targeting efficiency of 5.7%. Targeted clone R-Olig2 (like the other clones) retained pluripotency, typical hESC morphology, and a normal parental karyotype 46,XY. Most importantly, GFP expression recapitulated endogenous Olig2 expression when R-Olig2 was induced by sonic hedgehog and retinoic acid, and GFP-positive cells could be purified by fluorescence-activated cell sorting. Consistent with previous reports on rodents, early GFP-expressing cells appeared biased to a neuronal fate, whereas late GFP-expressing cells appeared biased to an oligodendrocytic fate. This was corroborated by myoblast coculture, transplantation into the rat spinal cords, and whole genome expression profiling. The present work reports an hESC reporter line generated by homologous recombination targeting a neural lineage-specific gene, which can be differentiated and sorted to obtain pure neural progenitor populations.

In Vitro Differentiation and Propagation of Urothelium from Pluripotent Stem Cell Lines.

PubMed

Osborn, Stephanie L; Kurzrock, Eric A

2018-01-01

Bioengineering of bladder tissue, particularly for those patients who have advanced bladder disease, requires a source of urothelium that is healthy, capable of significant proliferation in vitro and immunologically tolerated upon transplant. As pluripotent stem cells have the potential to fulfill such criteria, they provide a critical cell source from which urothelium might be derived in vitro and used clinically. Herein, we describe the in vitro differentiation of urothelium from the H9 human embryonic stem cell (hESC) line through the definitive endoderm (DE) phase via selective culture techniques. The protocol can be used to derive urothelium from other hESCs or human-induced pluripotent stem cells.

Switching of the core structures of glycosphingolipids from globo- and lacto- to ganglio-series upon human embryonic stem cell differentiation.

PubMed

Liang, Yuh-Jin; Kuo, Huan-Hsien; Lin, Chi-Hung; Chen, Yen-Ying; Yang, Bei-Chia; Cheng, Yuan-Yuan; Yu, Alice L; Khoo, Kay-Hooi; Yu, John

2010-12-28

A systematic survey of expression profiles of glycosphingolipids (GSLs) in two hESC lines and their differentiated embryoid body (EB) outgrowth with three germ layers was carried out using immunofluorescence, flow cytometry, and MALDI-MS and MS/MS analyses. In addition to the well-known hESC-specific markers stage-specific embryonic antigen 3 (SSEA-3) and SSEA-4, we identified several globosides and lacto-series GSLs, previously unrevealed in hESCs, including Gb(4)Cer, Lc(4)Cer, fucosyl Lc(4)Cer, Globo H, and disialyl Gb(5)Cer. During hESC differentiation into EBs, MS analysis revealed a clear-cut switch in the core structures of GSLs from globo- and lacto- to ganglio-series, which was not as evident by immunostaining with antibodies against SSEA-3 and SSEA-4, owing to their cross-reactivities with various glycosphingolipids. Such a switch was attributable to altered expression of key glycosyltransferases (GTs) in the biosynthetic pathways by the up-regulation of ganglio-series-related GTs with simultaneous down-regulation of globo- and lacto-series-related GTs. Thus, these results provide insights into the unique stage-specific transition and mechanism for alterations of GSL core structures during hESC differentiation. In addition, unique glycan structures uncovered by MS analyses may serve as surface markers for further delineation of hESCs and help identify of their functional roles not only in hESCs but also in cancers.

Establishment and characterization of a human uterine endometrial undifferentiated carcinoma cell line, TMG-L.

PubMed

Hasegawa, Kiyoshi; Suzuki, Machiko; Ishikawa, Kunimi; Yasue, Akira; Kato, Rina; Nakamura, Azumi; Kuroki, Jun; Udagawa, Yasuhiro

2003-03-01

A new cell line of human uterine endometrial undifferentiated carcinoma, designated as TMG-L, was established from the metastatic lymph node of 56-year-old patient TMG-L cells have been cultured with Ham's F-12 medium supplemented with 10% FCS and grew as a loosely adherent monolayer with polygonal or spindle-shaped cells exhibiting poor cell-cell contact and piled up against each other, showing a tendency to grow as floating cells. The doubling time of this cell line was about 48 hours, and chromosomal analysis revealed aneuploidy at passage 25. The cells formed tumors in SCID mouse, the histology of which was similar to that of undifferentiated carcinoma component of primary tumor. TMG-L cells showed the loss of expression and membranous localization of either E-cadherin or alpha-catenin, implied corresponding loss of their adhesive function. And this dysfunction implicated the biological aggressive behavior of uterine endometrial undifferentiated carcinoma. This cell line appears to provide a useful system for studying uterine undifferentiated carcinoma in vivo and in vitro.

Development of a Xeno-Free Feeder-Layer System from Human Umbilical Cord Mesenchymal Stem Cells for Prolonged Expansion of Human Induced Pluripotent Stem Cells in Culture

PubMed Central

Zou, Qing; Wu, Mingjun; Zhong, Liwu; Fan, Zhaoxin; Zhang, Bo; Chen, Qiang; Ma, Feng

2016-01-01

Various feeder layers have been extensively applied to support the prolonged growth of human pluripotent stem cells (hPSCs) for in vitro cultures. Among them, mouse embryonic fibroblast (MEF) and mouse fibroblast cell line (SNL) are most commonly used feeder cells for hPSCs culture. However, these feeder layers from animal usually cause immunogenic contaminations, which compromises the potential of hPSCs in clinical applications. In the present study, we tested human umbilical cord mesenchymal stem cells (hUC-MSCs) as a potent xeno-free feeder system for maintaining human induced pluripotent stem cells (hiPSCs). The hUC-MSCs showed characteristics of MSCs in xeno-free culture condition. On the mitomycin-treated hUC-MSCs feeder, hiPSCs maintained the features of undifferentiated human embryonic stem cells (hESCs), such as low efficiency of spontaneous differentiation, stable expression of stemness markers, maintenance of normal karyotypes, in vitro pluripotency and in vivo ability to form teratomas, even after a prolonged culture of more than 30 passages. Our study indicates that the xeno-free culture system may be a good candidate for growth and expansion of hiPSCs as the stepping stone for stem cell research to further develop better and safer stem cells. PMID:26882313

Development of a Xeno-Free Feeder-Layer System from Human Umbilical Cord Mesenchymal Stem Cells for Prolonged Expansion of Human Induced Pluripotent Stem Cells in Culture.

PubMed

Zou, Qing; Wu, Mingjun; Zhong, Liwu; Fan, Zhaoxin; Zhang, Bo; Chen, Qiang; Ma, Feng

2016-01-01

Various feeder layers have been extensively applied to support the prolonged growth of human pluripotent stem cells (hPSCs) for in vitro cultures. Among them, mouse embryonic fibroblast (MEF) and mouse fibroblast cell line (SNL) are most commonly used feeder cells for hPSCs culture. However, these feeder layers from animal usually cause immunogenic contaminations, which compromises the potential of hPSCs in clinical applications. In the present study, we tested human umbilical cord mesenchymal stem cells (hUC-MSCs) as a potent xeno-free feeder system for maintaining human induced pluripotent stem cells (hiPSCs). The hUC-MSCs showed characteristics of MSCs in xeno-free culture condition. On the mitomycin-treated hUC-MSCs feeder, hiPSCs maintained the features of undifferentiated human embryonic stem cells (hESCs), such as low efficiency of spontaneous differentiation, stable expression of stemness markers, maintenance of normal karyotypes, in vitro pluripotency and in vivo ability to form teratomas, even after a prolonged culture of more than 30 passages. Our study indicates that the xeno-free culture system may be a good candidate for growth and expansion of hiPSCs as the stepping stone for stem cell research to further develop better and safer stem cells.

YKL-40 is differentially expressed in human embryonic stem cells and in cell progeny of the three germ layers.

PubMed

Brøchner, Christian B; Johansen, Julia S; Larsen, Lars A; Bak, Mads; Mikkelsen, Hanne B; Byskov, Anne Grete; Andersen, Claus Yding; Møllgård, Kjeld

2012-03-01

The secreted glycoprotein YKL-40 participates in cell differentiation, inflammation, and cancer progression. High YKL-40 expression is reported during early human development, but its functions are unknown. Six human embryonic stem cell (hESC) lines were cultured in an atmosphere of low or high oxygen tension, in culture medium with or without basic fibroblast growth factor, and on feeder layers comprising mouse embryonic fibroblasts or human foreskin fibroblasts to evaluate whether hESCs and their progeny produced YKL-40 and to characterize YKL-40 expression during differentiation. Secreted YKL-40 protein and YKL-40 mRNA expression were measured by enzyme-linked immunosorbent assay (ELISA) and quantitative RT-PCR. Serial-sectioned colonies were stained for YKL-40 protein and for pluripotent hESC (OCT4, NANOG) and germ layer (HNF-3β, PDX1, CD34, p63, nestin, PAX6) markers. Double-labeling showed YKL-40 expression in OCT4-positive hESCs, PAX6-positive neuroectodermal cells, and HNF-3β-positive endodermal cells. The differentiating progeny showed strong YKL-40 expression. Abrupt transition between YKL-40 and OCT4-positive hESCs and YKL-40-positive ecto- and neuroectodermal lineages was observed within the same epithelial-like layer. YKL-40-positive cells within deeper layers lacked contact with OCT4-positive cells. YKL-40 may be important in initial cell differentiation from hESCs toward ectoderm and neuroectoderm, with retained epithelial morphology, whereas later differentiation into endoderm and mesoderm involves a transition into the deeper layers of the colony.

Embryonic stem cells and the next generation of developmental toxicity testing.

PubMed

Kugler, Josephine; Huhse, Bettina; Tralau, Tewes; Luch, Andreas

2017-08-01

The advent of stem cell technology has seen the establishment of embryonic stem cells (ESCs) as molecular model systems and screening tools. Although ESCs are nowadays widely used in research, regulatory implementation for developmental toxicity testing is pending. Areas Covered: This review evaluates the performance of current ESC, including human (h)ESC testing systems, trying to elucidate their potential for developmental toxicity testing. It shall discuss defining parameters and mechanisms, their relevance and contemplate what can realistically be expected. Crucially this includes the question of how to ascertain the quality of currently employed cell lines and tests based thereon. Finally, the use of hESCs will raise ethical concerns which should be addressed early on. Expert Opinion: While the suitability of (h)ESCs as tools for research and development goes undisputed, any routine use for developmental toxicity testing currently still seems premature. The reasons for this comprise inherent biological deficiencies as well as cell line quality and system validation. Overcoming these issues will require collaboration of scientists, test developers and regulators. Also, validation needs to be made worthwhile for academia. Finally we have to continuously rethink existing strategies, making room for improved testing and innovative approaches.

Pluripotent stem cell models of Shwachman-Diamond syndrome reveal a common mechanism for pancreatic and hematopoietic dysfunction

PubMed Central

Tulpule, Asmin; Kelley, James M.; Lensch, M. William; McPherson, Jade; Park, In Hyun; Hartung, Odelya; Nakamura, Tomoka; Schlaeger, Thorsten M.; Shimamura, Akiko; Daley, George Q.

2013-01-01

Summary Shwachman-Diamond syndrome (SDS), a rare autosomal recessive disorder characterized by exocrine pancreatic insufficiency and hematopoietic dysfunction, is caused by mutations in the Shwachman-Bodian-Diamond syndrome (SBDS) gene. We created human pluripotent stem cell models of SDS by knock-down of SBDS in human embryonic stem cells (hESCs) and generation of induced pluripotent stem cell (iPSC) lines from two SDS patients. SBDS-deficient hESCs and iPSCs manifest deficits in exocrine pancreatic and hematopoietic differentiation in vitro, enhanced apoptosis and elevated protease levels in culture supernatants, which could be reversed by restoring SBDS protein expression through transgene rescue or by supplementing culture media with protease inhibitors. Protease-mediated auto-digestion provides a mechanistic link between the pancreatic and hematopoietic phenotypes in SDS, highlighting the utility of hESCs and iPSCs in obtaining novel insights into human disease. PMID:23602541

Epigenetics changes caused by the fusion of human embryonic stem cell and ovarian cancer cells.

PubMed

He, Ke; Qu, Hu; Xu, Li-Nan; Gao, Jun; Cheng, Fu-Yi; Xiang, Peng; Zhou, Can-Quan

2016-10-01

To observe the effect of gene expression and tumorigenicity in hybrid cells of human embryonic stem cells (hESCs) and ovarian cancer cells in vitro and in vivo using a mouse model, and to determine its feasibility in reprogramming tumour cells growth and apoptosis, for a potential exploration of the role of hESCs and tumour cells fusion in the management of ovarian cancer. Stable transgenic hESCs (H1) and ovarian cancer cell line OVCAR-3 were established before fusion, and cell fusion system was established to analyse the related indicators. PTEN expression in HO-H1 cells was higher than those in the parental stem cells and lower than those in parental tumour cells; the growth of OV-H1 (RFP+GFP) hybrid cells with double fluorescence expressions were obviously slower than that of human embryonic stem cells and OVCAR-3 ovarian cancer cells. The apoptosis signal of the OV-H1 hybrid cells was significantly higher than that of the hESCs and OVCAR-3 ovarian cancer cells. In vivo results showed that compared with 7 days, 28 days and 35 days after inoculation of OV-H1 hybrid cells; also, apoptotic cell detection indicated that much stronger apoptotic signal was found in OV-H1 hybrid cells inoculated mouse. The hESCs can inhibit the growth of OVCAR-3 cells in vitro by suppressing p53 and PTEN expression to suppress the growth of tumour that may be achieved by inducing apoptosis of OVCAR-3 cells. The change of epigenetics after fusion of ovarian cancer cells and hESCs may become a novel direction for treatment of ovarian cancer. © 2016 The Author(s).

Enhanced expression of FNDC5 in human embryonic stem cell-derived neural cells along with relevant embryonic neural tissues.

PubMed

Ghahrizjani, Fatemeh Ahmadi; Ghaedi, Kamran; Salamian, Ahmad; Tanhaei, Somayeh; Nejati, Alireza Shoaraye; Salehi, Hossein; Nabiuni, Mohammad; Baharvand, Hossein; Nasr-Esfahani, Mohammad Hossein

2015-02-25

Availability of human embryonic stem cells (hESCs) has enhanced the capability of basic and clinical research in the context of human neural differentiation. Derivation of neural progenitor (NP) cells from hESCs facilitates the process of human embryonic development through the generation of neuronal subtypes. We have recently indicated that fibronectin type III domain containing 5 protein (FNDC5) expression is required for appropriate neural differentiation of mouse embryonic stem cells (mESCs). Bioinformatics analyses have shown the presence of three isoforms for human FNDC5 mRNA. To differentiate which isoform of FNDC5 is involved in the process of human neural differentiation, we have used hESCs as an in vitro model for neural differentiation by retinoic acid (RA) induction. The hESC line, Royan H5, was differentiated into a neural lineage in defined adherent culture treated by RA and basic fibroblast growth factor (bFGF). We collected all cell types that included hESCs, rosette structures, and neural cells in an attempt to assess the expression of FNDC5 isoforms. There was a contiguous increase in all three FNDC5 isoforms during the neural differentiation process. Furthermore, the highest level of expression of the isoforms was significantly observed in neural cells compared to hESCs and the rosette structures known as neural precursor cells (NPCs). High expression levels of FNDC5 in human fetal brain and spinal cord tissues have suggested the involvement of this gene in neural tube development. Additional research is necessary to determine the major function of FDNC5 in this process. Copyright © 2014 Elsevier B.V. All rights reserved.

Random Mutagenesis, Clonal Events, and Embryonic or Somatic Origin Determine the mtDNA Variant Type and Load in Human Pluripotent Stem Cells.

PubMed

Zambelli, Filippo; Mertens, Joke; Dziedzicka, Dominika; Sterckx, Johan; Markouli, Christina; Keller, Alexander; Tropel, Philippe; Jung, Laura; Viville, Stephane; Van de Velde, Hilde; Geens, Mieke; Seneca, Sara; Sermon, Karen; Spits, Claudia

2018-06-07

In this study, we deep-sequenced the mtDNA of human embryonic and induced pluripotent stem cells (hESCs and hiPSCs) and their source cells and found that the majority of variants pre-existed in the cells used to establish the lines. Early-passage hESCs carried few and low-load heteroplasmic variants, similar to those identified in oocytes and inner cell masses. The number and heteroplasmic loads of these variants increased with prolonged cell culture. The study of 120 individual cells of early- and late-passage hESCs revealed a significant diversity in mtDNA heteroplasmic variants at the single-cell level and that the variants that increase during time in culture are always passenger to the appearance of chromosomal abnormalities. We found that early-passage hiPSCs carry much higher loads of mtDNA variants than hESCs, which single-fibroblast sequencing proved pre-existed in the source cells. Finally, we show that these variants are stably transmitted during short-term differentiation. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Microfabricated Modular Scale-Down Device for Regenerative Medicine Process Development

PubMed Central

Reichen, Marcel; Macown, Rhys J.; Jaccard, Nicolas; Super, Alexandre; Ruban, Ludmila; Griffin, Lewis D.; Veraitch, Farlan S.; Szita, Nicolas

2012-01-01

The capacity of milli and micro litre bioreactors to accelerate process development has been successfully demonstrated in traditional biotechnology. However, for regenerative medicine present smaller scale culture methods cannot cope with the wide range of processing variables that need to be evaluated. Existing microfabricated culture devices, which could test different culture variables with a minimum amount of resources (e.g. expensive culture medium), are typically not designed with process development in mind. We present a novel, autoclavable, and microfabricated scale-down device designed for regenerative medicine process development. The microfabricated device contains a re-sealable culture chamber that facilitates use of standard culture protocols, creating a link with traditional small-scale culture devices for validation and scale-up studies. Further, the modular design can easily accommodate investigation of different culture substrate/extra-cellular matrix combinations. Inactivated mouse embryonic fibroblasts (iMEF) and human embryonic stem cell (hESC) colonies were successfully seeded on gelatine-coated tissue culture polystyrene (TC-PS) using standard static seeding protocols. The microfluidic chip included in the device offers precise and accurate control over the culture medium flow rate and resulting shear stresses in the device. Cells were cultured for two days with media perfused at 300 µl.h−1 resulting in a modelled shear stress of 1.1×10−4 Pa. Following perfusion, hESC colonies stained positively for different pluripotency markers and retained an undifferentiated morphology. An image processing algorithm was developed which permits quantification of co-cultured colony-forming cells from phase contrast microscope images. hESC colony sizes were quantified against the background of the feeder cells (iMEF) in less than 45 seconds for high-resolution images, which will permit real-time monitoring of culture progress in future experiments. The presented device is a first step to harness the advantages of microfluidics for regenerative medicine process development. PMID:23284952

Coculturing with endothelial cells promotes in vitro maturation and electrical coupling of human embryonic stem cell-derived cardiomyocytes.

PubMed

Pasquier, Jennifer; Gupta, Renuka; Rioult, Damien; Hoarau-Véchot, Jessica; Courjaret, Raphael; Machaca, Khaled; Al Suwaidi, Jassim; Stanley, Edouard G; Rafii, Shahin; Elliott, David A; Abi Khalil, Charbel; Rafii, Arash

2017-06-01

Pluripotent human embryonic stem cells (hESC) are a promising source of repopulating cardiomyocytes. We hypothesized that we could improve maturation of cardiomyocytes and facilitate electrical interconnections by creating a model that more closely resembles heart tissue; that is, containing both endothelial cells (ECs) and cardiomyocytes. We induced cardiomyocyte differentiation in the coculture of an hESC line expressing the cardiac reporter NKX2.5-green fluorescent protein (GFP), and an Akt-activated EC line (E4 + ECs). We quantified spontaneous beating rates, synchrony, and coordination between different cardiomyocyte clusters using confocal imaging of Fura Red-detected calcium transients and computer-assisted image analysis. After 8 days in culture, 94% ± 6% of the NKX2-5GFP + cells were beating when hESCs embryonic bodies were plated on E4 + ECs compared with 34% ± 12.9% for controls consisting of hESCs cultured on BD Matrigel (BD Biosciences) without ECs at Day 11 in culture. The spatial organization of beating areas in cocultures was different. The GFP + cardiomyocytes were close to the E4 + ECs. The average beats/min of the cardiomyocytes in coculture was faster and closer to physiologic heart rates compared with controls (50 ± 14 [n = 13] vs 25 ± 9 [n = 8]; p < 0.05). The coculture with ECs led to synchronized beating relying on the endothelial network, as illustrated by the loss of synchronization upon the disruption of endothelial bridges. The coculturing of differentiating cardiomyocytes with Akt-activated ECs but not EC-conditioned media results in (1) improved efficiency of the cardiomyocyte differentiation protocol and (2) increased maturity leading to better intercellular coupling with improved chronotropy and synchrony. Copyright © 2017. Published by Elsevier Inc.

Comparison of the early response of human embryonic stem cells and human induced pluripotent stem cells to ionizing radiation.

PubMed

Suchorska, Wiktoria Maria; Augustyniak, Ewelina; Łukjanow, Magdalena

2017-04-01

Despite the well-demonstrated efficacy of stem cell (SC) therapy, this approach has a number of key drawbacks. One important concern is the response of pluripotent SCs to treatment with ionizing radiation (IR), given that SCs used in regenerative medicine will eventually be exposed to IR for diagnostic or treatment‑associated purposes. Therefore, the aim of the present study was to examine and compare early IR‑induced responses of pluripotent SCs to assess their radioresistance and radiosensitivity. In the present study, 3 cell lines; human embryonic SCs (hESCs), human induced pluripotent SCs (hiPSCs) and primary human dermal fibroblasts (PHDFs); were exposed to IR at doses ranging from 0 to 15 gray (Gy). Double strand breaks (DSBs), and the gene expression of the following DNA repair genes were analyzed: P53; RAD51; BRCA2; PRKDC; and XRCC4. hiPSCs demonstrated greater radioresistance, as fewer DSBs were identified, compared with hESCs. Both pluripotent SC lines exhibited distinct gene expression profiles in the most common DNA repair genes that are involved in homologous recombination, non‑homologous end‑joining and enhanced DNA damage response following IR exposure. Although hESCs and hiPSCs are equivalent in terms of capacity for pluripotency and differentiation into 3 germ layers, the results of the present study indicate that these 2 types of SCs differ in gene expression following exposure to IR. Consequently, further research is required to determine whether hiPSCs and hESCs are equally safe for application in clinical practice. The present study contributes to a greater understanding of DNA damage response (DDR) mechanisms activated in pluripotent SCs and may aid in the future development of safe SC‑based clinical protocols.

Characterization of Tetraploid Somatic Cell Nuclear Transfer-Derived Human Embryonic Stem Cells.

PubMed

Shin, Dong-Hyuk; Lee, Jeoung-Eun; Eum, Jin Hee; Chung, Young Gie; Lee, Hoon Taek; Lee, Dong Ryul

2017-12-01

Polyploidy is occurred by the process of endomitosis or cell fusion and usually represent terminally differentiated stage. Their effects on the developmental process were mainly investigated in the amphibian and fishes, and only observed in some rodents as mammalian model. Recently, we have established tetraploidy somatic cell nuclear transfer-derived human embryonic stem cells (SCNT-hESCs) and examined whether it could be available as a research model for the polyploidy cells existed in the human tissues. Two tetraploid hESC lines were artificially acquired by reintroduction of remained 1st polar body during the establishment of SCNT-hESC using MII oocytes obtained from female donors and dermal fibroblasts (DFB) from a 35-year-old adult male. These tetraploid SCNT-hESC lines (CHA-NT1 and CHA-NT3) were identified by the cytogenetic genotyping (91, XXXY,-6, t[2:6] / 92,XXXY,-12,+20) and have shown of indefinite proliferation, but slow speed when compared to euploid SCNT-hESCs. Using the eight Short Tendem Repeat (STR) markers, it was confirmed that both CHA-NT1 and CHA-NT3 lines contain both nuclear and oocyte donor genotypes. These hESCs expressed pluripotency markers and their embryoid bodies (EB) also expressed markers of the three embryonic germ layers and formed teratoma after transplantation into immune deficient mice. This study showed that tetraploidy does not affect the activities of proliferation and differentiation in SCNT-hESC. Therefore, tetraploid hESC lines established after SCNT procedure could be differentiated into various types of cells and could be an useful model for the study of the polyploidy cells in the tissues.

Characterization of Tetraploid Somatic Cell Nuclear Transfer-Derived Human Embryonic Stem Cells

PubMed Central

Shin, Dong-Hyuk; Lee, Jeoung-Eun; Eum, Jin Hee; Chung, Young Gie; Lee, Hoon Taek; Lee, Dong Ryul

2017-01-01

ABSTRACT Polyploidy is occurred by the process of endomitosis or cell fusion and usually represent terminally differentiated stage. Their effects on the developmental process were mainly investigated in the amphibian and fishes, and only observed in some rodents as mammalian model. Recently, we have established tetraploidy somatic cell nuclear transfer-derived human embryonic stem cells (SCNT-hESCs) and examined whether it could be available as a research model for the polyploidy cells existed in the human tissues. Two tetraploid hESC lines were artificially acquired by reintroduction of remained 1st polar body during the establishment of SCNT-hESC using MII oocytes obtained from female donors and dermal fibroblasts (DFB) from a 35-year-old adult male. These tetraploid SCNT-hESC lines (CHA-NT1 and CHA-NT3) were identified by the cytogenetic genotyping (91, XXXY,-6, t[2:6] / 92,XXXY,-12,+20) and have shown of indefinite proliferation, but slow speed when compared to euploid SCNT-hESCs. Using the eight Short Tendem Repeat (STR) markers, it was confirmed that both CHA-NT1 and CHA-NT3 lines contain both nuclear and oocyte donor genotypes. These hESCs expressed pluripotency markers and their embryoid bodies (EB) also expressed markers of the three embryonic germ layers and formed teratoma after transplantation into immune deficient mice. This study showed that tetraploidy does not affect the activities of proliferation and differentiation in SCNT-hESC. Therefore, tetraploid hESC lines established after SCNT procedure could be differentiated into various types of cells and could be an useful model for the study of the polyploidy cells in the tissues. PMID:29359202

Human Embryonic and Induced Pluripotent Stem Cell Research Trends: Complementation and Diversification of the Field

PubMed Central

Kobold, Sabine; Guhr, Anke; Kurtz, Andreas; Löser, Peter

2015-01-01

Summary Research in human induced pluripotent stem cells (hiPSCs) is rapidly developing and there are expectations that this research may obviate the need to use human embryonic stem cells (hESCs), the ethics of which has been a subject of controversy for more than 15 years. In this study, we investigated approximately 3,400 original research papers that reported an experimental use of these types of human pluripotent stem cells (hPSCs) and were published from 2008 to 2013. We found that research into both cell types was conducted independently and further expanded, accompanied by a growing intersection of both research fields. Moreover, an in-depth analysis of papers that reported the use of both cell types indicates that hESCs are still being used as a “gold standard,” but in a declining proportion of publications. Instead, the expanding research field is diversifying and hESC and hiPSC lines are increasingly being used in more independent research and application areas. PMID:25866160

Induction of human embryonic and induced pluripotent stem cells into urothelium.

PubMed

Osborn, Stephanie L; Thangappan, Ravikumar; Luria, Ayala; Lee, Justin H; Nolta, Jan; Kurzrock, Eric A

2014-05-01

In vitro generation of human urothelium from stem cells would be a major advancement in the regenerative medicine field, providing alternate nonurologic and/or nonautologous tissue sources for bladder grafts. Such a model would also help decipher the mechanisms of urothelial differentiation and would facilitate investigation of deviated differentiation of normal progenitors into urothelial cancer stem cells, perhaps elucidating areas of intervention for improved treatments. Thus far, in vitro derivation of urothelium from human embryonic stem cells (hESCs) or human induced pluripotent stem (hiPS) cells has not been reported. The goal of this work was to develop an efficient in vitro protocol for the induction of hESCs into urothelium through an intermediary definitive endoderm step and free of matrices and cell contact. During directed differentiation in a urothelial-specific medium ("Uromedium"), hESCs produced up to 60% urothelium, as determined by uroplakin expression; subsequent propagation selected for 90% urothelium. Alteration of the epithelial and mesenchymal cell signaling contribution through noncell contact coculture or conditioned media did not enhance the production of urothelium. Temporospatial evaluation of transcription factors known to be involved in urothelial specification showed association of IRF1, GET1, and GATA4 with uroplakin expression. Additional hESC and hiPS cell lines could also be induced into urothelium using this in vitro system. These results demonstrate that derivation and propagation of urothelium from hESCs and hiPS cells can be efficiently accomplished in vitro in the absence of matrices, cell contact, or adult cell signaling and that the induction process appears to mimic normal differentiation.

Induction of Human Embryonic and Induced Pluripotent Stem Cells Into Urothelium

PubMed Central

Osborn, Stephanie L.; Thangappan, Ravikumar; Luria, Ayala; Lee, Justin H.; Nolta, Jan

2014-01-01

In vitro generation of human urothelium from stem cells would be a major advancement in the regenerative medicine field, providing alternate nonurologic and/or nonautologous tissue sources for bladder grafts. Such a model would also help decipher the mechanisms of urothelial differentiation and would facilitate investigation of deviated differentiation of normal progenitors into urothelial cancer stem cells, perhaps elucidating areas of intervention for improved treatments. Thus far, in vitro derivation of urothelium from human embryonic stem cells (hESCs) or human induced pluripotent stem (hiPS) cells has not been reported. The goal of this work was to develop an efficient in vitro protocol for the induction of hESCs into urothelium through an intermediary definitive endoderm step and free of matrices and cell contact. During directed differentiation in a urothelial-specific medium (“Uromedium”), hESCs produced up to 60% urothelium, as determined by uroplakin expression; subsequent propagation selected for 90% urothelium. Alteration of the epithelial and mesenchymal cell signaling contribution through noncell contact coculture or conditioned media did not enhance the production of urothelium. Temporospatial evaluation of transcription factors known to be involved in urothelial specification showed association of IRF1, GET1, and GATA4 with uroplakin expression. Additional hESC and hiPS cell lines could also be induced into urothelium using this in vitro system. These results demonstrate that derivation and propagation of urothelium from hESCs and hiPS cells can be efficiently accomplished in vitro in the absence of matrices, cell contact, or adult cell signaling and that the induction process appears to mimic normal differentiation. PMID:24657961

mAb C19 targets a novel surface marker for the isolation of human cardiac progenitor cells from human heart tissue and differentiated hESCs.

PubMed

Leung, Hau Wan; Moerkamp, Asja T; Padmanabhan, Jayanthi; Ng, Sze-Wai; Goumans, Marie-José; Choo, Andre

2015-05-01

Cardiac progenitor cells (CPCs) have been isolated from adult and developing hearts using an anti-mouse Sca-1 antibody. However, the absence of a human Sca-1 homologue has hampered the clinical application of the CPCs. Therefore, we generated novel monoclonal antibodies (mAbs) specifically raised against surface markers expressed by resident human CPCs. Here, we explored the suitability of one of these mAbs, mAb C19, for the identification, isolation and characterization of CPCs from fetal heart tissue and differentiating cultures of human embryonic stem cells (hESCs). Using whole-cell immunization, mAbs were raised against Sca-1+ CPCs and screened for reactivity to various CPC lines by flow cytometry. mAb C19 was found to be specific for Sca-1+ CPCs, with high cell surface binding capabilities. mAb C19 stained small stem-like cells in cardiac tissue sections. Moreover, during differentiation of hESCs towards cardiomyocytes, a transient population of cells with mAb C19 reactivity was identified and isolated using magnetic-activated cell sorting. Their cell fate was tracked and found to improve cardiomyocyte purity from hESC-derived cultures. mAb C19+ CPCs, from both hESC differentiation and fetal heart tissues, were maintained and expanded in culture, while retaining their CPC-like characteristics and their ability to further differentiate into cardiomyocytes by stimulation with TGFβ1. Finally, gene expression profiling of these mAb C19+ CPCs suggested a highly angiogenic nature, which was further validated by cell-based angiogenesis assays. mAb C19 is a new surface marker for the isolation of multipotent CPCs from both human heart tissues and differentiating hESCs. Copyright © 2015 Elsevier Ltd. All rights reserved.

Human fibroblast matrices bio-assembled under macromolecular crowding support stable propagation of human embryonic stem cells.

PubMed

Peng, Yanxian; Bocker, Michael Thomas; Holm, Jennifer; Toh, Wei Seong; Hughes, Christopher Stephen; Kidwai, Fahad; Lajoie, Gilles Andre; Cao, Tong; Lyko, Frank; Raghunath, Michael

2012-11-01

Stable pluripotent feeder-free propagation of human embryonic stem cells (hESCs) prior to their therapeutic applications remains a major challenge. Matrigel™ (BD Singapore) is a murine sarcoma-derived extracellular matrix (ECM) widely used as a cell-free support combined with conditioned or chemically defined media; however, inherent xenogenic and immunological threats invalidate it for clinical applications. Using human fibrogenic cells to generate ECM is promising but currently suffers from inefficient and time-consuming deposition in vitro. We recently showed that macromolecular crowding (MMC) accelerated ECM deposition substantially in vitro. In the current study, we used dextran sulfate 500 kDa as a macromolecular crowder to induce WI-38 fetal human lung fibroblasts at 0.5% serum condition to deposit human ECM in three days. After decellularization, the generated ECMs allowed stable propagation of H9 hESCs over 20 passages in chemically-defined medium (mTEsR1) with an overall improved outcome compared to Matrigel in terms of population doubling while retaining teratoma formation and differentiation capacity. Of significance, only ECMs generated by MMC allowed the successful propagation of hESCs. ECMs were highly complex and in contrast to Matrigel, contained no vitronectin but did contain collagen XII, ig-h3 and novel for hESC-supporting human matrices, substantial amounts of transglutaminase 2. Genome-wide analysis of promoter DNA methylation states revealed high overall similarity between human ECM- and Matrigel-cultured hESCs; however, distinct differences were observed with 49 genes associated with a variety of cellular functions. Thus, human ECMs deposited by MMC by selected fibroblast lines are a suitable human microenvironment for stable hESC propagation and clinically translational settings. Copyright © 2012 John Wiley & Sons, Ltd.

Cryopreserved mouse fetal liver stromal cells treated with mitomycin C are able to support the growth of human embryonic stem cells.

PubMed

Zhang, Wei; Hu, Jiabo; Ma, Quanhui; Hu, Sanqiang; Wang, Yanyan; Wen, Xiangmei; Ma, Yongbin; Xu, Hong; Qian, Hui; Xu, Wenrong

2014-09-01

An immortalized mouse fetal liver stromal cell line, named KM3, has demonstrated the potential to support the growth and maintenance of human embryonic stem cells (hESCs). In this study, the characteristics of KM3 cells were examined following cryopreservation at -70°C and in liquid nitrogen for 15, 30 and 60 days following treatment with 10 μg/ml mitomycin C. In addition, whether the KM3 cells were suitable for use as feeder cells to support the growth of hESCs was evaluated. The inhibition of mitosis without cell death was observed when the KM3 cells were treated with 10 μg/ml mitomycin C for 2 h. The morphology of the KM3 cells cryopreserved in liquid nitrogen for 60 days was not markedly changed, and the cell survival rate was 84.60±1.14%. By contrast, the survival rate of the KM3 cells was 66.40±2.88% following cryopreservation at -70°C for 60 days; the cells readily detached, were maintained for a shorter time, and had a reduced expression level of basic fibroblast growth factor. hESCs cultured on KM3 cells cryopreserved in liquid nitrogen for 60 days showed the typical bird's nest structure, with clear boundaries and a differentiation rate of 16.33±2.08%. The differentiation rate of hESCs cultured on KM3 cells cryopreserved at -70°C for 60 days was 37.67±3.51%. These results indicate that the cryopreserved KM3 cells treated with mitomycin C may be directly used in the subculture of hESCs, and the effect is relatively good with -70°C short-term or liquid nitrogen cryopreservation.

Reprogramming triggers endogenous L1 and Alu retrotransposition in human induced pluripotent stem cells.

PubMed

Klawitter, Sabine; Fuchs, Nina V; Upton, Kyle R; Muñoz-Lopez, Martin; Shukla, Ruchi; Wang, Jichang; Garcia-Cañadas, Marta; Lopez-Ruiz, Cesar; Gerhardt, Daniel J; Sebe, Attila; Grabundzija, Ivana; Merkert, Sylvia; Gerdes, Patricia; Pulgarin, J Andres; Bock, Anja; Held, Ulrike; Witthuhn, Anett; Haase, Alexandra; Sarkadi, Balázs; Löwer, Johannes; Wolvetang, Ernst J; Martin, Ulrich; Ivics, Zoltán; Izsvák, Zsuzsanna; Garcia-Perez, Jose L; Faulkner, Geoffrey J; Schumann, Gerald G

2016-01-08

Human induced pluripotent stem cells (hiPSCs) are capable of unlimited proliferation and can differentiate in vitro to generate derivatives of the three primary germ layers. Genetic and epigenetic abnormalities have been reported by Wissing and colleagues to occur during hiPSC derivation, including mobilization of engineered LINE-1 (L1) retrotransposons. However, incidence and functional impact of endogenous retrotransposition in hiPSCs are yet to be established. Here we apply retrotransposon capture sequencing to eight hiPSC lines and three human embryonic stem cell (hESC) lines, revealing endogenous L1, Alu and SINE-VNTR-Alu (SVA) mobilization during reprogramming and pluripotent stem cell cultivation. Surprisingly, 4/7 de novo L1 insertions are full length and 6/11 retrotransposition events occurred in protein-coding genes expressed in pluripotent stem cells. We further demonstrate that an intronic L1 insertion in the CADPS2 gene is acquired during hiPSC cultivation and disrupts CADPS2 expression. These experiments elucidate endogenous retrotransposition, and its potential consequences, in hiPSCs and hESCs.

Reprogramming triggers endogenous L1 and Alu retrotransposition in human induced pluripotent stem cells

PubMed Central

Klawitter, Sabine; Fuchs, Nina V.; Upton, Kyle R.; Muñoz-Lopez, Martin; Shukla, Ruchi; Wang, Jichang; Garcia-Cañadas, Marta; Lopez-Ruiz, Cesar; Gerhardt, Daniel J.; Sebe, Attila; Grabundzija, Ivana; Merkert, Sylvia; Gerdes, Patricia; Pulgarin, J. Andres; Bock, Anja; Held, Ulrike; Witthuhn, Anett; Haase, Alexandra; Sarkadi, Balázs; Löwer, Johannes; Wolvetang, Ernst J.; Martin, Ulrich; Ivics, Zoltán; Izsvák, Zsuzsanna; Garcia-Perez, Jose L.; Faulkner, Geoffrey J.; Schumann, Gerald G.

2016-01-01

Human induced pluripotent stem cells (hiPSCs) are capable of unlimited proliferation and can differentiate in vitro to generate derivatives of the three primary germ layers. Genetic and epigenetic abnormalities have been reported by Wissing and colleagues to occur during hiPSC derivation, including mobilization of engineered LINE-1 (L1) retrotransposons. However, incidence and functional impact of endogenous retrotransposition in hiPSCs are yet to be established. Here we apply retrotransposon capture sequencing to eight hiPSC lines and three human embryonic stem cell (hESC) lines, revealing endogenous L1, Alu and SINE-VNTR-Alu (SVA) mobilization during reprogramming and pluripotent stem cell cultivation. Surprisingly, 4/7 de novo L1 insertions are full length and 6/11 retrotransposition events occurred in protein-coding genes expressed in pluripotent stem cells. We further demonstrate that an intronic L1 insertion in the CADPS2 gene is acquired during hiPSC cultivation and disrupts CADPS2 expression. These experiments elucidate endogenous retrotransposition, and its potential consequences, in hiPSCs and hESCs. PMID:26743714

Conservative site-specific and single-copy transgenesis in human LINE-1 elements

PubMed Central

Vijaya Chandra, Shree Harsha; Makhija, Harshyaa; Peter, Sabrina; Myint Wai, Cho Mar; Li, Jinming; Zhu, Jindong; Ren, Zhonglu; D'Alcontres, Martina Stagno; Siau, Jia Wei; Chee, Sharon; Ghadessy, Farid John; Dröge, Peter

2016-01-01

Genome engineering of human cells plays an important role in biotechnology and molecular medicine. In particular, insertions of functional multi-transgene cassettes into suitable endogenous sequences will lead to novel applications. Although several tools have been exploited in this context, safety issues such as cytotoxicity, insertional mutagenesis and off-target cleavage together with limitations in cargo size/expression often compromise utility. Phage λ integrase (Int) is a transgenesis tool that mediates conservative site-specific integration of 48 kb DNA into a safe harbor site of the bacterial genome. Here, we show that an Int variant precisely recombines large episomes into a sequence, termed attH4X, found in 1000 human Long INterspersed Elements-1 (LINE-1). We demonstrate single-copy transgenesis through attH4X-targeting in various cell lines including hESCs, with the flexibility of selecting clones according to transgene performance and downstream applications. This is exemplified with pluripotency reporter cassettes and constitutively expressed payloads that remain functional in LINE1-targeted hESCs and differentiated progenies. Furthermore, LINE-1 targeting does not induce DNA damage-response or chromosomal aberrations, and neither global nor localized endogenous gene expression is substantially affected. Hence, this simple transgene addition tool should become particularly useful for applications that require engineering of the human genome with multi-transgenes. PMID:26673710

Growth regulation by estrogen in breast cancer 1 (GREB1) is a novel progesterone-responsive gene required for human endometrial stromal decidualization.

PubMed

Camden, Alison J; Szwarc, Maria M; Chadchan, Sangappa B; DeMayo, Francesco J; O'Malley, Bert W; Lydon, John P; Kommagani, Ramakrishna

2017-09-01

Is Growth Regulation by Estrogen in Breast Cancer 1 (GREB1) required for progesterone-driven endometrial stromal cell decidualization? GREB1 is a novel progesterone-responsive gene required for progesterone-driven human endometrial stromal cell (HESC) decidualization. Successful establishment of pregnancy requires HESCs to transform from fibroblastic to epithelioid cells in a process called decidualization. This process depends on the hormone progesterone, but the molecular mechanisms by which it occurs have not been determined. Primary and transformed HESCs in which GREB1 expression was knocked down were decidualized in culture for up to 6 days. Wild-type and progesterone receptor (PR) knockout mice were treated with progesterone, and their uteri were assessed for levels of GREB1 expression. Analysis of previous data included data mining of expression profile data sets and in silico transcription factor-binding analysis. Endometrial biopsies obtained from healthy women of reproductive age during the proliferative phase (Days 8-12) of their menstrual cycle were used for isolating HESCs. Experiments were carried out with early passage (no more than four passages) HESCs isolated from at least three subjects. Transcript levels of decidualization markers prolactin (PRL) and insulin-like growth factor-binding protein-1 (IGFBP-1) were detected by quantitative RT-PCR as readouts for HESC decidualization. Cells were also imaged by phase-contrast microscopy. To assess the requirement for GREB1, PR and SRC-2, cells were transfected with specifically targeted small interfering RNAs. Results are shown as mean and SE from three replicates of one representative patient-derived primary endometrial cell line. Experiments were also conducted with transformed HESCs. Progesterone treatment of mice and transformed HESCs led to an ~5-fold (5.6 ± 0.81, P < 0.05, and 5.2 ± 0.26, P < 0.01, respectively) increase in GREB1 transcript levels. This increase was significantly reduced in the uteri of PR knock-out mice (P < 0.01), in HESCs treated with the PR antagonist RU486 (P < 0.01), or in HESCs in which PR expression was knocked down (P < 0.05). When GREB1 expression was knocked down, progesterone-driven decidualization markers in both immortalized and primary HESCs was significantly reduced (P < 0.05 and P < 0.01). Finally, GREB1 knock down signficantly reduced expression of the PR target genes WNT4 and FOXOA1 (P < 0.05 and P < 0.01, respectively). This study used the Nuclear Receptor Signaling Atlas. Although in vitro cell culture studies indicate that GREB1 is required for endoemtrial decidualization, the in vivo role of GREB1 in endometrial function and dysfunction should be assessed by using knock-out mouse models. Identification and functional analysis of GREB1 as a key molecular mediator of decidualization may lead to improved diagnosis and clinical management of women with peri-implantation loss due to inadequate endometrial decidualization. This research was funded in part by: a National Institutes of Health (NIH)/ National Institute of Child Health and Human Development (NICHD) grant (R00 HD080742) and Washington University School of Medicine start-up funds to R.K., an NIH/NICHD grant (RO1 HD-07857) to B.W.O.M., and a NIH/NICHD grant (R01 HD-042311) to J.P.L. The authors declare no conflicts of interests. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Differentiation of human embryonic stem cells to HOXA+ hemogenic vasculature that resembles the aorta-gonad-mesonephros.

PubMed

Ng, Elizabeth S; Azzola, Lisa; Bruveris, Freya F; Calvanese, Vincenzo; Phipson, Belinda; Vlahos, Katerina; Hirst, Claire; Jokubaitis, Vanta J; Yu, Qing C; Maksimovic, Jovana; Liebscher, Simone; Januar, Vania; Zhang, Zhen; Williams, Brenda; Conscience, Aude; Durnall, Jennifer; Jackson, Steven; Costa, Magdaline; Elliott, David; Haylock, David N; Nilsson, Susan K; Saffery, Richard; Schenke-Layland, Katja; Oshlack, Alicia; Mikkola, Hanna K A; Stanley, Edouard G; Elefanty, Andrew G

2016-11-01

The ability to generate hematopoietic stem cells from human pluripotent cells would enable many biomedical applications. We find that hematopoietic CD34 + cells in spin embryoid bodies derived from human embryonic stem cells (hESCs) lack HOXA expression compared with repopulation-competent human cord blood CD34 + cells, indicating incorrect mesoderm patterning. Using reporter hESC lines to track the endothelial (SOX17) to hematopoietic (RUNX1C) transition that occurs in development, we show that simultaneous modulation of WNT and ACTIVIN signaling yields CD34 + hematopoietic cells with HOXA expression that more closely resembles that of cord blood. The cultures generate a network of aorta-like SOX17 + vessels from which RUNX1C + blood cells emerge, similar to hematopoiesis in the aorta-gonad-mesonephros (AGM). Nascent CD34 + hematopoietic cells and corresponding cells sorted from human AGM show similar expression of cell surface receptors, signaling molecules and transcription factors. Our findings provide an approach to mimic in vitro a key early stage in human hematopoiesis for the generation of AGM-derived hematopoietic lineages from hESCs.

Derivation of vascular endothelial cells from human embryonic stem cells under GMP-compliant conditions: towards clinical studies in ischaemic disease.

PubMed

Kaupisch, A; Kennedy, L; Stelmanis, V; Tye, B; Kane, N M; Mountford, J C; Courtney, A; Baker, A H

2012-10-01

Revascularisation of ischaemic tissue remains an area of substantial unmet clinical need in cardiovascular disease. Strategies to induce therapeutic angiogenesis are therefore attractive. Our recent focus has been on human embryonic stem cell (hESC) strategies since hESC can be maintained in a pluripotent state or differentiated into any desired cell type, including endothelial cells (EC), under defined differentiation culture conditions. We recently published a protocol for non-good manufacturing practice (GMP) feeder- and serum-free hESC-EC-directed monolayer differentiation to vascular EC demonstrating the potential to generate hESC-derived EC in a GMP-compliant manner suitable for use in clinical trials. In this study we modified that laboratory protocol to GMP compliance. EC production was confirmed by flow cytometry, qRT-PCR and production of vascular structures in Matrigel®, yielding approximately 30 % mature VE-cadherin(+)/PECAM-1(+) cells using the GMP-compliant hESC line RC13. In conclusion, we have successfully demonstrated the production of vascular EC under GMP-compliant conditions suitable for clinical evaluation.

Alternative sources of pluripotency: science, ethics, and stem cells.

PubMed

Kastenberg, Zachary J; Odorico, Jon S

2008-07-01

Despite many advances in human embryonic stem cell (hESC) technology the ethical dilemma involving the destruction of a human embryo is one factor that has limited the development of hESC based clinical therapies. Two recent reports describing the production of pluripotent stem cells following the in vitro reprogramming of human somatic cells with certain defined factors illustrate one potential method of bypassing the ethical debate surrounding hESCs (Yu J, Vodyanik MA, Smuga-Otto K, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007 Dec;318(5858):1917-1920; Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007 Nov;131(5): 861-872.). Other alternative methods include nuclear transfer, altered nuclear transfer, and parthenogenesis; each with its own set of advantages and disadvantages. This review discusses recent advances in these technologies with specific focus on the issues of embryo destruction, oocyte recovery, and the potential of each technology to produce large scale, patient specific cell transplantation therapies that would require little or no immunosuppression.

Rapid fibroblast removal from high density human embryonic stem cell cultures.

PubMed

Turner, William S; McCloskey, Kara E

2012-10-28

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation(1). This feeder system maintains hESCs from undergoing spontaneous differentiation during cell expansion. However, this co-culture method is labor intensive, requires highly trained personnel, and yields low hESC purity(4). Many laboratories have attempted to minimize the number of feeder cells in hESC cultures (i.e. incorporating matrix-coated dishes or other feeder cell types(5-8)). These modified culture systems have shown some promise, but have not supplanted the standard method for culturing hESCs with mitomycin C-treated mouse embyronic fibroblasts in order to retard unwanted spontaneous differentiation of the hESC cultures. Therefore, the feeder cells used in hESC expansion should be removed during differentiation experiments. Although several techniques are available for purifying the hESC colonies (FACS, MACS, or use of drug resistant vectors) from feeders, these techniques are labor intensive, costly and/or destructive to the hESC. The aim of this project was to invent a method of purification that enables the harvesting of a purer population of hESCs. We have observed that in a confluent hESC culture, the MEF population can be removed using a simple and rapid aspiration of the MEF sheet. This removal is dependent on several factors, including lateral cell-to-cell binding of MEFs that have a lower binding affinity to the styrene culture dish, and the ability of the stem cell colonies to push the fibroblasts outward during the generation of their own "niche". The hESC were then examined for SSEA-4, Oct3/4 and Tra 1-81 expression up to 10 days after MEF removal to ensure maintenance of pluripotency. Moreover, hESC colonies were able to continue growing from into larger formations after MEF removal, providing an additional level of hESC expansion.

Governing stem cell banks and registries: emerging issues.

PubMed

Isasi, Rosario M; Knoppers, Bartha M

2009-01-01

The expansion of national and international research efforts in stem cell research is increasingly paired with the trend of establishing stem cell banks and registries. In jurisdictions crossing the spectrum of restrictive to liberal stem cell policies, banks and registries are emerging as an essential resource for transnational access to quality-controlled and ethically sourced stem cell lines. In this study, we report the preliminary findings of a survey of stem cell banks participating in the International Stem Cell Forum's International Stem Cell Banking Initiative (ISCBI). The questionnaire circulated to all ISCBI members addressed both general issues surrounding research policies (e.g., national policies regulating the permissibility of conducting embryonic stem cell research (hESCR)) and, more specifically, issues relating to the governance of stem cell banking projects. The results of the questionnaire were complemented by scholarly research conducted by the authors. This article provides an overview of the current international hESC banking landscape (I). For this purpose, the policy and governance approaches adopted in the surveyed stem cell banks at the national level will be analyzed and areas of convergence and variance will be identified (II). It is beyond the scope of this paper to provide a comprehensive analysis of the wide range of possible governance approaches, policy responses, and their implications. However, we want to provide a starting point for discussion surrounding key questions and challenges as concerns provenance, access, and deposit of hESC lines (III). Finally, while our analysis is focused on research grade hESCs, the lessons to be gleaned from this examination will encourage further thought, analysis, and research into the issues raised in the banking and governance of other sources of stem cell lines (e.g., SCNT, parthenogenesis, iPs) (IV).

ELABELA Is an Endogenous Growth Factor that Sustains hESC Self-Renewal via the PI3K/AKT Pathway.

PubMed

Ho, Lena; Tan, Shawn Y X; Wee, Sheena; Wu, Yixuan; Tan, Sam J C; Ramakrishna, Navin B; Chng, Serene C; Nama, Srikanth; Szczerbinska, Iwona; Sczerbinska, Iwona; Chan, Yun-Shen; Avery, Stuart; Tsuneyoshi, Norihiro; Ng, Huck Hui; Gunaratne, Jayantha; Dunn, N Ray; Reversade, Bruno

2015-10-01

ELABELA (ELA) is a peptide hormone required for heart development that signals via the Apelin Receptor (APLNR, APJ). ELA is also abundantly secreted by human embryonic stem cells (hESCs), which do not express APLNR. Here we show that ELA signals in a paracrine fashion in hESCs to maintain self-renewal. ELA inhibition by CRISPR/Cas9-mediated deletion, shRNA, or neutralizing antibodies causes reduced hESC growth, cell death, and loss of pluripotency. Global phosphoproteomic and transcriptomic analyses of ELA-pulsed hESCs show that it activates PI3K/AKT/mTORC1 signaling required for cell survival. ELA promotes hESC cell-cycle progression and protein translation and blocks stress-induced apoptosis. INSULIN and ELA have partially overlapping functions in hESC medium, but only ELA can potentiate the TGFβ pathway to prime hESCs toward the endoderm lineage. We propose that ELA, acting through an alternate cell-surface receptor, is an endogenous secreted growth factor in human embryos and hESCs that promotes growth and pluripotency. Copyright © 2015 Elsevier Inc. All rights reserved.

Defining "research" in the US and EU: contrast of Sherley v. Sebelius and Brüstle v. Greenpeace rulings.

PubMed

Cuchiara, Maude L; Lawford Davies, James; Matthews, Kirstin R W

2013-12-01

In 2011, courts in both the United States and European Union handed down decisions related to human embryonic stem cell (hESC) research. In both cases, the definition of research was challenged - but the two courts reached different opinions. In the US case, Sherley v. Sebelius, research was defined as a specific project. The US District Court of Appeals did not link research utilizing existing hESC lines to the act of destroying a human embryo in order to create the line, which is not eligible for federal funding. In contrast, the Court of Justice of the European Union in the Brüstle v. Greenpeace case determined inventions related to hESCs were unpatentable since they resulted from research that involved the destruction of human embryos. In this article, we will compare and contrast these two court cases, the politics related to the rulings, and their impacts. We find that these cases significantly impacted current research and have the potential to negatively impact future stem cell research and development. However, the long-term effects of the cases remain to be seen, and there is a chance that these cases could actually strengthen this area of science. Ultimately, we feel that stem cell polices must be straightforward and supported by the public to prevent courts and judges from making decisions on science, which are disruptive to the progression of research.

Dosage and cell line dependent inhibitory effect of bFGF supplement in human pluripotent stem cell culture on inactivated human mesenchymal stem cells.

PubMed

Quang, Tara; Marquez, Maribel; Blanco, Giselle; Zhao, Yuanxiang

2014-01-01

Many different culture systems have been developed for expanding human pluripotent stem cells (hESCs and hiPSCs). In general, 4-10 ng/ml of bFGF is supplemented in culture media in feeder-dependent systems regardless of feeder cell types, whereas in feeder-free systems, up to 100 ng/ml of bFGF is required for maintaining long-term culture on various substrates. The amount of bFGF required in native hESCs growth niche is unclear. Here we report using inactivated adipose-derived human mesenchymal stem cells as feeder cells to examine long-term parallel cultures of two hESCs lines (H1 and H9) and one hiPSCs line (DF19-9-7T) in media supplemented with 0, 0.4 or 4 ng/ml of bFGF for up to 23 passages, as well as parallel cultures of H9 and DF19 in media supplemented with 4, 20 or 100 ng/ml bFGF for up to 13 passages for comparison. Across all cell lines tested, bFGF supplement demonstrated inhibitory effect over growth expansion, single cell colonization and recovery from freezing in a dosage dependent manner. In addition, bFGF exerted differential effects on different cell lines, inducing H1 and DF19 differentiation at 4 ng/ml or higher, while permitting long-term culture of H9 at the same concentrations with no apparent dosage effect. Pluripotency was confirmed for all cell lines cultured in 0, 0.4 or 4 ng/ml bFGF excluding H1-4 ng, as well as H9 cultured in 4, 20 and 100 ng/ml bFGF. However, DF19 demonstrated similar karyotypic abnormality in both 0 and 4 ng/ml bFGF media while H1 and H9 were karyotypically normal in 0 ng/ml bFGF after long-term culture. Our results indicate that exogenous bFGF exerts dosage and cell line dependent effect on human pluripotent stem cells cultured on mesenchymal stem cells, and implies optimal use of bFGF in hESCs/hiPSCs culture should be based on specific cell line and its culture system.

Dosage and Cell Line Dependent Inhibitory Effect of bFGF Supplement in Human Pluripotent Stem Cell Culture on Inactivated Human Mesenchymal Stem Cells

PubMed Central

Quang, Tara; Marquez, Maribel; Blanco, Giselle; Zhao, Yuanxiang

2014-01-01

Many different culture systems have been developed for expanding human pluripotent stem cells (hESCs and hiPSCs). In general, 4–10 ng/ml of bFGF is supplemented in culture media in feeder-dependent systems regardless of feeder cell types, whereas in feeder-free systems, up to 100 ng/ml of bFGF is required for maintaining long-term culture on various substrates. The amount of bFGF required in native hESCs growth niche is unclear. Here we report using inactivated adipose-derived human mesenchymal stem cells as feeder cells to examine long-term parallel cultures of two hESCs lines (H1 and H9) and one hiPSCs line (DF19-9-7T) in media supplemented with 0, 0.4 or 4 ng/ml of bFGF for up to 23 passages, as well as parallel cultures of H9 and DF19 in media supplemented with 4, 20 or 100 ng/ml bFGF for up to 13 passages for comparison. Across all cell lines tested, bFGF supplement demonstrated inhibitory effect over growth expansion, single cell colonization and recovery from freezing in a dosage dependent manner. In addition, bFGF exerted differential effects on different cell lines, inducing H1 and DF19 differentiation at 4 ng/ml or higher, while permitting long-term culture of H9 at the same concentrations with no apparent dosage effect. Pluripotency was confirmed for all cell lines cultured in 0, 0.4 or 4 ng/ml bFGF excluding H1-4 ng, as well as H9 cultured in 4, 20 and 100 ng/ml bFGF. However, DF19 demonstrated similar karyotypic abnormality in both 0 and 4 ng/ml bFGF media while H1 and H9 were karyotypically normal in 0 ng/ml bFGF after long-term culture. Our results indicate that exogenous bFGF exerts dosage and cell line dependent effect on human pluripotent stem cells cultured on mesenchymal stem cells, and implies optimal use of bFGF in hESCs/hiPSCs culture should be based on specific cell line and its culture system. PMID:24465853

Development and production of good manufacturing practice grade human embryonic stem cell lines as source material for clinical application.

PubMed

De Sousa, P A; Downie, J M; Tye, B J; Bruce, K; Dand, P; Dhanjal, S; Serhal, P; Harper, J; Turner, M; Bateman, M

2016-09-01

From 2006 to 2011, Roslin Cells Ltd derived 17 human embryonic stem cells (hESC) while developing (RCM1, RC-2 to -8, -10) and implementing (RC-9, -11 to -17) quality assured standards of operation in a facility operating in compliance with European Union (EU) directives and United Kingdom (UK) licensure for procurement, processing and storage of human cells as source material for clinical application, and targeted to comply with an EU Good Manufacturing Practice specification. Here we describe the evolution and specification of the facility, its operation and outputs, complementing hESC resource details communicated in Stem Cell Research Lab Resources. Copyright © 2016. Published by Elsevier B.V.

Rapid micropatterning of cell lines and human pluripotent stem cells on elastomeric membranes.

PubMed

Paik, Isha; Scurr, David J; Morris, Bryan; Hall, Graham; Denning, Chris; Alexander, Morgan R; Shakesheff, Kevin M; Dixon, James E

2012-10-01

Tissue function during development and in regenerative medicine completely relies on correct cell organization and patterning at micro and macro scales. We describe a rapid method for patterning mammalian cells including human embryonic stem cells (HESCs) and induced pluripotent stem cells (iPSCs) on elastomeric membranes such that micron-scale control of cell position can be achieved over centimeter-length scales. Our method employs surface engineering of hydrophobic polydimethylsiloxane (PDMS) membranes by plasma polymerization of allylamine. Deposition of plasma polymerized allylamine (ppAAm) using our methods may be spatially restricted using a micro-stencil leaving faithful hydrophilic ppAAm patterns. We employed airbrushing to create aerosols which deposit extracellular matrix (ECM) proteins (such as fibronectin and Matrigel™) onto the same patterned ppAAm rich regions. Cell patterns were created with a variety of well characterized cell lines (e.g., NIH-3T3, C2C12, HL1, BJ6, HESC line HUES7, and HiPSC line IPS2). Individual and multiple cell line patterning were also achieved. Patterning remains faithful for several days and cells are viable and proliferate. To demonstrate the utility of our technique we have patterned cells in a variety of configurations. The ability to rapidly pattern cells at high resolution over macro scales should aid future tissue engineering efforts for regenerative medicine applications and in creating in vitro stem cell niches. Copyright © 2012 Wiley Periodicals, Inc.

Stem Cell Therapy in Injured Vocal Folds: A Three-Month Xenograft Analysis of Human Embryonic Stem Cells

PubMed Central

Svensson, Bengt; Nagubothu, Srinivasa R.; Nord, Christoffer; Cedervall, Jessica; Hultman, Isabell; Ährlund-Richter, Lars; Tolf, Anna; Hertegård, Stellan

2015-01-01

We have previously shown that human embryonic stem cell (hESC) therapy to injured rabbit vocal folds (VFs) induces human tissue generation with regained VF vibratory capacity. The aims of this study were to test the sustainability of such effect and to what extent derivatives of the transplanted hESCs are propagated in the VFs. The VFs of 14 New Zealand rabbits were injured by a localized resection. HESCs were transplanted to 22 VFs which were analyzed for persistence of hESCs after six weeks and after three months. At three months, the VFs were also analyzed for viscoelasticity, measured as dynamic viscosity and elastic modulus, for the lamina propria (Lp) thickness and relative content of collagen type I. Three months after hESC cell therapy, the dynamic viscosity and elastic modulus of the hESC treated VFs were similar to normal controls and lower than untreated VFs (p ≤ 0.011). A normalized VF architecture, reduction in collagen type I, and Lp thickness were found compared with untreated VFs (p ≤ 0.031). At three months, no derivatives of hESCs were detected. HESCs transplanted to injured rabbit VFs restored the vibratory characteristics of the VFs, with maintained restored function for three months without remaining hESCs or derivatives. PMID:26557696

Stem Cell Therapy in Injured Vocal Folds: A Three-Month Xenograft Analysis of Human Embryonic Stem Cells.

PubMed

Svensson, Bengt; Nagubothu, Srinivasa R; Nord, Christoffer; Cedervall, Jessica; Hultman, Isabell; Ährlund-Richter, Lars; Tolf, Anna; Hertegård, Stellan

2015-01-01

We have previously shown that human embryonic stem cell (hESC) therapy to injured rabbit vocal folds (VFs) induces human tissue generation with regained VF vibratory capacity. The aims of this study were to test the sustainability of such effect and to what extent derivatives of the transplanted hESCs are propagated in the VFs. The VFs of 14 New Zealand rabbits were injured by a localized resection. HESCs were transplanted to 22 VFs which were analyzed for persistence of hESCs after six weeks and after three months. At three months, the VFs were also analyzed for viscoelasticity, measured as dynamic viscosity and elastic modulus, for the lamina propria (Lp) thickness and relative content of collagen type I. Three months after hESC cell therapy, the dynamic viscosity and elastic modulus of the hESC treated VFs were similar to normal controls and lower than untreated VFs (p ≤ 0.011). A normalized VF architecture, reduction in collagen type I, and Lp thickness were found compared with untreated VFs (p ≤ 0.031). At three months, no derivatives of hESCs were detected. HESCs transplanted to injured rabbit VFs restored the vibratory characteristics of the VFs, with maintained restored function for three months without remaining hESCs or derivatives.

ATM-independent, high-fidelity nonhomologous end joining predominates in human embryonic stem cells

PubMed Central

Adams, Bret R.; Hawkins, Amy J.; Povirk, Lawrence F.; Valerie, Kristoffer

2010-01-01

We recently demonstrated that human embryonic stem cells (hESCs) utilize homologous recombination repair (HRR) as primary means of double-strand break (DSB) repair. We now show that hESCs also use nonhomologous end joining (NHEJ). NHEJ kinetics were several-fold slower in hESCs and neural progenitors (NPs) than in astrocytes derived from hESCs. ATM and DNA-PKcs inhibitors were ineffective or partially effective, respectively, at inhibiting NHEJ in hESCs, whereas progressively more inhibition was seen in NPs and astrocytes. The lack of any major involvement of DNA-PKcs in NHEJ in hESCs was supported by siRNA-mediated DNA-PKcs knockdown. Expression of a truncated XRCC4 decoy or XRCC4 knock-down reduced NHEJ by more than half suggesting that repair is primarily canonical NHEJ. Poly(ADP-ribose) polymerase (PARP) was dispensable for NHEJ suggesting that repair is largely independent of backup NHEJ. Furthermore, as hESCs differentiated a progressive decrease in the accuracy of NHEJ was observed. Altogether, we conclude that NHEJ in hESCs is largely independent of ATM, DNA-PKcs, and PARP but dependent on XRCC4 with repair fidelity several-fold greater than in astrocytes. PMID:20844317

Differential impact of science policy on subfields of human embryonic stem cell research.

PubMed

Moon, Seongwuk; Cho, Seong Beom

2014-01-01

In this research, we examine how restrictive policy influenced performance in human embryonic stem cell research (hESC) between 1998 and 2008. In previous research, researchers argued whether restrictive policy decreased the performance of stem cell research in some nations, especially in the US. Here, we hypothesize that this policy influenced specific subfields of the hESC research. To investigate the selective policy effects, we categorize hESC research publications into three subfields-derivation, differentiation, and medical application research. Our analysis shows that restrictive policy had different effects on different subfields. In general, the US outperformed in overall hESC research throughout these periods. In the derivation of hESC, however, the US almost lost its competence under restrictive policy. Interestingly, the US scientific community showed prominent resilience in hESC research through international collaboration. We concluded that the US resilience and performance stemmed from the wide breadth of research portfolio of US scientists across the hESC subfields, combined with their strategic efforts to collaborate internationally on derivation research.

Enzyme-mediated hyaluronic acid-tyramine hydrogels for the propagation of human embryonic stem cells in 3D.

PubMed

Xu, Keming; Narayanan, Karthikeyan; Lee, Fan; Bae, Ki Hyun; Gao, Shujun; Kurisawa, Motoichi

2015-09-01

The propagation of human embryonic stem cells (hESCs) in three-dimensional (3D) scaffolds facilitates the cell expansion process and supplies pluripotent cells of high quality for broad-spectrum applications in regenerative medicine. Herein, we report an enzyme-mediated hyaluronic acid-tyramine (HA-Tyr) hydrogel that encapsulated and propagated hESCs in 3D. HA-Tyr hydrogels were formed by crosslinking the tyramine moieties with horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). By changing the HRP and H2O2 concentration, we prepared HA-Tyr hydrogels of different mechanical strength and studied the self-renewal properties of hESCs in these scaffolds. We observed that both the chemical composition and mechanical strength of substrates were important factors affecting cell proliferation and pluripotency. The HA-Tyr hydrogel with a compressive modulus of ∼350Pa supported the proliferation of hESCs at the pluripotent state in both mTeSR1 medium and mouse embryonic fibroblast (MEF)-conditioned medium. Immunohistochemical analyses revealed that hESCs proliferated well and formed spheroid structures in 3D, without undergoing apoptosis. The hESCs cultured in HA-Tyr hydrogels showed high expression of CD44 and pluripotency markers. These cells exhibited the capability to form cell derivatives of all three embryonic germ layers in vitro and in vivo. In addition, the genetic integrity of the hESCs was unaffected in the 3D cultivation system. The scope of this study is to provide a stable 3D cultivation system for the expansion of human embryonic stem cells (hESCs) towards clinical applications. We report an enzyme mediated hyaluronic acid-tyramine (HA-Tyr) hydrogel that encapsulated and propagated hESCs in 3D. Unlike other HA-based photo-crosslinked hydrogel systems reported, we investigated the effects of mechanical strength of hydrogels on the self-renewal properties of hESCs in 3D. Then, we characterized hESCs cultured in hydrogels with lower mechanical strength that best supported the self-renewal of hESCs. Hence, we demonstrated a reliable approach for the controlled propagation of hESCs in 3D. We believe that such an approach would facilitate the development of stem cell-based therapy towards clinical applications. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Polyphenol-Rich Extract of Syzygium cumini Leaf Dually Improves Peripheral Insulin Sensitivity and Pancreatic Islet Function in Monosodium L-Glutamate-Induced Obese Rats

PubMed Central

Sanches, Jonas R.; França, Lucas M.; Chagas, Vinicyus T.; Gaspar, Renato S.; dos Santos, Kayque A.; Gonçalves, Luciana M.; Sloboda, Deborah M.; Holloway, Alison C.; Dutra, Richard P.; Carneiro, Everardo M.; Cappelli, Ana Paula G.; Paes, Antonio Marcus de A.

2016-01-01

Syzygium cumini (L.) Skeels (Myrtaceae) has been traditionally used to treat a number of illnesses. Ethnopharmacological studies have particularly addressed antidiabetic and metabolic-related effects of extracts prepared from its different parts, especially seed, and pulp-fruit, however. there is a lack of studies on phytochemical profile and biological properties of its leaf. As there is considerable interest in bioactive compounds to treat metabolic syndrome and its clustered risk factors, we sought to characterize the metabolic effects of hydroethanolic extract of S. cumini leaf (HESc) on lean and monosodium L-glutamate (MSG)-induced obese rats. HPLC-MS/MS characterization of the HESc polyphenolic profile, at 254 nm, identified 15 compounds pertaining to hydrolysable tannin and flavanol subclasses. At 60 days of age, both groups were randomly assigned to receive HESc (500 mg/kg) or vehicle for 30 days. At the end of treatment, obese+HESc exhibited significantly lower body weight gain, body mass index, and white adipose tissue mass, compared to obese rats receiving vehicle. Obese rats treated with HESc showed a twofold increase in lipolytic activity in the periepididymal fat pad, as well as, brought triglyceride levels in serum, liver and skeletal muscle back to levels close those found in lean animals. Furthermore, HESc also improved hyperinsulinemia and insulin resistance in obese+HESc rats, which resulted in partial reversal of glucose intolerance, as compared to obese rats. HESc had no effect in lean rats. Assessment of ex vivo glucose-stimulated insulin secretion showed HESc potentiated pancreatic function in islets isolated from both lean and obese rats treated with HESc. In addition, HESc (10–1000 μg/mL) increased glucose stimulated insulin secretion from both isolated rat islets and INS-1E β-cells. These data demonstrate that S. cumini leaf improved peripheral insulin sensitivity via stimulating/modulating β-cell insulin release, which was associated with improvements in metabolic outcomes in MSG-induced obese rats. PMID:27014062

Polyphenol-Rich Extract of Syzygium cumini Leaf Dually Improves Peripheral Insulin Sensitivity and Pancreatic Islet Function in Monosodium L-Glutamate-Induced Obese Rats.

PubMed

Sanches, Jonas R; França, Lucas M; Chagas, Vinicyus T; Gaspar, Renato S; Dos Santos, Kayque A; Gonçalves, Luciana M; Sloboda, Deborah M; Holloway, Alison C; Dutra, Richard P; Carneiro, Everardo M; Cappelli, Ana Paula G; Paes, Antonio Marcus de A

2016-01-01

Syzygium cumini (L.) Skeels (Myrtaceae) has been traditionally used to treat a number of illnesses. Ethnopharmacological studies have particularly addressed antidiabetic and metabolic-related effects of extracts prepared from its different parts, especially seed, and pulp-fruit, however. there is a lack of studies on phytochemical profile and biological properties of its leaf. As there is considerable interest in bioactive compounds to treat metabolic syndrome and its clustered risk factors, we sought to characterize the metabolic effects of hydroethanolic extract of S. cumini leaf (HESc) on lean and monosodium L-glutamate (MSG)-induced obese rats. HPLC-MS/MS characterization of the HESc polyphenolic profile, at 254 nm, identified 15 compounds pertaining to hydrolysable tannin and flavanol subclasses. At 60 days of age, both groups were randomly assigned to receive HESc (500 mg/kg) or vehicle for 30 days. At the end of treatment, obese+HESc exhibited significantly lower body weight gain, body mass index, and white adipose tissue mass, compared to obese rats receiving vehicle. Obese rats treated with HESc showed a twofold increase in lipolytic activity in the periepididymal fat pad, as well as, brought triglyceride levels in serum, liver and skeletal muscle back to levels close those found in lean animals. Furthermore, HESc also improved hyperinsulinemia and insulin resistance in obese+HESc rats, which resulted in partial reversal of glucose intolerance, as compared to obese rats. HESc had no effect in lean rats. Assessment of ex vivo glucose-stimulated insulin secretion showed HESc potentiated pancreatic function in islets isolated from both lean and obese rats treated with HESc. In addition, HESc (10-1000 μg/mL) increased glucose stimulated insulin secretion from both isolated rat islets and INS-1E β-cells. These data demonstrate that S. cumini leaf improved peripheral insulin sensitivity via stimulating/modulating β-cell insulin release, which was associated with improvements in metabolic outcomes in MSG-induced obese rats.

Laser-assisted selection and passaging of human pluripotent stem cell colonies.

PubMed

Terstegge, Stefanie; Rath, Barbara H; Laufenberg, Iris; Limbach, Nina; Buchstaller, Andrea; Schütze, Karin; Brüstle, Oliver

2009-09-10

The derivation of somatic cell products from human embryonic stem cells (hESCs) requires a highly standardized production process with sufficient throughput. To date, the most common technique for hESC passaging is the manual dissection of colonies, which is a gentle, but laborious and time-consuming process and is consequently inappropriate for standardized maintenance of hESC. Here, we present a laser-based technique for the contact-free dissection and isolation of living hESCs (laser microdissection and pressure catapulting, LMPC). Following LMPC treatment, 80.6+/-8.7% of the cells remained viable as compared to 88.6+/-1.7% of manually dissected hESCs. Furthermore, there was no significant difference in the expression of pluripotency-associated markers when compared to the control. Flow cytometry revealed that 83.8+/-4.1% of hESCs isolated by LMPC expressed the surface marker Tra-1-60 (control: 83.9+/-3.6%). In vitro differentiation potential of LMPC treated hESCs as determined by embryoid body formation and multi-germlayer formation was not impaired. Moreover, we could not detect any overt karyotype alterations as a result of the LMPC process. Our data demonstrate the feasibility of standardized laser-based passaging of hESC cultures. This technology should facilitate both colony selection and maintenance culture of pluripotent stem cells.

Multivariate Calibration Approach for Quantitative Determination of Cell-Line Cross Contamination by Intact Cell Mass Spectrometry and Artificial Neural Networks.

PubMed

Valletta, Elisa; Kučera, Lukáš; Prokeš, Lubomír; Amato, Filippo; Pivetta, Tiziana; Hampl, Aleš; Havel, Josef; Vaňhara, Petr

2016-01-01

Cross-contamination of eukaryotic cell lines used in biomedical research represents a highly relevant problem. Analysis of repetitive DNA sequences, such as Short Tandem Repeats (STR), or Simple Sequence Repeats (SSR), is a widely accepted, simple, and commercially available technique to authenticate cell lines. However, it provides only qualitative information that depends on the extent of reference databases for interpretation. In this work, we developed and validated a rapid and routinely applicable method for evaluation of cell culture cross-contamination levels based on mass spectrometric fingerprints of intact mammalian cells coupled with artificial neural networks (ANNs). We used human embryonic stem cells (hESCs) contaminated by either mouse embryonic stem cells (mESCs) or mouse embryonic fibroblasts (MEFs) as a model. We determined the contamination level using a mass spectra database of known calibration mixtures that served as training input for an ANN. The ANN was then capable of correct quantification of the level of contamination of hESCs by mESCs or MEFs. We demonstrate that MS analysis, when linked to proper mathematical instruments, is a tangible tool for unraveling and quantifying heterogeneity in cell cultures. The analysis is applicable in routine scenarios for cell authentication and/or cell phenotyping in general.

Multivariate Calibration Approach for Quantitative Determination of Cell-Line Cross Contamination by Intact Cell Mass Spectrometry and Artificial Neural Networks

PubMed Central

Prokeš, Lubomír; Amato, Filippo; Pivetta, Tiziana; Hampl, Aleš; Havel, Josef; Vaňhara, Petr

2016-01-01

Cross-contamination of eukaryotic cell lines used in biomedical research represents a highly relevant problem. Analysis of repetitive DNA sequences, such as Short Tandem Repeats (STR), or Simple Sequence Repeats (SSR), is a widely accepted, simple, and commercially available technique to authenticate cell lines. However, it provides only qualitative information that depends on the extent of reference databases for interpretation. In this work, we developed and validated a rapid and routinely applicable method for evaluation of cell culture cross-contamination levels based on mass spectrometric fingerprints of intact mammalian cells coupled with artificial neural networks (ANNs). We used human embryonic stem cells (hESCs) contaminated by either mouse embryonic stem cells (mESCs) or mouse embryonic fibroblasts (MEFs) as a model. We determined the contamination level using a mass spectra database of known calibration mixtures that served as training input for an ANN. The ANN was then capable of correct quantification of the level of contamination of hESCs by mESCs or MEFs. We demonstrate that MS analysis, when linked to proper mathematical instruments, is a tangible tool for unraveling and quantifying heterogeneity in cell cultures. The analysis is applicable in routine scenarios for cell authentication and/or cell phenotyping in general. PMID:26821236

Identification of distinct topographical surface microstructures favoring either undifferentiated expansion or differentiation of murine embryonic stem cells.

PubMed

Markert, Lotte D'Andrea; Lovmand, Jette; Foss, Morten; Lauridsen, Rune Hoff; Lovmand, Michael; Füchtbauer, Ernst-Martin; Füchtbauer, Annette; Wertz, Karin; Besenbacher, Flemming; Pedersen, Finn Skou; Duch, Mogens

2009-11-01

The potential of embryonic stem (ES) cells for both self-renewal and differentiation into cells of all three germ layers has generated immense interest in utilizing these cells for tissue engineering or cell-based therapies. However, the ability to culture undifferentiated ES cells without the use of feeder cells as well as means to obtain homogeneous, differentiated cell populations devoid of residual pluripotent ES cells still remain major challenges. Here we have applied murine ES cells to topographically microstructured surface libraries, BioSurface Structure Arrays (BSSA), and investigated whether these could be used to (i) identify topographically microstructured growth supports alleviating the need for feeder cells for expansion of undifferentiated ES cells and (ii) identify specific types of microstructures enforcing differentiation of ES cells. The BSSA surfaces arrays consisted of 504 different topographical microstructures each located in a tester field of 3 x 3 mm. The murine ES cell lines CJ7 and KH2 were seeded upon the BSSA libraries and specific topographical structures facilitating either undifferentiated ES cell growth or enhancing spreading indicative of differentiation of the ES cells were identified. Secondly serial passage of undifferentiated CJ7 ES cells on selected microstructures, identified in the screening of these BSSA libraries, showed that these cells had retained germ-line potential. These results indicate that one specific type of topographical surface microstructures, identified by the BSSA technology, can substitute for feeder cells and that another subset may be used to eliminate undifferentiated ES cells from a population of differentiated ES cells.

Small molecule screening with laser cytometry can be used to identify pro-survival molecules in human embryonic stem cells.

PubMed

Sherman, Sean P; Pyle, April D

2013-01-01

Differentiated cells from human embryonic stem cells (hESCs) provide an unlimited source of cells for use in regenerative medicine. The recent derivation of human induced pluripotent cells (hiPSCs) provides a potential supply of pluripotent cells that avoid immune rejection and could provide patient-tailored therapy. In addition, the use of pluripotent cells for drug screening could enable routine toxicity testing and evaluation of underlying disease mechanisms. However, prior to establishment of patient specific cells for cell therapy it is important to understand the basic regulation of cell fate decisions in hESCs. One critical issue that hinders the use of these cells is the fact that hESCs survive poorly upon dissociation, which limits genetic manipulation because of poor cloning efficiency of individual hESCs, and hampers production of large-scale culture of hESCs. To address the problems associated with poor growth in culture and our lack of understanding of what regulates hESC signaling, we successfully developed a screening platform that allows for large scale screening for small molecules that regulate survival. In this work we developed the first large scale platform for hESC screening using laser scanning cytometry and were able to validate this platform by identifying the pro-survival molecule HA-1077. These small molecules provide targets for both improving our basic understanding of hESC survival as well as a tool to improve our ability to expand and genetically manipulate hESCs for use in regenerative applications.

Concise Review: One Stone for Multiple Birds: Generating Universally Compatible Human Embryonic Stem Cells.

PubMed

Zheng, Dejin; Wang, Xiaofang; Xu, Ren-He

2016-09-01

With ongoing clinical trials, human embryonic stem cells (hESCs) have shown substantial potential for regenerative medicine. However, due to the mismatch of human leukocyte antigens (HLAs) between hESC-derived allografts and recipients, immunosuppressant regimens must be used to prevent immune rejection of the grafts. Considerable efforts have been devoted to overcoming this hurdle via the derivation and banking of human nuclear transfer ESCs, parthenogenetic ESCs, and induced pluripotent stem cells. However, ethical and safety concerns remain, hindering the application of these types of pluripotent cells. Other approaches have recently been explored to generate universally compatible hESCs through the silencing or deletion of HLAs or genes essential for HLA expression, including β-2-microglobulin and class-II MHC transactivator, as well as the induction of immunosuppression via the ectopic expression of non-classical HLAs (e.g., HLA-E and -G), cytotoxic T lymphocyte antigen 4 fused with immunoglobulin, and programmed death ligand-1. In this review, we introduce developments in this line of research and discuss strategies to reduce the tumorigenic concerns regarding hESCs, especially after they acquire the capability to escape immune surveillance. Stem Cells 2016;34:2269-2275. © 2016 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Prospectively isolated NGN3-expressing progenitors from human embryonic stem cells give rise to pancreatic endocrine cells.

PubMed

Cai, Qing; Bonfanti, Paola; Sambathkumar, Rangarajan; Vanuytsel, Kim; Vanhove, Jolien; Gysemans, Conny; Debiec-Rychter, Maria; Raitano, Susanna; Heimberg, Harry; Ordovas, Laura; Verfaillie, Catherine M

2014-04-01

Pancreatic endocrine progenitors obtained from human embryonic stem cells (hESCs) represent a promising source to develop cell-based therapies for diabetes. Although endocrine pancreas progenitor cells have been isolated from mouse pancreata on the basis of Ngn3 expression, human endocrine progenitors have not been isolated yet. As substantial differences exist between human and murine pancreas biology, we investigated whether it is possible to isolate pancreatic endocrine progenitors from differentiating hESC cultures by lineage tracing of NGN3. We targeted the 3' end of NGN3 using zinc finger nuclease-mediated homologous recombination to allow selection of NGN3eGFP(+) cells without disrupting the coding sequence of the gene. Isolated NGN3eGFP(+) cells express PDX1, NKX6.1, and chromogranin A and differentiate in vivo toward insulin, glucagon, and somatostatin single hormone-expressing cells but not to ductal or exocrine pancreatic cells or other endodermal, mesodermal, or ectodermal lineages. This confirms that NGN3(+) cells represent pancreatic endocrine progenitors in humans. In addition, this hESC reporter line constitutes a unique tool that may aid in gaining insight into the developmental mechanisms underlying fate choices in human pancreas and in developing cell-based therapies.

Comparative Proteomic Analysis of Supportive and Unsupportive Extracellular Matrix Substrates for Human Embryonic Stem Cell Maintenance*

PubMed Central

Soteriou, Despina; Iskender, Banu; Byron, Adam; Humphries, Jonathan D.; Borg-Bartolo, Simon; Haddock, Marie-Claire; Baxter, Melissa A.; Knight, David; Humphries, Martin J.; Kimber, Susan J.

2013-01-01

Human embryonic stem cells (hESCs) are pluripotent cells that have indefinite replicative potential and the ability to differentiate into derivatives of all three germ layers. hESCs are conventionally grown on mitotically inactivated mouse embryonic fibroblasts (MEFs) or feeder cells of human origin. In addition, feeder-free culture systems can be used to support hESCs, in which the adhesive substrate plays a key role in the regulation of stem cell self-renewal or differentiation. Extracellular matrix (ECM) components define the microenvironment of the niche for many types of stem cells, but their role in the maintenance of hESCs remains poorly understood. We used a proteomic approach to characterize in detail the composition and interaction networks of ECMs that support the growth of self-renewing hESCs. Whereas many ECM components were produced by supportive and unsupportive MEF and human placental stromal fibroblast feeder cells, some proteins were only expressed in supportive ECM, suggestive of a role in the maintenance of pluripotency. We show that identified candidate molecules can support attachment and self-renewal of hESCs alone (fibrillin-1) or in combination with fibronectin (perlecan, fibulin-2), in the absence of feeder cells. Together, these data highlight the importance of specific ECM interactions in the regulation of hESC phenotype and provide a resource for future studies of hESC self-renewal. PMID:23658023

Dynamic dependence on ATR and ATM for double-strand break repair in human embryonic stem cells and neural descendants.

PubMed

Adams, Bret R; Golding, Sarah E; Rao, Raj R; Valerie, Kristoffer

2010-04-02

The DNA double-strand break (DSB) is the most toxic form of DNA damage. Studies aimed at characterizing DNA repair during development suggest that homologous recombination repair (HRR) is more critical in pluripotent cells compared to differentiated somatic cells in which nonhomologous end joining (NHEJ) is dominant. We have characterized the DNA damage response (DDR) and quality of DNA double-strand break (DSB) repair in human embryonic stem cells (hESCs), and in vitro-derived neural cells. Resolution of ionizing radiation-induced foci (IRIF) was used as a surrogate for DSB repair. The resolution of gamma-H2AX foci occurred at a slower rate in hESCs compared to neural progenitors (NPs) and astrocytes perhaps reflective of more complex DSB repair in hESCs. In addition, the resolution of RAD51 foci, indicative of active homologous recombination repair (HRR), showed that hESCs as well as NPs have high capacity for HRR, whereas astrocytes do not. Importantly, the ATM kinase was shown to be critical for foci formation in astrocytes, but not in hESCs, suggesting that the DDR is different in these cells. Blocking the ATM kinase in astrocytes not only prevented the formation but also completely disassembled preformed repair foci. The ability of hESCs to form IRIF was abrogated with caffeine and siRNAs targeted against ATR, implicating that hESCs rely on ATR, rather than ATM for regulating DSB repair. This relationship dynamically changed as cells differentiated. Interestingly, while the inhibition of the DNA-PKcs kinase (and presumably non-homologous endjoining [NHEJ]) in astrocytes slowed IRIF resolution it did not in hESCs, suggesting that repair in hESCs does not utilize DNA-PKcs. Altogether, our results show that hESCs have efficient DSB repair that is largely ATR-dependent HRR, whereas astrocytes critically depend on ATM for NHEJ, which, in part, is DNA-PKcs-independent.

Sensitivity of human embryonic stem cells to different conditions during cryopreservation.

PubMed

Xu, Yanqing; Zhang, Liang; Xu, Jiandong; Wei, Yuping; Xu, Xia

2015-12-01

Low cell recovery rate of human embryonic stem cells (hESCs) resulting from cryopreservation damages leads to the difficulty in their successful commercialization of clinical applications. Hence in this study, sensitivity of human embryonic stem cells (hESCs) to different cooling rates, ice seeding and cryoprotective agent (CPA) types was compared and cell viability and recovery after cryopreservation under different cooling conditions were assessed. Both extracellular and intracellular ice formation were observed. Reactive oxidative species (ROS) accumulation of hESCs was determined. Cryopreservation of hESCs at 1 °C/min with the ice seeding and at the theoretically predicted optimal cooling rate (TPOCR) led to lower level of intracellular ROS, and prevented irregular and big ice clump formation compared with cryopreservation at 1 °C/min. This strategy further resulted in a significant increase in the hESC recovery when glycerol and 1,2-propanediol were used as the CPAs, but no increase for Me2SO. hESCs after cryopreservation under all the tested conditions still maintained their pluripotency. Our results provide guidance for improving the hESC cryopreservation recovery through the combination of CPA type, cooling rate and ice seeding. Copyright © 2015 Elsevier Inc. All rights reserved.

Generating hypoimmunogenic human embryonic stem cells by the disruption of beta 2-microglobulin.

PubMed

Lu, Pengfei; Chen, Jijun; He, Lixiazi; Ren, Jiangtao; Chen, Haide; Rao, Lingjun; Zhuang, Qinggang; Li, Hui; Li, Lei; Bao, Lei; He, Ji; Zhang, Wei; Zhu, Faming; Cui, Chun; Xiao, Lei

2013-12-01

Immune rejection hinders the application of human embryonic stem cells (hESCs) in transplantation therapy. Human leukocyte antigens (HLAs) on the cell surface are the major cause of graft rejection. In this study, we generated HLA class I-deficient hESCs via disruption of beta 2-microglobulin (β2m), the light chain of HLA Class I. We found that HLA class I proteins were not present on the cell surface of β2m-null hESCs. These cells showed the same pluripotency as wildtype hESCs and demonstrated hypoimmunogenicity. Thus, HLA class I-deficient hESCs might serve as an unlimited cell source for the generation of universally compatible "off-the-shelf" cell grafts, tissues or organs in the future.

Constructing an ethical framework for embryo donation to research: is it time for a restricted consent policy?

PubMed

Ehrich, Kathryn; Farsides, Bobbie; Williams, Clare; Scott, Rosamund

2011-06-01

An Ethics & Policy Workshop was held with 20 invited UK stakeholders to consider whether embryo donors should be able to restrict the future use of human embryonic stem cells (hESCs) created from their embryos. Participants cited tensions between pure altruism and a more reciprocal basis for donation; and between basic research (in which genetic material would never form part of another living being) and treatment applications. Two restriction models were suggested to acknowledge specific ethical issues raised by hESCs' use in research and treatments: (1) a two tier system: hESCs with unrestricted consent could go to the UK Stem Cell Bank; those with restricted consent could be used in individual labs which could guarantee to honour the restrictions, and Bank deposit would not be required. (2) a three category system: restrictions could include (i) basic hESC research; (ii) hESC research and treatment; no gamete derivation (iii) 'unrestricted' hESC research and treatment.

Wnt/β-catenin signaling promotes self-renewal and inhibits the primed state transition in naïve human embryonic stem cells.

PubMed

Xu, Zhuojin; Robitaille, Aaron M; Berndt, Jason D; Davidson, Kathryn C; Fischer, Karin A; Mathieu, Julie; Potter, Jennifer C; Ruohola-Baker, Hannele; Moon, Randall T

2016-10-18

In both mice and humans, pluripotent stem cells (PSCs) exist in at least two distinct states of pluripotency, known as the naïve and primed states. Our understanding of the intrinsic and extrinsic factors that enable PSCs to self-renew and to transition between different pluripotent states is important for understanding early development. In mouse embryonic stem cells (mESCs), Wnt proteins stimulate mESC self-renewal and support the naïve state. In human embryonic stem cells (hESCs), Wnt/β-catenin signaling is active in naïve-state hESCs and is reduced or absent in primed-state hESCs. However, the role of Wnt/β-catenin signaling in naïve hESCs remains largely unknown. Here, we demonstrate that inhibition of the secretion of Wnts or inhibition of the stabilization of β-catenin in naïve hESCs reduces cell proliferation and colony formation. Moreover, we show that addition of recombinant Wnt3a partially rescues cell proliferation in naïve hESCs caused by inhibition of Wnt secretion. Notably, inhibition of Wnt/β-catenin signaling in naïve hESCs did not cause differentiation. Instead, it induced primed hESC-like proteomic and metabolic profiles. Thus, our results suggest that naïve hESCs secrete Wnts that activate autocrine or paracrine Wnt/β-catenin signaling to promote efficient self-renewal and inhibit the transition to the primed state.

Patient-specific embryonic stem cells derived from human SCNT blastocysts.

PubMed

Hwang, Woo Suk; Roh, Sung Il; Lee, Byeong Chun; Kang, Sung Keun; Kwon, Dae Kee; Kim, Sue; Kim, Sun Jong; Park, Sun Woo; Kwon, Hee Sun; Lee, Chang Kyu; Lee, Jung Bok; Kim, Jin Mee; Ahn, Curie; Paek, Sun Ha; Chang, Sang Sik; Koo, Jung Jin; Yoon, Hyun Soo; Hwang, Jung Hye; Hwang, Youn Young; Park, Ye Soo; Oh, Sun Kyung; Kim, Hee Sun; Park, Jong Hyuk; Moon, Shin Yong; Schatten, Gerald

2005-06-17

Patient-specific, immune-matched human embryonic stem cells (hESCs) are anticipated to be of great biomedical importance for studies of disease and development and to advance clinical deliberations regarding stem cell transplantation. Eleven hESC lines were established by somatic cell nuclear transfer (SCNT) of skin cells from patients with disease or injury into donated oocytes. These lines, nuclear transfer (NT)-hESCs, grown on human feeders from the same NT donor or from genetically unrelated individuals, were established at high rates, regardless of NT donor sex or age. NT-hESCs were pluripotent, chromosomally normal, and matched the NT patient's DNA. The major histocompatibility complex identity of each NT-hESC when compared to the patient's own showed immunological compatibility, which is important for eventual transplantation. With the generation of these NT-hESCs, evaluations of genetic and epigenetic stability can be made. Additional work remains to be done regarding the development of reliable directed differentiation and the elimination of remaining animal components. Before clinical use of these cells can occur, preclinical evidence is required to prove that transplantation of differentiated NT-hESCs can be safe, effective, and tolerated.

Lectin binding profiles of SSEA-4 enriched, pluripotent human embryonic stem cell surfaces

PubMed Central

Venable, Alison; Mitalipova, Maisam; Lyons, Ian; Jones, Karen; Shin, Soojung; Pierce, Michael; Stice, Steven

2005-01-01

Background Pluripotent human embryonic stem cells (hESCs) have the potential to form every cell type in the body. These cells must be appropriately characterized prior to differentiation studies or when defining characteristics of the pluripotent state. Some developmentally regulated cell surface antigens identified by monoclonal antibodies in a variety of species and stem cell types have proven to be side chains of membrane glycolipids and glycoproteins. Therefore, to examine hESC surfaces for other potential pluripotent markers, we used a panel of 14 lectins, which were chosen based on their specificity for a variety of carbohydrates and carbohydrate linkages, along with stage specific embryonic antigen-4 (SSEA-4), to determine binding quantitation by flow cytometry and binding localization in adherent colonies by immunocytochemistry. Results Enriching cells for SSEA-4 expression increased the percentage of SSEA-4 positive cells to 98–99%. Using enriched high SSEA-4-expressing hESCs, we then analyzed the binding percentages of selected lectins and found a large variation in binding percentages ranging from 4% to 99% binding. Lycopersicon (tomato)esculetum lectin (TL), Ricinus communis agglutinin (RCA), and Concanavalin A (Con A) bound to SSEA-4 positive regions of hESCs and with similar binding percentages as SSEA-4. In contrast, we found Dolichos biflorus agglutinin (DBA) and Lotus tetragonolobus lectin (LTL) did not bind to hESCs while Phaseolus vulgaris leuco-agglutinin (PHA-L), Vicia villosa agglutinin (VVA), Ulex europaeus agglutinin (UEA), Phaseolus vulgaris erythro-agglutinin (PHA-E), and Maackia amurensis agglutinin (MAA) bound partially to hESCs. These binding percentages correlated well with immunocytochemistry results. Conclusion Our results provide information about types of carbohydrates and carbohydrate linkages found on pluripotent hESC surfaces. We propose that TL, RCA and Con A may be used as markers that are associated with the pluripotent state of hESCs because binding percentages and binding localization of these lectins are similar to those of SSEA-4. Non-binding lectins, DBA and LTL, may identify differentiated cell types; however, we did not find these lectins to bind to pluripotent SSEA-4 positive hESCs. This work represents a fundamental base to systematically classify pluripotent hESCs, and in future studies these lectins may be used to distinguish differentiated hESC types based on glycan presentation that accompanies differentiation. PMID:16033656

An In Vitro Mechanism Study on the Proliferation and Pluripotency of Human Embryonic Stems Cells in Response to Magnesium Degradation

PubMed Central

Nguyen, Thanh Yen; Liew, Chee Gee; Liu, Huinan

2013-01-01

Magnesium (Mg) is a promising biodegradable metallic material for applications in cellular/tissue engineering and biomedical implants/devices. To advance clinical translation of Mg-based biomaterials, we investigated the effects and mechanisms of Mg degradation on the proliferation and pluripotency of human embryonic stem cells (hESCs). We used hESCs as the in vitro model system to study cellular responses to Mg degradation because they are sensitive to toxicants and capable of differentiating into any cell types of interest for regenerative medicine. In a previous study when hESCs were cultured in vitro with either polished metallic Mg (99.9% purity) or pre-degraded Mg, cell death was observed within the first 30 hours of culture. Excess Mg ions and hydroxide ions induced by Mg degradation may have been the causes for the observed cell death; hence, their respective effects on hESCs were investigated for the first time to reveal the potential mechanisms. For this purpose, the mTeSR®1 hESC culture media was either modified to an alkaline pH of 8.1 or supplemented with 0.4–40 mM of Mg ions. We showed that the initial increase of media pH to 8.1 had no adverse effect on hESC proliferation. At all tested Mg ion dosages, the hESCs grew to confluency and retained pluripotency as indicated by the expression of OCT4, SSEA3, and SOX2. When the supplemental Mg ion dosages increased to greater than 10 mM, however, hESC colony morphology changed and cell counts decreased. These results suggest that Mg-based implants or scaffolds are promising in combination with hESCs for regenerative medicine applications, providing their degradation rate is moderate. Additionally, the hESC culture system could serve as a standard model for cytocompatibility studies of Mg in vitro, and an identified 10 mM critical dosage of Mg ions could serve as a design guideline for safe degradation of Mg-based implants/scaffolds. PMID:24146887

An Efficient Method for Generation of Knockout Human Embryonic Stem Cells Using CRISPR/Cas9 System.

PubMed

Bohaciakova, Dasa; Renzova, Tereza; Fedorova, Veronika; Barak, Martin; Kunova Bosakova, Michaela; Hampl, Ales; Cajanek, Lukas

2017-11-01

Human embryonic stem cells (hESCs) represent a promising tool to study functions of genes during development, to model diseases, and to even develop therapies when combined with gene editing techniques such as CRISPR/CRISPR-associated protein-9 nuclease (Cas9) system. However, the process of disruption of gene expression by generation of null alleles is often inefficient and tedious. To circumvent these limitations, we developed a simple and efficient protocol to permanently downregulate expression of a gene of interest in hESCs using CRISPR/Cas9. We selected p53 for our proof of concept experiments. The methodology is based on series of hESC transfection, which leads to efficient downregulation of p53 expression even in polyclonal population (p53 Low cells), here proven by a loss of regulation of the expression of p53 target gene, microRNA miR-34a. We demonstrate that our approach achieves over 80% efficiency in generating hESC clonal sublines that do not express p53 protein. Importantly, we document by a set of functional experiments that such genetically modified hESCs do retain typical stem cells characteristics. In summary, we provide a simple and robust protocol to efficiently target expression of gene of interest in hESCs that can be useful for laboratories aiming to employ gene editing in their hESC applications/protocols.

Microencapsulation Technology: A Powerful Tool for Integrating Expansion and Cryopreservation of Human Embryonic Stem Cells

PubMed Central

Malpique, Rita; Brito, Catarina; Jensen, Janne; Bjorquist, Petter; Carrondo, Manuel J. T.; Alves, Paula M.

2011-01-01

The successful implementation of human embryonic stem cells (hESCs)-based technologies requires the production of relevant numbers of well-characterized cells and their efficient long-term storage. In this study, cells were microencapsulated in alginate to develop an integrated bioprocess for expansion and cryopreservation of pluripotent hESCs. Different three-dimensional (3D) culture strategies were evaluated and compared, specifically, microencapsulation of hESCs as: i) single cells, ii) aggregates and iii) immobilized on microcarriers. In order to establish a scalable bioprocess, hESC-microcapsules were cultured in stirred tank bioreactors. The combination of microencapsulation and microcarrier technology resulted in a highly efficient protocol for the production and storage of pluripotent hESCs. This strategy ensured high expansion ratios (an approximately twenty-fold increase in cell concentration) and high cell recovery yields (>70%) after cryopreservation. When compared with non-encapsulated cells, cell survival post-thawing demonstrated a three-fold improvement without compromising hESC characteristics. Microencapsulation also improved the culture of hESC aggregates by protecting cells from hydrodynamic shear stress, controlling aggregate size and maintaining cell pluripotency for two weeks. This work establishes that microencapsulation technology may prove a powerful tool for integrating the expansion and cryopreservation of pluripotent hESCs. The 3D culture strategy developed herein represents a significant breakthrough towards the implementation of hESCs in clinical and industrial applications. PMID:21850261

Variations in Glycogen Synthesis in Human Pluripotent Stem Cells with Altered Pluripotent States

PubMed Central

Chen, Richard J.; Zhang, Guofeng; Garfield, Susan H.; Shi, Yi-Jun; Chen, Kevin G.; Robey, Pamela G.; Leapman, Richard D.

2015-01-01

Human pluripotent stem cells (hPSCs) represent very promising resources for cell-based regenerative medicine. It is essential to determine the biological implications of some fundamental physiological processes (such as glycogen metabolism) in these stem cells. In this report, we employ electron, immunofluorescence microscopy, and biochemical methods to study glycogen synthesis in hPSCs. Our results indicate that there is a high level of glycogen synthesis (0.28 to 0.62 μg/μg proteins) in undifferentiated human embryonic stem cells (hESCs) compared with the glycogen levels (0 to 0.25 μg/μg proteins) reported in human cancer cell lines. Moreover, we found that glycogen synthesis was regulated by bone morphogenetic protein 4 (BMP-4) and the glycogen synthase kinase 3 (GSK-3) pathway. Our observation of glycogen bodies and sustained expression of the pluripotent factor Oct-4 mediated by the potent GSK-3 inhibitor CHIR-99021 reveals an altered pluripotent state in hPSC culture. We further confirmed glycogen variations under different naïve pluripotent cell growth conditions based on the addition of the GSK-3 inhibitor BIO. Our data suggest that primed hPSCs treated with naïve growth conditions acquire altered pluripotent states, similar to those naïve-like hPSCs, with increased glycogen synthesis. Furthermore, we found that suppression of phosphorylated glycogen synthase was an underlying mechanism responsible for altered glycogen synthesis. Thus, our novel findings regarding the dynamic changes in glycogen metabolism provide new markers to assess the energetic and various pluripotent states in hPSCs. The components of glycogen metabolic pathways offer new assays to delineate previously unrecognized properties of hPSCs under different growth conditions. PMID:26565809

Generation of iPSC line iPSC-FH2.1 in hypoxic conditions from human foreskin fibroblasts.

PubMed

Questa, María; Romorini, Leonardo; Blüguermann, Carolina; Solari, Claudia María; Neiman, Gabriel; Luzzani, Carlos; Scassa, María Élida; Sevlever, Gustavo Emilio; Guberman, Alejandra Sonia; Miriuka, Santiago Gabriel

2016-03-01

Human foreskin fibroblasts were used to generate the iPSC line iPSC-FH2.1 using the EF1a-hSTEMCCA-loxP vector expressing OCT4, SOX2, c-MYC and KLF4, in 5% O2 culture conditions. Stemness was confirmed, as was pluripotency both in vivo and in vitro, in normoxia and hypoxia. Human Embryonic Stem Cell (hESC) line WA-09 and reprogrammed fibroblast primary culture HFF-FM were used as controls. Copyright © 2015 University of Texas at Austin Dell Medical School. Published by Elsevier B.V. All rights reserved.

Nutrient supplemented serum-free medium increases cardiomyogenesis efficiency of human pluripotent stem cells.

PubMed

Ting, Sherwin; Lecina, Marti; Chan, Yau-Chi; Tse, Hung Fat; Reuveny, Shaul; Oh, Steve Kw

2013-07-26

To development of an improved p38 MAPK inhibitor-based serum-free medium for embryoid body cardiomyocyte differentiation of human pluripotent stem cells. Human embryonic stem cells (hESC) differentiated to cardiomyocytes (CM) using a p38 MAPK inhibitor (SB203580) based serum-free medium (SB media). Nutrient supplements known to increase cell viability were added to SB medium. The ability of these supplements to improve cardiomyogenesis was evaluated by measurements of cell viability, total cell count, and the expression of cardiac markers via flow cytometry. An improved medium containing Soy hydrolysate (HySoy) and bovine serum albumin (BSA) (SupSB media) was developed and tested on 2 additional cell lines (H1 and Siu-hiPSC). Characterization of the cardiomyocytes was done by immunohistochemistry, electrophysiology and quantitative real-time reverse transcription-polymerase chain reaction. hESC cell line, HES-3, differentiating in SB medium for 16 d resulted in a cardiomyocyte yield of 0.07 ± 0.03 CM/hESC. A new medium (SupSB media) was developed with the addition of HySoy and BSA to SB medium. This medium resulted in 2.6 fold increase in cardiomyocyte yield (0.21 ± 0.08 CM/hESC). The robustness of SupSB medium was further demonstrated using two additional pluripotent cell lines (H1, hESC and Siu1, hiPSC), showing a 15 and 9 fold increase in cardiomyocyte yield respectively. The age (passage number) of the pluripotent cells did not affect the cardiomyocyte yields. Embryoid body (EB) cardiomyocytes formed in SupSB medium expressed canonical cardiac markers (sarcomeric α-actinin, myosin heavy chain and troponin-T) and demonstrated all three major phenotypes: nodal-, atrial- and ventricular-like. Electrophysiological characteristics (maximum diastolic potentials and action potential durations) of cardiomyocytes derived from SB and SupSB media were similar. The nutrient supplementation (HySoy and BSA) leads to increase in cell viability, cell yield and cardiac marker expression during cardiomyocyte differentiation, translating to an overall increase in cardiomyocyte yield.

Human pluripotent stem cells recurrently acquire and expand dominant negative P53 mutations

PubMed Central

Kamitaki, Nolan; Mitchell, Jana; Avior, Yishai; Mello, Curtis; Kashin, Seva; Mekhoubad, Shila; Ilic, Dusko; Charlton, Maura; Saphier, Genevieve; Handsaker, Robert E.; Genovese, Giulio; Bar, Shiran; Benvenisty, Nissim; McCarroll, Steven A.; Eggan, Kevin

2017-01-01

Human pluripotent stem cells (hPSCs) can self-renew indefinitely, making them an attractive source for regenerative therapies. This expansion potential has been linked with acquisition of large copy number variants (CNVs) that provide mutant cells with a growth advantage in culture1–3. However, the nature, extent, and functional impact of other acquired genome sequence mutations in cultured hPSCs is not known. Here, we sequenced the protein-coding genes (exomes) of 140 independent human embryonic stem cell (hESC) lines, including 26 lines prepared for potential clinical use4. We then applied computational strategies for identifying mutations present in a subset of cells5. Though such mosaic mutations were generally rare, we identified five unrelated hESC lines that carried six mutations in the TP53 gene that encodes the tumor suppressor P53. Notably, the TP53 mutations we observed are dominant negative and are the mutations most commonly seen in human cancers. We used droplet digital PCR to demonstrate that the TP53 mutant allelic fraction increased with passage number under standard culture conditions, suggesting that P53 mutation confers selective advantage. When we then mined published RNA sequencing data from 117 hPSC lines, we observed another nine TP53 mutations, all resulting in coding changes in the DNA binding domain of P53. Strikingly, in three lines, the allelic fraction exceeded 50%, suggesting additional selective advantage resulting from loss of heterozygosity at the TP53 locus. As the acquisition and favored expansion of cancer-associated mutations in hPSCs may go unnoticed during most applications, we suggest that careful genetic characterization of hPSCs and their differentiated derivatives should be carried out prior to clinical use. PMID:28445466

Alcohol-Induced Molecular Dysregulation in Human Embryonic Stem Cell-Derived Neural Precursor Cells

PubMed Central

Kim, Yi Young; Roubal, Ivan; Lee, Youn Soo; Kim, Jin Seok; Hoang, Michael; Mathiyakom, Nathan; Kim, Yong

2016-01-01

Adverse effect of alcohol on neural function has been well documented. Especially, the teratogenic effect of alcohol on neurodevelopment during embryogenesis has been demonstrated in various models, which could be a pathologic basis for fetal alcohol spectrum disorders (FASDs). While the developmental defects from alcohol abuse during gestation have been described, the specific mechanisms by which alcohol mediates these injuries have yet to be determined. Recent studies have shown that alcohol has significant effect on molecular and cellular regulatory mechanisms in embryonic stem cell (ESC) differentiation including genes involved in neural development. To test our hypothesis that alcohol induces molecular alterations during neural differentiation we have derived neural precursor cells from pluripotent human ESCs in the presence or absence of ethanol treatment. Genome-wide transcriptomic profiling identified molecular alterations induced by ethanol exposure during neural differentiation of hESCs into neural rosettes and neural precursor cell populations. The Database for Annotation, Visualization and Integrated Discovery (DAVID) functional analysis on significantly altered genes showed potential ethanol’s effect on JAK-STAT signaling pathway, neuroactive ligand-receptor interaction, Toll-like receptor (TLR) signaling pathway, cytokine-cytokine receptor interaction and regulation of autophagy. We have further quantitatively verified ethanol-induced alterations of selected candidate genes. Among verified genes we further examined the expression of P2RX3, which is associated with nociception, a peripheral pain response. We found ethanol significantly reduced the level of P2RX3 in undifferentiated hESCs, but induced the level of P2RX3 mRNA and protein in hESC-derived NPCs. Our result suggests ethanol-induced dysregulation of P2RX3 along with alterations in molecules involved in neural activity such as neuroactive ligand-receptor interaction may be a molecular event associated with alcohol-related peripheral neuropathy of an enhanced nociceptive response. PMID:27682028

Energy Metabolism in Human Pluripotent Stem Cells and Their Differentiated Counterparts

PubMed Central

Moura, Michelle B.; Momcilovic, Olga; Easley, Charles A.; Ramalho-Santos, João; Van Houten, Bennett; Schatten, Gerald

2011-01-01

Background Human pluripotent stem cells have the ability to generate all cell types present in the adult organism, therefore harboring great potential for the in vitro study of differentiation and for the development of cell-based therapies. Nonetheless their use may prove challenging as incomplete differentiation of these cells might lead to tumoregenicity. Interestingly, many cancer types have been reported to display metabolic modifications with features that might be similar to stem cells. Understanding the metabolic properties of human pluripotent stem cells when compared to their differentiated counterparts can thus be of crucial importance. Furthermore recent data has stressed distinct features of different human pluripotent cells lines, namely when comparing embryo-derived human embryonic stem cells (hESCs) and induced pluripotent stem cells (IPSCs) reprogrammed from somatic cells. Methodology/Principal Findings We compared the energy metabolism of hESCs, IPSCs, and their somatic counterparts. Focusing on mitochondria, we tracked organelle localization and morphology. Furthermore we performed gene expression analysis of several pathways related to the glucose metabolism, including glycolysis, the pentose phosphate pathway and the tricarboxylic acid (TCA) cycle. In addition we determined oxygen consumption rates (OCR) using a metabolic extracellular flux analyzer, as well as total intracellular ATP levels by high performance liquid chromatography (HPLC). Finally we explored the expression of key proteins involved in the regulation of glucose metabolism. Conclusions/Findings Our results demonstrate that, although the metabolic signature of IPSCs is not identical to that of hESCs, nonetheless they cluster with hESCs rather than with their somatic counterparts. ATP levels, lactate production and OCR revealed that human pluripotent cells rely mostly on glycolysis to meet their energy demands. Furthermore, our work points to some of the strategies which human pluripotent stem cells may use to maintain high glycolytic rates, such as high levels of hexokinase II and inactive pyruvate dehydrogenase (PDH). PMID:21698063

Novel developmental biology-based protocol of embryonic stem cell differentiation to morphologically sound and functional yet immature hepatocytes.

PubMed

Bukong, Terence N; Lo, Tracie; Szabo, Gyongyi; Dolganiuc, Angela

2012-05-01

Liver diseases are common in the United States and often require liver transplantation; however, donated organs are limited and thus alternative sources for liver cells are in high demand. Embryonic stem cells (ESC) can provide a continuous and readily available source of liver cells. ESC differentiation to liver cells is yet to be fully understood and comprehensive differentiation protocols are yet to be defined. Here, we aimed to achieve human (h)ESC differentiation into mature hepatocytes using defined recombinant differentiation factors and metabolites. Embryonic stem cell H1 line was sub-cultured on feeder layer. We induced hESCs into endodermal differentiation succeeded by early/late hepatic specification and finally into hepatocyte maturation using step combinations of Activin A and fibroblast growth factor (FGF)-2 for 7 days; followed by FGF-4 and bone morphogenic protein 2 (BMP2) for 7 days, succeeded by FGF-10 + hepatocyte growth factor 4 + epidermal growth factor for 14 days. Specific inhibitors/stimulators were added sequentially throughout differentiation. Cells were analysed by PCR, flow cytometry, microscopy or functional assays. Our hESC differentiation protocol resulted in viable cells with hepatocyte shape and morphology. We observed gradual changes in cell transcriptome, including up-regulation of differentiation-promoting GATA4, GATA6, POU5F1 and HNF4 transcription factors, steady levels of stemness-promoting SOX-2 and low levels of Nanog, as defined by PCR. The hESC-derived hepatocytes expressed alpha-antitrypsin, CD81, cytokeratin 8 and low density lipoprotein (LDL) receptor. The levels of alpha-fetoprotein and proliferation marker Ki-67 in hESC-derived hepatocytes remained elevated. Unlike stem cells, the hESC-derived hepatocytes performed LDL uptake, produced albumin and alanine aminotransferase and had functional alcohol dehydrogenase. We report a novel protocol for hESC differentiation into morphological and functional yet immature hepatocytes as an alternative method for hepatocyte generation. © 2012 John Wiley & Sons A/S.

Safety paradigm: genetic evaluation of therapeutic grade human embryonic stem cells.

PubMed

Stephenson, Emma; Ogilvie, Caroline Mackie; Patel, Heema; Cornwell, Glenda; Jacquet, Laureen; Kadeva, Neli; Braude, Peter; Ilic, Dusko

2010-12-06

The use of stem cells for regenerative medicine has captured the imagination of the public, with media attention contributing to rising expectations of clinical benefits. Human embryonic stem cells (hESCs) are the best model for capital investment in stem cell therapy and there is a clear need for their robust genetic characterization before scaling-up cell expansion for that purpose. We have to be certain that the genome of the starting material is stable and normal, but the limited resolution of conventional karyotyping is unable to give us such assurance. Advanced molecular cytogenetic technologies such as array comparative genomic hybridization for identifying chromosomal imbalances, and single nucleotide polymorphism analysis for identifying ethnic background and loss of heterozygosity should be introduced as obligatory diagnostic tests for each newly derived hESC line before it is deposited in national stem cell banks. If this new quality standard becomes a requirement, as we are proposing here, it would facilitate and accelerate the banking process, since end-users would be able to select the most appropriate line for their particular application, thus improving efficiency and streamlining the route to manufacturing therapeutics. The pharmaceutical industry, which may use hESC-derived cells for drug screening, should not ignore their genomic profile as this may risk misinterpretation of results and significant waste of resources.

An intermittent rocking platform for integrated expansion and differentiation of human pluripotent stem cells to cardiomyocytes in suspended microcarrier cultures.

PubMed

Ting, Sherwin; Chen, Allen; Reuveny, Shaul; Oh, Steve

2014-09-01

The development of novel platforms for large scale production of human embryonic stem cells (hESC) derived cardiomyocytes (CM) becomes more crucial as the demand for CMs in preclinical trials, high throughput cardio toxicity assays and future regenerative therapeutics rises. To this end, we have designed a microcarrier (MC) suspension agitated platform that integrates pluripotent hESC expansion followed by CM differentiation in a continuous, homogenous process. Hydrodynamic shear stresses applied during the hESC expansion and CM differentiation steps drastically reduced the capability of the cells to differentiate into CMs. Applying vigorous stirring during pluripotent hESC expansion on Cytodex 1 MC in spinner cultures resulted in low CM yields in the following differentiation step (cardiac troponin-T (cTnT): 22.83±2.56%; myosin heavy chain (MHC): 19.30±5.31%). Whereas the lower shear experienced in side to side rocker (wave type) platform resulted in higher CM yields (cTNT: 47.50±7.35%; MHC: 42.85±2.64%). The efficiency of CM differentiation is also affected by the hydrodynamic shear stress applied during the first 3days of the differentiation stage. Even low shear applied continuously by side to side rocker agitation resulted in very low CM differentiation efficiency (cTnT<5%; MHC<2%). Simply by applying intermittent agitation during these 3days followed by continuous agitation for the subsequent 9days, CM differentiation efficiency can be substantially increased (cTNT: 65.73±10.73%; MHC: 59.73±9.17%). These yields are 38.3% and 39.3% higher (for cTnT and MHC respectively) than static culture control. During the hESC expansion phase, cells grew on continuously agitated rocker platform as pluripotent cell/MC aggregates (166±88×10(5)μm(2)) achieving a cell concentration of 3.74±0.55×10(6)cells/mL (18.89±2.82 fold expansion) in 7days. These aggregates were further differentiated into CMs using a WNT modulation differentiation protocol for the subsequent 12days on a rocking platform with an intermittent agitation regime during the first 3days. Collectively, the integrated MC rocker platform produced 190.5±58.8×10(6) CMs per run (31.75±9.74 CM/hESC seeded). The robustness of the system was demonstrated by using 2 cells lines, hESC (HES-3) and human induced pluripotent stem cell (hiPSC) IMR-90. The CM/MC aggregates formed extensive sarcomeres that exhibited cross-striations confirming cardiac ontogeny. Functionality of the CMs was demonstrated by monitoring the effect of inotropic drug, Isoproterenol on beating frequency. In conclusion, we have developed a simple robust and scalable platform that integrates both hESC expansion and CM differentiation in one unit process which is capable of meeting the need for large amounts of CMs. Copyright © 2014. Published by Elsevier B.V.

Neural Differentiation of Human Pluripotent Stem Cells for Nontherapeutic Applications: Toxicology, Pharmacology, and In Vitro Disease Modeling.

PubMed

Yap, May Shin; Nathan, Kavitha R; Yeo, Yin; Lim, Lee Wei; Poh, Chit Laa; Richards, Mark; Lim, Wei Ling; Othman, Iekhsan; Heng, Boon Chin

2015-01-01

Human pluripotent stem cells (hPSCs) derived from either blastocyst stage embryos (hESCs) or reprogrammed somatic cells (iPSCs) can provide an abundant source of human neuronal lineages that were previously sourced from human cadavers, abortuses, and discarded surgical waste. In addition to the well-known potential therapeutic application of these cells in regenerative medicine, these are also various promising nontherapeutic applications in toxicological and pharmacological screening of neuroactive compounds, as well as for in vitro modeling of neurodegenerative and neurodevelopmental disorders. Compared to alternative research models based on laboratory animals and immortalized cancer-derived human neural cell lines, neuronal cells differentiated from hPSCs possess the advantages of species specificity together with genetic and physiological normality, which could more closely recapitulate in vivo conditions within the human central nervous system. This review critically examines the various potential nontherapeutic applications of hPSC-derived neuronal lineages and gives a brief overview of differentiation protocols utilized to generate these cells from hESCs and iPSCs.

Donor Dependent Variations in Hematopoietic Differentiation among Embryonic and Induced Pluripotent Stem Cell Lines

PubMed Central

Féraud, Olivier; Valogne, Yannick; Melkus, Michael W.; Zhang, Yanyan; Oudrhiri, Noufissa; Haddad, Rima; Daury, Aurélie; Rocher, Corinne; Larbi, Aniya; Duquesnoy, Philippe; Divers, Dominique; Gobbo, Emilie; Brunet de la Grange, Philippe; Louache, Fawzia; Bennaceur-Griscelli, Annelise; Mitjavila-Garcia, Maria Teresa

2016-01-01

Hematopoiesis generated from human embryonic stem cells (ES) and induced pluripotent stem cells (iPS) are unprecedented resources for cell therapy. We compared hematopoietic differentiation potentials from ES and iPS cell lines originated from various donors and derived them using integrative and non-integrative vectors. Significant differences in differentiation toward hematopoietic lineage were observed among ES and iPS. The ability of engraftment of iPS or ES-derived cells in NOG mice varied among the lines with low levels of chimerism. iPS generated from ES cell-derived mesenchymal stem cells (MSC) reproduce a similar hematopoietic outcome compared to their parental ES cell line. We were not able to identify any specific hematopoietic transcription factors that allow to distinguish between good versus poor hematopoiesis in undifferentiated ES or iPS cell lines. There is a relatively unpredictable variation in hematopoietic differentiation between ES and iPS cell lines that could not be predicted based on phenotype or gene expression of the undifferentiated cells. These results demonstrate the influence of genetic background in variation of hematopoietic potential rather than the reprogramming process. PMID:26938212

Ethanol Inactivated Mouse Embryonic Fibroblasts Maintain the Self-Renew and Proliferation of Human Embryonic Stem Cells.

PubMed

Huang, Boxian; Ning, Song; Zhuang, Lili; Jiang, Chunyan; Cui, Yugui; Fan, Guoping; Qin, Lianju; Liu, Jiayin

2015-01-01

Conventionally, mouse embryonic fibroblasts (MEFs) inactivated by mitomycin C or irradiation were applied to support the self-renew and proliferation of human embryonic stem cells (hESCs). To avoid the disadvangtages of mitomycin C and irradiation, here MEFs were treated by ethanol (ET). Our data showed that 10% ET-inactivated MEFs (eiMEFs) could well maintain the self-renew and proliferation of hESCs. hESCs grown on eiMEFs expressed stem cell markers of NANOG, octamer-binding protein 4 (OCT4), stage-specific embryonic antigen-4 (SSEA4) and tumour related antigen-1-81 (TRA-1-81), meanwhile maintained normal karyotype after long time culture. Also, hESCs cocultured with eiMEFs were able to form embryoid body (EB) in vitro and develop teratoma in vivo. Moreover, eiMEFs could keep their nutrient functions after long time cryopreservation. Our results indicate that the application of eiMEF in hESCs culture is safe, economical and convenient, thus is a better choice.

SCL/TAL1-mediated transcriptional network enhances megakaryocytic specification of human embryonic stem cells.

PubMed

Toscano, Miguel G; Navarro-Montero, Oscar; Ayllon, Veronica; Ramos-Mejia, Veronica; Guerrero-Carreno, Xiomara; Bueno, Clara; Romero, Tamara; Lamolda, Mar; Cobo, Marien; Martin, Francisco; Menendez, Pablo; Real, Pedro J

2015-01-01

Human embryonic stem cells (hESCs) are a unique in vitro model for studying human developmental biology and represent a potential source for cell replacement strategies. Platelets can be generated from cord blood progenitors and hESCs; however, the molecular mechanisms and determinants controlling the in vitro megakaryocytic specification of hESCs remain elusive. We have recently shown that stem cell leukemia (SCL) overexpression accelerates the emergence of hemato-endothelial progenitors from hESCs and promotes their subsequent differentiation into blood cells with higher clonogenic potential. Given that SCL participates in megakaryocytic commitment, we hypothesized that it may potentiate megakaryopoiesis from hESCs. We show that ectopic SCL expression enhances the emergence of megakaryocytic precursors, mature megakaryocytes (MKs), and platelets in vitro. SCL-overexpressing MKs and platelets respond to different activating stimuli similar to their control counterparts. Gene expression profiling of megakaryocytic precursors shows that SCL overexpression renders a megakaryopoietic molecular signature. Connectivity Map analysis reveals that trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA), both histone deacetylase (HDAC) inhibitors, functionally mimic SCL-induced effects. Finally, we confirm that both TSA and SAHA treatment promote the emergence of CD34(+) progenitors, whereas valproic acid, another HDAC inhibitor, potentiates MK and platelet production. We demonstrate that SCL and HDAC inhibitors are megakaryopoiesis regulators in hESCs.

Human embryonic stem cell science and policy: The case of Iran☆

PubMed Central

Saniei, Mansooreh

2013-01-01

The paper is based on a large qualitative study of ethics, policy and regulation of human embryonic stem cell (hESC) science in Iran. This case study in five academic research centres used semi-structured interviews to examine in depth the views of stem cell scientists, embryologists and ethics committee members on hESC research policy in this Shia Muslim country. Although Iran's policy approach has been considered 'intermediate', what is described here seems to be a 'more flexible' policy on hESC science. This article describes three arguments to explain why Iran has shaped such a policy. These are: (1) a flexibility of the Shia tradition has allowed for hESC science; (2) permissive policy related to other fields of biomedicine, such as new assisted reproductive technologies, facilitated approval of hESC research; and (3) a lack of public debate of bioscience in Iran influences how its hESC research policy is perceived. Based on the empirical data, this paper then expands and refines the conceptual bioethical basis for the co-production of science, policy, and society in Iran. The notion of co-production implies that scientists, policy-makers, and sometimes other societal actors cooperate in the exchange, production, and application of knowledge to make science policy. PMID:24230960

Human embryonic stem cell science and policy: the case of Iran.

PubMed

Saniei, Mansooreh

2013-12-01

The paper is based on a large qualitative study of ethics, policy and regulation of human embryonic stem cell (hESC) science in Iran. This case study in five academic research centres used semi-structured interviews to examine in depth the views of stem cell scientists, embryologists and ethics committee members on hESC research policy in this Shia Muslim country. Although Iran's policy approach has been considered 'intermediate', what is described here seems to be a 'more flexible' policy on hESC science. This article describes three arguments to explain why Iran has shaped such a policy. These are: (1) a flexibility of the Shia tradition has allowed for hESC science; (2) permissive policy related to other fields of biomedicine, such as new assisted reproductive technologies, facilitated approval of hESC research; and (3) a lack of public debate of bioscience in Iran influences how its hESC research policy is perceived. Based on the empirical data, this paper then expands and refines the conceptual bioethical basis for the co-production of science, policy, and society in Iran. The notion of co-production implies that scientists, policy-makers, and sometimes other societal actors cooperate in the exchange, production, and application of knowledge to make science policy. Copyright © 2013 Elsevier Ltd. All rights reserved.

A Human Pluripotent Stem Cell Model of Facioscapulohumeral Muscular Dystrophy-Affected Skeletal Muscles.

PubMed

Caron, Leslie; Kher, Devaki; Lee, Kian Leong; McKernan, Robert; Dumevska, Biljana; Hidalgo, Alejandro; Li, Jia; Yang, Henry; Main, Heather; Ferri, Giulia; Petek, Lisa M; Poellinger, Lorenz; Miller, Daniel G; Gabellini, Davide; Schmidt, Uli

2016-09-01

: Facioscapulohumeral muscular dystrophy (FSHD) represents a major unmet clinical need arising from the progressive weakness and atrophy of skeletal muscles. The dearth of adequate experimental models has severely hampered our understanding of the disease. To date, no treatment is available for FSHD. Human embryonic stem cells (hESCs) potentially represent a renewable source of skeletal muscle cells (SkMCs) and provide an alternative to invasive patient biopsies. We developed a scalable monolayer system to differentiate hESCs into mature SkMCs within 26 days, without cell sorting or genetic manipulation. Here we show that SkMCs derived from FSHD1-affected hESC lines exclusively express the FSHD pathogenic marker double homeobox 4 and exhibit some of the defects reported in FSHD. FSHD1 myotubes are thinner when compared with unaffected and Becker muscular dystrophy myotubes, and differentially regulate genes involved in cell cycle control, oxidative stress response, and cell adhesion. This cellular model will be a powerful tool for studying FSHD and will ultimately assist in the development of effective treatments for muscular dystrophies. This work describes an efficient and highly scalable monolayer system to differentiate human pluripotent stem cells (hPSCs) into skeletal muscle cells (SkMCs) and demonstrates disease-specific phenotypes in SkMCs derived from both embryonic and induced hPSCs affected with facioscapulohumeral muscular dystrophy. This study represents the first human stem cell-based cellular model for a muscular dystrophy that is suitable for high-throughput screening and drug development. ©AlphaMed Press.

High-Dose Fluoride Impairs the Properties of Human Embryonic Stem Cells via JNK Signaling.

PubMed

Fu, Xin; Xie, Fang-Nan; Dong, Ping; Li, Qiu-Chen; Yu, Guang-Yan; Xiao, Ran

2016-01-01

Fluoride is a ubiquitous natural substance that is often used in dental products to prevent dental caries. The biphasic actions of fluoride imply that excessive systemic exposure to fluoride can cause harmful effects on embryonic development in both animal models and humans. However, insufficient information is available on the effects of fluoride on human embryonic stem cells (hESCs), which is a novel in vitro humanized model for analyzing the embryotoxicities of chemical compounds. Therefore, we investigated the effects of sodium fluoride (NaF) on the proliferation, differentiation and viability of H9 hESCs. For the first time, we showed that 1 mM NaF did not significantly affect the proliferation of hESCs but did disturb the gene expression patterns of hESCs during embryoid body (EB) differentiation. Higher doses of NaF (2 mM and above) markedly decreased the viability and proliferation of hESCs. The mode and underlying mechanism of high-dose NaF-induced cell death were further investigated by assessing the sub-cellular morphology, mitochondrial membrane potential (MMP), caspase activities, cellular reactive oxygen species (ROS) levels and activation of mitogen-activated protein kinases (MAPKs). High-dose NaF caused the death of hESCs via apoptosis in a caspase-mediated but ROS-independent pathway, coupled with an increase in the phospho-c-Jun N-terminal kinase (p-JNK) levels. Pretreatment with a p-JNK-specific inhibitor (SP600125) could effectively protect hESCs from NaF-induced cell death in a concentration- and time-dependent manner. These findings suggest that NaF might interfere with early human embryogenesis by disturbing the specification of the three germ layers as well as osteogenic lineage commitment and that high-dose NaF could cause apoptosis through a JNK-dependent pathway in hESCs.

Acute erythroblastic leukemia presenting as acute undifferentiated leukemia: a report of two cases with ultrastructural features.

PubMed

Reiffers, J; Bernard, P; Larrue, J; Dachary, D; David, B; Boisseau, M; Broustet, A

1985-01-01

This report describes two elderly patients with acute leukemia in which blast cells were undifferentiated with conventional light microscopy (L.M.) and cytochemistry. Blast cells were identified as belonging to the erythroblastic line by their ultrastructural features: glycogen deposits, lipidic vacuoles, cytoplasmic ferritin molecules and rhopheocytotic invagination. Moreover, blast cells were surrounding a central macrophage. Thus, these two patients had acute erythroblastic leukemia which differs from erythroleukemia (M6 of FAB classification) in which blast cells present myeloblastic characteristics.

Developing de novo human artificial chromosomes in embryonic stem cells using HSV-1 amplicon technology.

PubMed

Moralli, Daniela; Monaco, Zoia L

2015-02-01

De novo artificial chromosomes expressing genes have been generated in human embryonic stem cells (hESc) and are maintained following differentiation into other cell types. Human artificial chromosomes (HAC) are small, functional, extrachromosomal elements, which behave as normal chromosomes in human cells. De novo HAC are generated following delivery of alpha satellite DNA into target cells. HAC are characterized by high levels of mitotic stability and are used as models to study centromere formation and chromosome organisation. They are successful and effective as gene expression vectors since they remain autonomous and can accommodate larger genes and regulatory regions for long-term expression studies in cells unlike other viral gene delivery vectors currently used. Transferring the essential DNA sequences for HAC formation intact across the cell membrane has been challenging for a number of years. A highly efficient delivery system based on HSV-1 amplicons has been used to target DNA directly to the ES cell nucleus and HAC stably generated in human embryonic stem cells (hESc) at high frequency. HAC were detected using an improved protocol for hESc chromosome harvesting, which consistently produced high-quality metaphase spreads that could routinely detect HAC in hESc. In tumour cells, the input DNA often integrated in the host chromosomes, but in the host ES genome, it remained intact. The hESc containing the HAC formed embryoid bodies, generated teratoma in mice, and differentiated into neuronal cells where the HAC were maintained. The HAC structure and chromatin composition was similar to the endogenous hESc chromosomes. This review will discuss the technological advances in HAC vector delivery using HSV-1 amplicons and the improvements in the identification of de novo HAC in hESc.

Differential effects of the extracellular microenvironment on human embryonic stem cell differentiation into keratinocytes and their subsequent replicative life span.

PubMed

Movahednia, Mohammad Mehdi; Kidwai, Fahad Karim; Zou, Yu; Tong, Huei Jinn; Liu, Xiaochen; Islam, Intekhab; Toh, Wei Seong; Raghunath, Michael; Cao, Tong

2015-04-01

Culture microenvironment plays a critical role in the propagation and differentiation of human embryonic stem cells (hESCs) and their differentiated progenies. Although high efficiency of hESC differentiation to keratinocytes (hESC-Kert) has been achieved, little is known regarding the effects of early culture microenvironment and pertinent extracellular matrix (ECM) interactions during epidermal commitment on subsequent proliferative capacity of hESC-Kert. The aim of this study is to evaluate the effects of the different ECM microenvironments during hESC differentiation on subsequent replicative life span of hESC-Kert. In doing so, H1-hESCs were differentiated to keratinocytes (H1-Kert) in two differentiation systems. The first system employed autologous fibroblast feeder support, in which keratinocytes (H1-Kert(ACC)) were derived by coculture of hESCs with hESC-derived fibroblasts (H1-ebFs). The second system employed a novel decellularized matrix from H1-ebFs to create a dermoepidermal junction-like (DEJ) matrix. H1-Kert(AFF) were derived by differentiation of hESCs on the feeder-free system employing the DEJ matrix. Our study indicated that the feeder-free system with the use of DEJ matrix was more efficient in differentiation of hESCs toward epidermal progenitors. However, the feeder-free system was not sufficient to support the subsequent replicative capacity of differentiated keratinocytes. Of note, H1-Kert(AFF) showed limited replicative capacity with reduced telomere length and early cellular senescence. We further showed that the lack of cell-cell interactions during epidermal commitment led to heightened production of TGF-β1 by hESC-Kert during extended culture, which in turn was responsible for resulting in the limited replicative life span with cellular senescence of hESC-Kert derived under the feeder-free culture system. This study highlights for the first time the importance of the culture microenvironment and cell-ECM interactions during differentiation of hESCs on subsequent replicative life span and cellular senescence of the differentiated keratinocytes, with implications for use of these cells for applications in tissue engineering and regenerative medicine.

Effect of a feeder layer composed of mouse embryonic and human foreskin fibroblasts on the proliferation of human embryonic stem cells.

PubMed

Yang, Hua; Qiu, Ying; Zeng, Xianghui; Ding, Yan; Zeng, Jianye; Lu, Kehuan; Li, Dongsheng

2016-06-01

The aim of the present study was to investigate the effects of feeder layers composed of various ratios of mouse embryonic fibroblasts (MEFs) and human foreskin fibroblasts (hFFs) on the growth of human embryonic stem cells (hESCs). In addition, the secretion levels of basic fibroblast growth factor (bFGF) by the feeder layers was detected. MEFs and hFFs were treated with mitomycin C and seeded onto gelatin-coated plates at a density of 1×10 8 cells/l. The hFFs and MEFs were combined and plated at the following ratios: 0:1, 1:2, 1:1, 2:1 and 1:0. The secretion of bFGF by the various hFF/MEF ratio feeder layers was detected using an enzyme-linked immunosorbent assay. Subsequently, hESCs were cultured on top of the various feeder layers. The differences in the cellular morphology of the hESCs were observed using microscopy, and the expression levels alkaline phosphatase (AKP) and octamer-binding transcription factor 4 (OCT-4) were detected using immunohistochemical analysis as indicators of differentiation status. The results showed that the hFFs secreted substantial quantities of bFGF, while no bFGF was secreted by the MEFs. The clones of hESC growing on the feeder layer containing MEF or hFF alone were flat. By contrast, hESC clones grown on a mixed feeder layer containing hFFs + MEFs at a ratio of 1:1 exhibited an accumulated growth with a clear edge, as compared with the other ratios. In addition, hESCs growing on the feeder layer were positive for the expression of AKP and OCT-4. In summary, feeder layer hFFs secreted bFGF, while MEFs did not, indicating that bFGF is not the only factor that supports the growth and differentiation of hESCs. The optimal growth of hESCs was achieved using a mixed feeder layer composed of hFFs + MEFs at a ratio of 1:1.

Endogenous APOBEC3B restricts LINE-1 retrotransposition in transformed cells and human embryonic stem cells.

PubMed

Wissing, Silke; Montano, Mauricio; Garcia-Perez, Jose Luis; Moran, John V; Greene, Warner C

2011-10-21

Members of the APOBEC3 (A3) family of cytidine deaminase enzymes act as host defense mechanisms limiting both infections by exogenous retroviruses and mobilization of endogenous retrotransposons. Previous studies revealed that the overexpression of some A3 proteins could restrict engineered human Long INterspersed Element-1 (LINE-1 or L1) retrotransposition in HeLa cells. However, whether endogenous A3 proteins play a role in restricting L1 retrotransposition remains largely unexplored. Here, we show that HeLa cells express endogenous A3B and A3C, whereas human embryonic stem cells (hESCs) express A3B, A3C, A3DE, A3F, and A3G. To study the relative contribution of endogenous A3 proteins in restricting L1 retrotransposition, we first generated small hairpin RNAs (shRNAs) to suppress endogenous A3 mRNA expression, and then assessed L1 mobility using a cell-based L1 retrotransposition assay. We demonstrate that in both HeLa and hESCs, shRNA-based knockdown of A3B promotes a ∼2-3.7-fold increase in the retrotransposition efficiency of an engineered human L1. Knockdown of the other A3s produced no significant increase in L1 activity. Thus, A3B appears to restrict engineered L1 retrotransposition in a broad range of cell types, including pluripotent cells.

The Abbreviated Pluripotent Cell Cycle

PubMed Central

Kapinas, Kristina; Grandy, Rodrigo; Ghule, Prachi; Medina, Ricardo; Becker, Klaus; Pardee, Arthur; Zaidi, Sayyed K.; Lian, Jane; Stein, Janet; van Wijnen, Andre; Stein, Gary

2013-01-01

Human embryonic stem cells and induced pluripotent stem cells proliferate rapidly and divide symmetrically producing equivalent progeny cells. In contrast, lineage committed cells acquire an extended symmetrical cell cycle. Self-renewal of tissue-specific stem cells is sustained by asymmetric cell division where one progeny cell remains a progenitor while the partner progeny cell exits the cell cycle and differentiates. There are three principal contexts for considering the operation and regulation of the pluripotent cell cycle: temporal, regulatory andstructural. The primary temporal context that the pluripotent self-renewal cell cycle of human embryonic stem cells (hESCs) is a short G1 period without reducing periods of time allocated to S phase, G2, and mitosis. The rules that govern proliferation in hESCs remain to be comprehensively established. However, several lines of evidence suggest a key role for the naïve transcriptome of hESCs, which is competent to stringently regulate the ESC cell cycle. This supports the requirements of pluripotent cells to self propagate while suppressing expression of genes that confer lineage commitment and/or tissue specificity. However, for the first time, we consider unique dimensions to the architectural organization and assembly of regulatory machinery for gene expression in nuclear microenviornments that define parameters of pluripotency. From both fundamental biological and clinical perspectives, understanding control of the abbreviated embryonic stem cell cycle can provide options to coordinate control of proliferation versus differentiation. Wound healing, tissue engineering, and cell-based therapy to mitigate developmental aberrations illustrate applications that benefit from knowledge of the biology of the pluripotent cell cycle. PMID:22552993

Effects of Excess Copper Ions on Decidualization of Human Endometrial Stromal Cells.

PubMed

Li, Ying; Kang, Zhen-Long; Qiao, Na; Hu, Lian-Mei; Ma, Yong-Jiang; Liang, Xiao-Huan; Liu, Ji-Long; Yang, Zeng-Ming

2017-05-01

The aim of this study was to investigate the effects of copper ions on decidualization of human endometrial stromal cells (HESCs) cultured in vitro. Firstly, non-toxic concentrations of copper D-gluconate were screened in HESCs based on cell activity. Then, the effects of non-toxic concentrations of copper ions (0~250 μM) were examined on decidualization of human endometrial stromal cells. Our data demonstrated that the mRNA expressions of insulin-like growth factor binding protein (IGFBP-1), prolactin (PRL), Mn-SOD, and FOXO1were down-regulated during decidualization following the treatments with 100 or 250 μM copper ions. Meanwhile, the amount of malonaldehyde (MDA) in the supernatant of HESCs was increased. These results showed that in vitro decidualization of HESCs was impaired by copper treatment.

Size-dependent Toxicity of Gold Nanoparticles on Human Embryonic Stem Cells and Their Neural Derivatives

PubMed Central

Senut, Marie-Claude; Zhang, Yanhua; Liu, Fangchao; Sen, Arko; Ruden, Douglas M.; Mao, Guangzhao

2016-01-01

This study explores the use of human embryonic stem cells (hESCs) for assessing nanotoxicology, specifically, the effect of gold nanoparticles (AuNPs) of different core sizes (1.5 nm, 4 nm, and 14 nm) on the viability, pluripotency, neuronal differentiation, and DNA methylation of hESCs. The hESCs exposed to 1.5 nm thiolate-capped AuNPs exhibited loss of cohesiveness and detachment suggesting ongoing cell death at concentrations as low as 0.1 µg/mL. The cells exposed to 1.5 nm AuNPs at this concentration did not form embryoid bodies but rather disintegrated into single cells within 48 hours. Cell death caused by 1.5 nm AuNPs also occurred in hESC-derived neural progenitor cells. None of the other nanoparticles exhibited toxic effects on the hESCs at concentrations as high as 10 µg/mL during a 19 day neural differentiation period. Thiolate-capped 4 nm AuNPs at 10 µg/mL caused a dramatic decrease in global DNA methylation (5mC) and a corresponding increase in global DNA hydroxymethylation (5hmC) of the hESC’s DNA in only 24 hours. This work identifies a type of AuNPs highly toxic to hESCs and demonstrates the potential of hESCs in predicting nanotoxicity and characterizing their ability to alter the DNA methylation and hydroxymethylation patterns in the cells. PMID:26676601

Genome-wide identification of microRNA targets in human ES cells reveals a role for miR-302 in modulating BMP response

PubMed Central

Lipchina, Inna; Elkabetz, Yechiel; Hafner, Markus; Sheridan, Robert; Mihailovic, Aleksandra; Tuschl, Thomas; Sander, Chris; Studer, Lorenz; Betel, Doron

2011-01-01

MicroRNAs are important regulators in many cellular processes, including stem cell self-renewal. Recent studies demonstrated their function as pluripotency factors with the capacity for somatic cell reprogramming. However, their role in human embryonic stem (ES) cells (hESCs) remains poorly understood, partially due to the lack of genome-wide strategies to identify their targets. Here, we performed comprehensive microRNA profiling in hESCs and in purified neural and mesenchymal derivatives. Using a combination of AGO cross-linking and microRNA perturbation experiments, together with computational prediction, we identified the targets of the miR-302/367 cluster, the most abundant microRNAs in hESCs. Functional studies identified novel roles of miR-302/367 in maintaining pluripotency and regulating hESC differentiation. We show that in addition to its role in TGF-β signaling, miR-302/367 promotes bone morphogenetic protein (BMP) signaling by targeting BMP inhibitors TOB2, DAZAP2, and SLAIN1. This study broadens our understanding of microRNA function in hESCs and is a valuable resource for future studies in this area. PMID:22012620

ROCK inhibitor is not required for embryoid body formation from singularized human embryonic stem cells.

PubMed

Pettinato, Giuseppe; Vanden Berg-Foels, Wendy S; Zhang, Ning; Wen, Xuejun

2014-01-01

We report a technology to form human embryoid bodies (hEBs) from singularized human embryonic stem cells (hESCs) without the use of the p160 rho-associated coiled-coil kinase inhibitor (ROCKi) or centrifugation (spin). hEB formation was tested under four conditions: +ROCKi/+spin, +ROCKi/-spin, -ROCKi/+spin, and -ROCKi/-spin. Cell suspensions of BG01V/hOG and H9 hESC lines were pipetted into non-adherent hydrogel substrates containing defined microwell arrays. hEBs of consistent size and spherical geometry can be formed in each of the four conditions, including the -ROCKi/-spin condition. The hEBs formed under the -ROCKi/-spin condition differentiated to develop the three embryonic germ layers and tissues derived from each of the germ layers. This simplified hEB production technique offers homogeneity in hEB size and shape to support synchronous differentiation, elimination of the ROCKi xeno-factor and rate-limiting centrifugation treatment, and low-cost scalability, which will directly support automated, large-scale production of hEBs and hESC-derived cells needed for clinical, research, or therapeutic applications.

Neural Differentiation of Human Pluripotent Stem Cells for Nontherapeutic Applications: Toxicology, Pharmacology, and In Vitro Disease Modeling

PubMed Central

Yap, May Shin; Nathan, Kavitha R.; Yeo, Yin; Poh, Chit Laa; Richards, Mark; Lim, Wei Ling; Othman, Iekhsan; Heng, Boon Chin

2015-01-01

Human pluripotent stem cells (hPSCs) derived from either blastocyst stage embryos (hESCs) or reprogrammed somatic cells (iPSCs) can provide an abundant source of human neuronal lineages that were previously sourced from human cadavers, abortuses, and discarded surgical waste. In addition to the well-known potential therapeutic application of these cells in regenerative medicine, these are also various promising nontherapeutic applications in toxicological and pharmacological screening of neuroactive compounds, as well as for in vitro modeling of neurodegenerative and neurodevelopmental disorders. Compared to alternative research models based on laboratory animals and immortalized cancer-derived human neural cell lines, neuronal cells differentiated from hPSCs possess the advantages of species specificity together with genetic and physiological normality, which could more closely recapitulate in vivo conditions within the human central nervous system. This review critically examines the various potential nontherapeutic applications of hPSC-derived neuronal lineages and gives a brief overview of differentiation protocols utilized to generate these cells from hESCs and iPSCs. PMID:26089911

Repair of full-thickness tendon injury using connective tissue progenitors efficiently derived from human embryonic stem cells and fetal tissues.

PubMed

Cohen, Shahar; Leshansky, Lucy; Zussman, Eyal; Burman, Michael; Srouji, Samer; Livne, Erella; Abramov, Natalie; Itskovitz-Eldor, Joseph

2010-10-01

The use of stem cells for tissue engineering (TE) encourages scientists to design new platforms in the field of regenerative and reconstructive medicine. Human embryonic stem cells (hESC) have been proposed to be an important cell source for cell-based TE applications as well as an exciting tool for investigating the fundamentals of human development. Here, we describe the efficient derivation of connective tissue progenitors (CTPs) from hESC lines and fetal tissues. The CTPs were significantly expanded and induced to generate tendon tissues in vitro, with ultrastructural characteristics and biomechanical properties typical of mature tendons. We describe a simple method for engineering tendon grafts that can successfully repair injured Achilles tendons and restore the ankle joint extension movement in mice. We also show the CTP's ability to differentiate into bone, cartilage, and fat both in vitro and in vivo. This study offers evidence for the possibility of using stem cell-derived engineered grafts to replace missing tissues, and sets a basic platform for future cell-based TE applications in the fields of orthopedics and reconstructive surgery.

Robust Induction of DARPP32-Expressing GABAergic Striatal Neurons from Human Pluripotent Stem Cells.

PubMed

Fjodorova, Marija; Li, Meng

2018-01-01

Efficient generation of disease relevant neuronal subtypes from human pluripotent stem cells (PSCs) is fundamental for realizing their promise in disease modeling, pharmaceutical drug screening and cell therapy. Here we describe a step-by-step protocol for directing the differentiation of human embryonic and induced PSCs (hESCs and hiPSCs, respectively) toward medium spiny neurons, the type of cells that are preferentially lost in Huntington's disease patients. This method is based on a novel concept of Activin A-dependent induction of the lateral ganglionic/striatal fate using a simple monolayer culture paradigm under chemically defined conditions. Transplantable medium spiny neuron progenitors amenable for cryopreservation are produced in less than 20 days, which differentiate and mature into a high yield of dopamine- and cAMP-regulated phosphoprotein, Mr 32 kDa (DARPP32) expressing gamma-aminobutyric acid (GABA)-ergic neurons in vitro and in the adult rat brain after transplantation. This method has been validated in multiple hESC and hiPSC lines, and is independent of the regime for PSC maintenance.

Metabolic Profiling and Flux Analysis of MEL-2 Human Embryonic Stem Cells during Exponential Growth at Physiological and Atmospheric Oxygen Concentrations

PubMed Central

Titmarsh, Drew; Krömer, Jens O.; Kao, Li-Pin; Nielsen, Lars; Wolvetang, Ernst; Cooper-White, Justin

2014-01-01

As human embryonic stem cells (hESCs) steadily progress towards regenerative medicine applications there is an increasing emphasis on the development of bioreactor platforms that enable expansion of these cells to clinically relevant numbers. Surprisingly little is known about the metabolic requirements of hESCs, precluding the rational design and optimisation of such platforms. In this study, we undertook an in-depth characterisation of MEL-2 hESC metabolic behaviour during the exponential growth phase, combining metabolic profiling and flux analysis tools at physiological (hypoxic) and atmospheric (normoxic) oxygen concentrations. To overcome variability in growth profiles and the problem of closing mass balances in a complex environment, we developed protocols to accurately measure uptake and production rates of metabolites, cell density, growth rate and biomass composition, and designed a metabolic flux analysis model for estimating internal rates. hESCs are commonly considered to be highly glycolytic with inactive or immature mitochondria, however, whilst the results of this study confirmed that glycolysis is indeed highly active, we show that at least in MEL-2 hESC, it is supported by the use of oxidative phosphorylation within the mitochondria utilising carbon sources, such as glutamine to maximise ATP production. Under both conditions, glycolysis was disconnected from the mitochondria with all of the glucose being converted to lactate. No difference in the growth rates of cells cultured under physiological or atmospheric oxygen concentrations was observed nor did this cause differences in fluxes through the majority of the internal metabolic pathways associated with biogenesis. These results suggest that hESCs display the conventional Warburg effect, with high aerobic activity despite high lactate production, challenging the idea of an anaerobic metabolism with low mitochondrial activity. The results of this study provide new insight that can be used in rational bioreactor design and in the development of novel culture media for hESC maintenance and expansion. PMID:25412279

miRNA Signature and Dicer Requirement during Human Endometrial Stromal Decidualization In Vitro

PubMed Central

Estella, Carlos; Herrer, Isabel; Moreno-Moya, Juan Manuel; Quiñonero, Alicia; Martínez, Sebastián; Pellicer, Antonio; Simón, Carlos

2012-01-01

Decidualization is a morphological and biochemical transformation of endometrial stromal fibroblast into differentiated decidual cells, which is critical for embryo implantation and pregnancy establishment. The complex regulatory networks have been elucidated at both the transcriptome and the proteome levels, however very little is known about the post-transcriptional regulation of this process. miRNAs regulate multiple physiological pathways and their de-regulation is associated with human disorders including gynaecological conditions such as endometriosis and preeclampsia. In this study we profile the miRNAs expression throughout human endometrial stromal (hESCs) decidualization and analyze the requirement of the miRNA biogenesis enzyme Dicer during this process. A total of 26 miRNAs were upregulated and 17 miRNAs downregulated in decidualized hESCs compared to non-decidualized hESCs. Three miRNAs families, miR-181, miR-183 and miR-200, are down-regulated during the decidualization process. Using miRNAs target prediction algorithms we have identified the potential targets and pathways regulated by these miRNAs. The knockdown of Dicer has a minor effect on hESCs during in vitro decidualization. We have analyzed a battery of decidualization markers such as cell morphology, Prolactin, IGFBP-1, MPIF-1 and TIMP-3 secretion as well as HOXA10, COX2, SP1, C/EBPß and FOXO1 expression in decidualized hESCs with decreased Dicer function. We found decreased levels of HOXA10 and altered intracellular organization of actin filaments in Dicer knockdown decidualized hESCs compared to control. Our results provide the miRNA signature of hESC during the decidualization process in vitro. We also provide the first functional characterization of Dicer during human endometrial decidualization although surprisingly we found that Dicer plays a minor role regulating this process suggesting that alternative biogenesis miRNAs pathways must be involved in human endometrial decidualization. PMID:22911744

A protocol describing the use of a recombinant protein-based, animal product-free medium (APEL) for human embryonic stem cell differentiation as spin embryoid bodies.

PubMed

Ng, Elizabeth S; Davis, Richard; Stanley, Edouard G; Elefanty, Andrew G

2008-01-01

In order to promote the uniform and reproducible differentiation of human embryonic stem cells (HESCs) in response to exogenously added growth factors, we have developed a method (spin embryoid bodies (EBs)) that uses a recombinant protein-based, animal product-free medium in which HESCs are aggregated by centrifugation to form EBs. In this protocol we describe the formulation of this medium, denoted APEL (Albumin Polyvinylalcohol Essential Lipids), and its use in spin EB differentiation of HESCs. We also describe a more economical variant, BPEL (Bovine Serum Albumin (BSA) Polyvinylalchohol Essential Lipids), in which BSA replaces the recombinant human albumin. The integration of a medium that includes only defined and recombinant components with a defined number of cells to initiate EB formation results in a generally applicable, robust platform for growth factor-directed HESC differentiation.

Stem Cell Banking: A Global View.

PubMed

Stacey, Glyn

2017-01-01

Stem cell banking has been a topic of discussion and debate for more than a decade since the first public services to supply human embryonic stem cells (hESCs) were established in the USA and the UK. This topic has received a recent revival with numerous ambitious programmes announced to deliver large collections of human induced pluripotency cell (hiPSC) lines. This chapter will provide a brief overview charting the development of stem cell banks, their value, and their likely role in the future.

Optimization of flowrate for expansion of human embryonic stem cells in perfusion microbioreactors.

PubMed

Titmarsh, Drew; Hidalgo, Alejandro; Turner, Jennifer; Wolvetang, Ernst; Cooper-White, Justin

2011-12-01

Microfluidic systems create significant opportunities to establish highly controlled microenvironmental conditions for screening pluripotent stem cell fate. However, since cell fate is crucially dependent on this microenvironment, it remains unclear as to whether continual perfusion of culture medium supports pluripotent stem cell maintenance in feeder-free, chemically defined conditions, and further, whether optimum perfusion conditions exist for subsequent use of human embryonic stem cell (hESCs) in other microfludic systems. To investigate this, we designed microbioreactors based on resistive flow to screen hESCs under a linear range of flowrates. We report that at low rates (conditions where glucose transport is convection-limited with Péclet number <1), cells are affected by apparent nutrient depletion and waste accumulation, evidenced by reduced cell expansion and altered morphology. At higher rates, cells are spontaneously washed out, and display morphological changes which may be indicative of early-stage differentiation. However, between these thresholds exists a narrow range of flowrates in which hESCs expand comparably to the equivalent static culture system, with regular morphology and maintenance of the pluripotency marker TG30 in >95% of cells over 7 days. For MEL1 hESCs the optimum flowrate also coincided with the time-averaged medium exchange rate in static cultures, which may therefore provide a good first estimate of appropriate perfusion rates. Overall, we demonstrate hESCs can be maintained in microbioreactors under continual flow for up to 7 days, a critical outcome for the future development of microbioreactor-based screening systems and assays for hESC culture. Copyright © 2011 Crown in the right of Canada.

Mechanisms of Normal and Abnormal Endometrial Bleeding

PubMed Central

Lockwood, Charles J.

2011-01-01

Expression of tissue factor (TF), the primary initiator of coagulation, is enhanced in decidualized human endometrial stromal cells (HESC) during the progesterone-dominated luteal phase. Progesterone also augments a second HESC hemostatic factor, plasminogen activator inhibitor-1 (PAI-1). In contrast, progestins inhibit HESC matrix metalloproteinase (MMP)-1, 3 and 9 expression to stabilize endometrial stromal and vascular extracellular matrix. Through these mechanisms decidualized endometrium is rendered both hemostatic and resistant to excess trophoblast invasion in the mid-luteal phase and throughout gestation to prevent hemorrhage and accreta. In non-fertile cycles, progesterone withdrawal results in decreased HESC TF and PAI-expression and increased MMP activity and inflammatory cytokine production promoting the controlled hemorrhage of menstruation and related tissue sloughing. In contrast to these well ordered biochemical processes, unpredictable endometrial bleeding associated with anovulation reflects absence of progestational effects on TF, PAI-1 and MMP activity as well as unrestrained angiogenesis rendering the endometrium non-hemostatic, proteolytic and highly vascular. Abnormal bleeding associated with long-term progestin-only contraceptives results not from impaired hemostasis but from unrestrained angiogenesis leading to large fragile endometrial vessels. This abnormal angiogenesis reflects progestational inhibition of endometrial blood flow promoting local hypoxia and generation of reactive oxygen species that increase production of angiogenic factors such as vascular endothelial growth factor (VEGF) in HESCs and Angiopoietin-2 (Ang-2) in endometrial endothelial cells while decreasing HESC expression of angiostatic, Ang-1. The resulting vessel fragility promotes bleeding. Aberrant angiogenesis also underlies abnormal bleeding associated with myomas and endometrial polyps however there are gaps in our understanding of this pathology. PMID:21499503

Protein kinase A inhibitor, H89, enhances survival and clonogenicity of dissociated human embryonic stem cells through Rho-associated coiled-coil containing protein kinase (ROCK) inhibition.

PubMed

Zhang, Liang; Xu, Yanqing; Xu, Jiandong; Wei, Yuping; Xu, Xia

2016-04-01

Can cell survival of dissociated human embryonic stem cells (hESCs) be increased during culture? A protein kinase A (PKA) inhibitor, H89, can significantly enhance survival and clonogenicity of dissociated hESCs without affecting their pluripotency. hESCs are vulnerable to massive cell death upon cellular detachment and dissociation. hESCs were dissociated into single cells and then cultured in feeder-dependent and -independent manners. H89 was added to the culture medium at different concentrations for 1 day. The statistical results were obtained from at least three independent experiments (n ≥ 4). The group without treatment was used as the negative control. 4 µM H89 was added in the culture medium to promote cell survival and colony formation of dissociated hESCs. MTT method and propidium iodide (PI) staining were used to determine cell proliferation, cell death and cell cycle, respectively. To count colony formation, alkaline phosphatase (AP) staining was carried out. Western blot was performed to determine protein expression. Except AP staining, immunofluorescence, RT-PCR and karyotype analysis were used to confirm the pluripotent state of H89 treated hESCs. H89 inhibits the dissociation-induced phosphorylation of PKA and two substrates of Rho-associated coiled-coil containing protein kinase (ROCK), myosin light chain (MLC2) and myosin phosphatase target subunit 1 (MYPT1), significantly increases cell survival and colony formation, and strongly depresses dissociation-induced cell death and cell blebbing without affecting the pluripotency of hESCs and their differentiation in vitro. Appropriate H89 concentration should be used and 1 day of H89 treatment is sufficient for promoting survival and colony formation of dissociated hESCs. These results provide an alternative for human pluripotent stem cell (hPSC) culture, broaden the scope of participants in the cell death of single hES cells after dissociation and further enlighten clues to understand the mechanism of dissociation-induced cell death. This research was supported by the National Natural Science Foundation of China (21176238, 21576266), and Chinese Academy of Sciences. There is no conflict of interest to declare. Nil. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

NADPH oxidase-mediated generation of reactive oxygen species is critically required for survival of undifferentiated human promyelocytic leukemia cell line HL-60.

PubMed

Dong, Jing-Mei; Zhao, Sheng-Guo; Huang, Guo-Yin; Liu, Qing

2004-06-01

Nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) mediated generation of reactive oxygen species (ROS) was originally identified as the powerful host defense machinery against microorganism in phagocytes. But recent reports indicated that some non-phagocytic cells also have the NADPH oxidase activity, and the ROS produced by it may act as cell signal molecule. But as far as today, whether the NADPH oxidase also plays similar role in phagocyte has not been paid much attention. Utilizing the undifferentiated HL-60 promyelocytic leukemia cells as a model, the aim of the present study was to determine whether NADPH oxidase plays a role on ROS generation in undifferentiated HL-60, and the ROS mediated by it was essential for cell's survival. For the first time, we verified that the release of ROS in undifferentiated HL-60 was significantly increased by the stimulation with Calcium ionophore or opsonized zymosan, which are known to trigger respiration burst in phagocytes by NADPH oxidase pathway. Diphenylene iodonium (DPI) or apocynin (APO), two inhibitors of NADPH oxidase, significantly suppressed the increasing of ROS caused by opsonized zymosan. Cell survival assay and fluorescence double dyeing with acridine orange and ethidium bromide showed that DPI and APO, as well as superoxide dismutase (SOD) and catalase (CAT) concentration-dependently decreased the viability of undifferentiated HL-60 cells, whereas exogenous H2O2 can rescue the cells from death obviously. Our results suggested that the ROS, generated by NADPH oxidase play an essential role in the survival of undifferentiated HL-60 cells.

Effects of Aminoglycoside Antibiotics on Human Embryonic Stem Cell Viability during Differentiation In Vitro

PubMed Central

Varghese, Divya S.; Parween, Shama; Ardah, Mustafa T.; Emerald, Bright Starling

2017-01-01

Human embryonic stem cells (hESCs) are being used extensively in array of studies to understand different mechanisms such as early human embryogenesis, drug toxicity testing, disease modeling, and cell replacement therapy. The protocols for the directed differentiation of hESCs towards specific cell types often require long-term cell cultures. To avoid bacterial contamination, these protocols include addition of antibiotics such as pen-strep and gentamicin. Although aminoglycosides, streptomycin, and gentamicin have been shown to cause cytotoxicity in various animal models, the effect of these antibiotics on hESCs is not clear. In this study, we found that antibiotics, pen-strep, and gentamicin did not affect hESC cell viability or expression of pluripotency markers. However, during directed differentiation towards neural and hepatic fate, significant cell death was noted through the activation of caspase cascade. Also, the expression of neural progenitor markers Pax6, Emx2, Otx2, and Pou3f2 was significantly reduced suggesting that gentamicin may adversely affect early embryonic neurogenesis whereas no effect was seen on the expression of endoderm or hepatic markers during differentiation. Our results suggest that the use of antibiotics in cell culture media for the maintenance and differentiation of hESCs needs thorough investigation before use to avoid erroneous results. PMID:29147115

A Simple and Robust Method for Culturing Human-Induced Pluripotent Stem Cells in an Undifferentiated State Using Botulinum Hemagglutinin.

PubMed

Kim, Mee-Hae; Matsubara, Yoshifumi; Fujinaga, Yukako; Kino-Oka, Masahiro

2018-02-01

Clinical and industrial applications of human-induced pluripotent stem cells (hiPSCs) is hindered by the lack of robust culture strategies capable of sustaining a culture in an undifferentiated state. Here, a simple and robust hiPSC-culture-propagation strategy incorporating botulinum hemagglutinin (HA)-mediated selective removal of cells deviating from an undifferentiated state is developed. After HA treatment, cell-cell adhesion is disrupted, and deviated cells detached from the central region of the colony to subsequently form tight monolayer colonies following prolonged incubation. The authors find that the temporal and dose-dependent activity of HA regulated deviated-cell removal and recoverability after disruption of cell-cell adhesion in hiPSC colonies. The effects of HA are confirmed under all culture conditions examined, regardless of hiPSC line and feeder-dependent or -free culture conditions. After routine application of our HA-treatment paradigm for serial passages, hiPSCs maintains expression of pluripotent markers and readily forms embryoid bodies expressing markers for all three germ-cell layers. This method enables highly efficient culturing of hiPSCs and use of entire undifferentiated portions without having to pick deviated cells manually. This simple and readily reproducible culture strategy is a potentially useful tool for improving the robust and scalable maintenance of undifferentiated hiPSC cultures. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Bioprinting of human pluripotent stem cells and their directed differentiation into hepatocyte-like cells for the generation of mini-livers in 3D.

PubMed

Faulkner-Jones, Alan; Fyfe, Catherine; Cornelissen, Dirk-Jan; Gardner, John; King, Jason; Courtney, Aidan; Shu, Wenmiao

2015-10-21

We report the first investigation into the bioprinting of human induced pluripotent stem cells (hiPSCs), their response to a valve-based printing process as well as their post-printing differentiation into hepatocyte-like cells (HLCs). HLCs differentiated from both hiPSCs and human embryonic stem cells (hESCs) sources were bioprinted and examined for the presence of hepatic markers to further validate the compatibility of the valve-based bioprinting process with fragile cell transfer. Examined cells were positive for nuclear factor 4 alpha and were demonstrated to secrete albumin and have morphology that was also found to be similar to that of hepatocytes. Both hESC and hiPSC lines were tested for post-printing viability and pluripotency and were found to have negligible difference in terms of viability and pluripotency between the printed and non-printed cells. hESC-derived HLCs were 3D printed using alginate hydrogel matrix and tested for viability and albumin secretion during the remaining differentiation and were found to be hepatic in nature. 3D printed with 40-layer of HLC-containing alginate structures reached peak albumin secretion at day 21 of the differentiation protocol. This work demonstrates that the valve-based printing process is gentle enough to print human pluripotent stem cells (hPSCs) (both hESCs and hiPSCs) while either maintaining their pluripotency or directing their differentiation into specific lineages. The ability to bioprint hPSCs will pave the way for producing organs or tissues on demand from patient specific cells which could be used for animal-free drug development and personalized medicine.

Enhanced cyclooxygenase-2 expression levels and metalloproteinase 2 and 9 activation by Hexachlorobenzene in human endometrial stromal cells.

PubMed

Chiappini, Florencia; Bastón, Juan Ignacio; Vaccarezza, Agustina; Singla, José Javier; Pontillo, Carolina; Miret, Noelia; Farina, Mariana; Meresman, Gabriela; Randi, Andrea

2016-06-01

Hexachlorobenzene (HCB) is an organochlorine pesticide that induces toxic reproductive effects in laboratory animals. It is a dioxin-like compound and a weak ligand of the aryl hydrocarbon receptor (AhR). Endometriosis is characterized by the presence of functional endometrial tissues outside the uterine cavity. Experimental studies indicate that exposure to organochlorines can interfere with both hormonal regulation and immune function to promote endometriosis. Altered expression of metalloproteinases (MMPs) in patients with endometriosis, suggests that MMPs may play a critical role. In the endometriotic lesions, prostaglandin E2 (PGE2) produced by cyclooxygenase-2 (COX-2), binds to its EP4 receptor (EP4), and via c-Src kinase induces MMPs activation, promoting endometriosis. We examined the HCB action on MMP-2 and MMP-9 activities and expression, COX-2 levels, PGE2 signaling, and the AhR involvement in HCB-induced effects. We have used different in vitro models: (1) human endometrial stromal cell line T-HESC, (2) primary cultures of Human Uterine Fibroblast (HUF), and (3) primary cultures of endometrial stromal cells from eutopic endometrium of control (CESC) and subjects with endometriosis (EESC). Our results show that HCB enhances MMP-2 and MMP-9 activities in T-HESC, HUF and ESC cells. The MMP-9 levels were elevated in all models, while the MMP-2 expression only increased in ESC cells. HCB enhanced COX-2 and EP4 expression, PGE2 secretion and the c-Src kinase activation in T-HESC. Besides, we observed that AhR is implicated in these HCB-induced effects. In conclusion, our results show that HCB exposure could contribute to endometriosis development, affecting inflammation and invasion parameters of human endometrial cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Preparation, characterization, and banking of clinical-grade cells for neural transplantation: Scale up, fingerprinting, and genomic stability of stem cell lines.

PubMed

Natalwala, Ammar; Kunath, Tilo

2017-01-01

Parkinson's disease is a complex and progressive neurodegenerative condition that is characterized by the severe loss of midbrain dopaminergic (mDA) neurons, which innervate the striatum. Cell transplantation therapies to rebuild this dopaminergic network have been attempted for over 30 years. The most promising outcomes were observed when human fetal mesencephalic tissue was used as the source of cells for transplantation. However, reliance on terminations for a Parkinson's therapy presents significant logistical and ethical hurdles. An alternative source of transplantable mDA neurons is urgently needed, and the solution may come from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). Protocols to differentiate hESCs/iPSCs toward mDA neurons are now robust and efficient, and upon grafting the cells rescue preclinical animal models of Parkinson's disease. The challenge now is to apply Good Manufacturing Practice (GMP) to the academic discoveries and protocols to produce clinical-grade transplantable mDA cells. Major technical and logistical considerations include (i) source of hESC or iPSC line, (ii) GMP compliance of the differentiation protocol and all reagents, (iii) characterization of the cell product in terms of identity, safety, and efficacy, (iv) characterization of genomic state and stability, and (v) banking of a transplantation-ready cell product. Approaches and solutions to these challenges are reviewed here. © 2017 Elsevier B.V. All rights reserved.

Endogenous APOBEC3B Restricts LINE-1 Retrotransposition in Transformed Cells and Human Embryonic Stem Cells*

PubMed Central

Wissing, Silke; Montano, Mauricio; Garcia-Perez, Jose Luis; Moran, John V.; Greene, Warner C.

2011-01-01

Members of the APOBEC3 (A3) family of cytidine deaminase enzymes act as host defense mechanisms limiting both infections by exogenous retroviruses and mobilization of endogenous retrotransposons. Previous studies revealed that the overexpression of some A3 proteins could restrict engineered human Long INterspersed Element-1 (LINE-1 or L1) retrotransposition in HeLa cells. However, whether endogenous A3 proteins play a role in restricting L1 retrotransposition remains largely unexplored. Here, we show that HeLa cells express endogenous A3B and A3C, whereas human embryonic stem cells (hESCs) express A3B, A3C, A3DE, A3F, and A3G. To study the relative contribution of endogenous A3 proteins in restricting L1 retrotransposition, we first generated small hairpin RNAs (shRNAs) to suppress endogenous A3 mRNA expression, and then assessed L1 mobility using a cell-based L1 retrotransposition assay. We demonstrate that in both HeLa and hESCs, shRNA-based knockdown of A3B promotes a ∼2–3.7-fold increase in the retrotransposition efficiency of an engineered human L1. Knockdown of the other A3s produced no significant increase in L1 activity. Thus, A3B appears to restrict engineered L1 retrotransposition in a broad range of cell types, including pluripotent cells. PMID:21878639

Enhanced differentiation of human embryonic stem cells into cardiomyocytes by combining hanging drop culture and 5-azacytidine treatment.

PubMed

Yoon, Byung Sun; Yoo, Seung Jun; Lee, Jeoung Eun; You, Seungkwon; Lee, Hoon Taek; Yoon, Hyun Soo

2006-04-01

Cell replacement therapy is a promising approach for the treatment of cardiac diseases. It is, however, challenged by a limited supply of appropriate cells. Therefore, we have investigated whether functional cardiomyocytes can be efficiently generated from human embryonic stem cells (hESCs). In this study, we developed an efficient protocol for the generation of functional cardiomyocytes from hESCs by combining hanging drop culture and 5-azacytidine, a well-known demethylating agent, and then evaluated the expression of cardiac-specific markers. hESCs were cultured both in the medium without or with 0.1, 1, or 10 microM of 5-azacytidine under a hanging drop culture. The expression of several cardiac-specific markers was determined by real-time PCR, RT-PCR, immunofluorescence, and confocal microscopy. To verify the structural and functional properties of hESC-derived cardiomyocytes, we performed electron microscopy and electrophysiological recording. The efficiency of beating cell generation was significantly improved in the hanging drop culture compared with that in suspension culture. Treatment of hESCs with 0.1 microM of 5-azacytidine for 1-3 days significantly increased the number of beating cells and simultaneously enhanced the expression of cardiac-specific markers. Transmission electron microscopy and electrophysiological recording showed that hESC-derived cardiomyocytes acquired structural and functional properties of cardiomyocytes. In conclusion, these results suggest that differentiation of hESCs into cardiomyocytes can be enhanced by the combination of hanging drop culture and 5-azacytidine treatment. Also the methylation status of genes related to cardiomyocyte development may play an important role in the differentiation of hESCs into cardiomyocytes.

Thrombin impairs human endometrial endothelial angiogenesis; implications for progestin-only contraceptive-induced abnormal uterine bleeding.

PubMed

Shapiro, John P; Guzeloglu-Kayisli, Ozlem; Kayisli, Umit A; Semerci, Nihan; Huang, S Joseph; Arlier, Sefa; Larsen, Kellie; Fadda, Paolo; Schatz, Frederick; Lockwood, Charles J

2017-06-01

Progestin-only contraceptives induce abnormal uterine bleeding, accompanied by prothrombin leakage from dilated endometrial microvessels and increased thrombin generation by human endometrial stromal cell (HESC)-expressed tissue factor. Initial studies of the thrombin-treated HESC secretome identified elevated levels of cleaved chondroitin sulfate proteoglycan 4 (CSPG4), impairing pericyte-endothelial interactions. Thus, we investigated direct and CSPG4-mediated effects of thrombin in eliciting abnormal uterine bleeding by disrupting endometrial angiogenesis. Liquid chromatography/tandem mass spectrometry, enzyme-linked immunosorbent assay (ELISA) and quantitative real-time-polymerase chain reaction (PCR) evaluated conditioned medium supernatant and cell lysates from control versus thrombin-treated HESCs. Pre- and post-Depo medroxyprogesterone acetate (DMPA)-administered endometria were immunostained for CSPG4. Proliferation, apoptosis and tube formation were assessed in human endometrial endothelial cells (HEECs) incubated with recombinant human (rh)-CSPG4 or thrombin or both. Thrombin induced CSPG4 protein expression in cultured HESCs as detected by mass spectrometry and ELISA (p<.02, n=3). Compared to pre-DMPA endometria (n=5), stromal cells in post-DMPA endometria (n=5) displayed stronger CSPG4 immunostaining. In HEEC cultures (n=3), total tube-formed mesh area was significantly higher in rh-CSPG4 versus control (p<.05). However, thrombin disrupted HEEC tube formation by a concentration- and time-dependent reduction of angiogenic parameters (p<.05), whereas CSPG4 co-treatment did not reverse these thrombin-mediated effects. These results suggest that disruption of HEEC tube formation by thrombin induces aberrant angiogenesis and abnormal uterine bleeding in DMPA users. Mass spectrometry analysis identified several HESC-secreted proteins regulated by thrombin. Therapeutic agents blocking angiogenic effects of thrombin in HESCs can prevent or minimize progestin-only contraceptive-induced abnormal uterine bleeding. Copyright © 2017. Published by Elsevier Inc.

No DNA damage response and negligible genome-wide transcriptional changes in human embryonic stem cells exposed to terahertz radiation

PubMed Central

Bogomazova, A. N.; Vassina, E. M.; Goryachkovskaya, T. N.; Popik, V. M.; Sokolov, A. S.; Kolchanov, N. A.; Lagarkova, M. A.; Kiselev, S. L.; Peltek, S. E.

2015-01-01

Terahertz (THz) radiation was proposed recently for use in various applications, including medical imaging and security scanners. However, there are concerns regarding the possible biological effects of non-ionising electromagnetic radiation in the THz range on cells. Human embryonic stem cells (hESCs) are extremely sensitive to environmental stimuli, and we therefore utilised this cell model to investigate the non-thermal effects of THz irradiation. We studied DNA damage and transcriptome responses in hESCs exposed to narrow-band THz radiation (2.3 THz) under strict temperature control. The transcription of approximately 1% of genes was subtly increased following THz irradiation. Functional annotation enrichment analysis of differentially expressed genes revealed 15 functional classes, which were mostly related to mitochondria. Terahertz irradiation did not induce the formation of γH2AX foci or structural chromosomal aberrations in hESCs. We did not observe any effect on the mitotic index or morphology of the hESCs following THz exposure. PMID:25582954

SMAD7 directly converts human embryonic stem cells to telencephalic fate by a default mechanism

PubMed Central

Ozair, Mohammad Zeeshan; Noggle, Scott; Warmflash, Aryeh; Krzyspiak, Joanna Ela; Brivanlou, Ali H.

2013-01-01

Human embryonic stem cells (hESCs) provide a valuable window into the dissection of the molecular circuitry underlying the early formation of the human forebrain. However, dissection of signaling events in forebrain development using current protocols is complicated by non-neural contamination and fluctuation of extrinsic influences. Here we show that SMAD7, a cell-intrinsic inhibitor of TGFβ signaling, is sufficient to directly convert pluripotent hESCs to an anterior neural fate. Time-course gene expression revealed down-regulation of MAPK components, and combining MEK1/2 inhibition with SMAD7-mediated TGFβ inhibition promoted telencephalic conversion. FGF-MEK and TGFβ-SMAD signaling maintain hESCs by promoting pluripotency genes and repressing neural genes. Our findings suggest that in the absence of these cues, pluripotent cells simply revert to a program of neural conversion. Hence the “primed” state of hESCs requires inhibition of the “default” state of neural fate acquisition. This has parallels in amphibians, suggesting an evolutionarily conserved mechanism. PMID:23034881

Quantitative proteomics analysis highlights the role of redox hemostasis and energy metabolism in human embryonic stem cell differentiation to neural cells.

PubMed

Fathi, Ali; Hatami, Maryam; Vakilian, Haghighat; Han, Chia-Li; Chen, Yu-Ju; Baharvand, Hossein; Salekdeh, Ghasem Hosseini

2014-04-14

Neural differentiation of human embryonic stem cells (hESCs) is a unique opportunity for in vitro analyses of neurogenesis in humans. Extrinsic cues through neural plate formation are well described in the hESCs although intracellular mechanisms underlying neural development are largely unknown. Proteome analysis of hESC differentiation to neural cells will help to further define molecular mechanisms involved in neurogenesis in humans. Using a two-dimensional differential gel electrophoresis (2D-DIGE) system, we analyzed the proteome of hESC differentiation to neurons at three stages, early neural differentiation, neural ectoderm and mature neurons. Out of 137 differentially accumulated protein spots, 118 spots were identified using MALDI-TOF/TOF and LC MS/MS. We observed that proteins involved in redox hemostasis, vitamin and energy metabolism and ubiquitin dependent proteolysis were more abundant in differentiated cells, whereas the abundance of proteins associated with RNA processing and protein folding was higher in hESCs. Higher abundance of proteins involved in maintaining cellular redox state suggests the importance of redox hemostasis in neural differentiation. Furthermore, our results support the concept of a coupling mechanism between neuronal activity and glucose utilization. The protein network analysis showed that the majority of the interacting proteins were associated with the cell cycle and cellular proliferation. These results enhanced our understanding of the molecular dynamics that underlie neural commitment and differentiation. In highlighting the role of redox and unique metabolic properties of neuronal cells, the present findings add insight to our understanding of hESC differentiation to neurons. The abundance of fourteen proteins involved in maintaining cellular redox state, including 10 members of peroxiredoxin (Prdx) family, mainly increased during differentiation, thus highlighting a link of neural differentiation to redox. Our results revealed markedly higher expression of genes encoding enzymes involved in the glycolysis and amino acid synthesis during differentiation. Protein network analysis predicted a number of critical mediators in hESC differentiation. These proteins included TP53, CTNNB1, SMARCA4, TNF, TERT, E2F1, MYC, RB1, and AR. Copyright © 2014 Elsevier B.V. All rights reserved.

Cytoskeleton changes following differentiation of N1E-115 neuroblastoma cell line.

PubMed

Oh, J-E; Karlmark Raja, K; Shin, J-H; Pollak, A; Hengstschläger, M; Lubec, G

2006-10-01

No systematic approach to detect expression of differentiation-related elements was published so far. The undifferentiated N1E-115 neuroblastoma cell line was switched into a neuronal phenotype by DMSO treatment and used for proteomic experiments. We used two-dimensional gel electrophoresis followed by unambiguous mass spectrometrical identification of proteins to generate a map of cytoskeleton proteins (CPs), i.e., to search for differentiation-related structures. Alpha-actin, actin-like protein 6A, gamma-tubulin complex component 2, tubulin alpha 3/alpha 7, CLIP associating protein 2, B4 integrin interactor homolog were detectable in the undifferentiated cell line exclusively and neuron-specific CPs drebrin and presynaptic density protein 95, actin-related protein 2/3, alpha and beta-centractin, PDZ-domain actin binding protein, actinin alpha 1, profilin II, ezrin, coactosin-like protein, transgelin 2, myosin light polypeptide 6, tubulin alpha 2, 6 and 7, beta tubulin (94% similar with tubulin beta-2), tubulin beta 3, tubulin tyrosine ligase-like protein 1, lamin B1 and keratin 20 were observed in the differentiated cell line only. We herein identified differentiation-related expressional patterns thus providing new evidence for the role of CPs in the process of neuronal differentiation.

Human embryonic stem cell derived islet progenitors mature inside an encapsulation device without evidence of increased biomass or cell escape.

PubMed

Kirk, Kaitlyn; Hao, Ergeng; Lahmy, Reyhaneh; Itkin-Ansari, Pamela

2014-05-01

There are several challenges to successful implementation of a cell therapy for insulin dependent diabetes derived from human embryonic stem cells (hESC). Among these are development of functional insulin producing cells, a clinical delivery method that eliminates the need for chronic immunosuppression, and assurance that hESC derived tumors do not form in the patient. We and others have shown that encapsulation of cells in a bilaminar device (TheraCyte) provides immunoprotection in rodents and primates. Here we monitored human insulin secretion and employed bioluminescent imaging (BLI) to evaluate the maturation, growth, and containment of encapsulated islet progenitors derived from CyT49 hESC, transplanted into mice. Human insulin was detectable by 7 weeks post-transplant and increased 17-fold over the course of 8 weeks, yet during this period the biomass of encapsulated cells remained constant. Remarkably, by 20 weeks post-transplant encapsulated cells secreted sufficient levels of human insulin to ameliorate alloxan induced diabetes. Further, bioluminescent imaging revealed for the first time that hESCs remained fully contained in encapsulation devices for up to 150 days, the longest period tested. Collectively, the data suggest that encapsulated hESC derived islet progenitors hold great promise as an effective and safe cell replacement therapy for insulin dependent diabetes. Copyright © 2014. Published by Elsevier B.V.

Total cellular glycomics allows characterizing cells and streamlining the discovery process for cellular biomarkers.

PubMed

Fujitani, Naoki; Furukawa, Jun-ichi; Araki, Kayo; Fujioka, Tsuyoshi; Takegawa, Yasuhiro; Piao, Jinhua; Nishioka, Taiki; Tamura, Tomohiro; Nikaido, Toshio; Ito, Makoto; Nakamura, Yukio; Shinohara, Yasuro

2013-02-05

Although many of the frequently used pluripotency biomarkers are glycoconjugates, a glycoconjugate-based exploration of novel cellular biomarkers has proven difficult due to technical difficulties. This study reports a unique approach for the systematic overview of all major classes of oligosaccharides in the cellular glycome. The proposed method enabled mass spectrometry-based structurally intensive analyses, both qualitatively and quantitatively, of cellular N- and O-linked glycans derived from glycoproteins, glycosaminoglycans, and glycosphingolipids, as well as free oligosaccharides of human embryonic stem cells (hESCs), induced pluripotent stem cells (hiPSCs), and various human cells derived from normal and carcinoma cells. Cellular total glycomes were found to be highly cell specific, demonstrating their utility as unique cellular descriptors. Structures of glycans of all classes specifically observed in hESCs and hiPSCs tended to be immature in general, suggesting the presence of stem cell-specific glycosylation spectra. The current analysis revealed the high similarity of the total cellular glycome between hESCs and hiPSCs, although it was suggested that hESCs are more homogeneous than hiPSCs from a glycomic standpoint. Notably, this study enabled a priori identification of known pluripotency biomarkers such as SSEA-3, -4, and -5 and Tra-1-60/81, as well as a panel of glycans specifically expressed by hESCs and hiPSCs.

Transplant of Hepatocytes, Undifferentiated Mesenchymal Stem Cells, and In Vitro Hepatocyte-Differentiated Mesenchymal Stem Cells in a Chronic Liver Failure Experimental Model: A Comparative Study.

PubMed

El Baz, Hanan; Demerdash, Zeinab; Kamel, Manal; Atta, Shimaa; Salah, Faten; Hassan, Salwa; Hammam, Olfat; Khalil, Heba; Meshaal, Safa; Raafat, Inas

2018-02-01

Liver transplant is the cornerstone line of treatment for chronic liver diseases; however, the long list of complications and obstacles stand against this operation. Searching for new modalities for treatment of chronic liver illness is a must. In the present research, we aimed to compare the effects of transplant of undifferentiated human mesenchymal stem cells, in vitro differentiated mesenchymal stem cells, and adult hepatocytes in an experimental model of chronic liver failure. Undifferentiated human cord blood mesenchymal stem cells were isolated, pro-pagated, and characterized by morphology, gene expression analysis, and flow cytometry of surface markers and in vitro differentiated into hepatocyte-like cells. Rat hepatocytes were isolated by double perfusion technique. An animal model of chronic liver failure was developed, and undifferentiated human cord blood mesenchymal stem cells, in vitro hepato-genically differentiated mesenchymal stem cells, or freshly isolated rat hepatocytes were transplanted into a CCL4 cirrhotic experimental model. Animals were killed 3 months after transplant, and liver functions and histopathology were assessed. Compared with the cirrhotic control group, the 3 cell-treated groups showed improved alanine aminotransferase, aspartate aminotransferase, albumin, and bilirubin levels, with best results shown in the hepatocyte-treated group. Histopathologic examination of the treated groups showed improved fibrosis, with best results obtained in the undifferentiated mesenchymal stem cell-treated group. Both adult hepatocytes and cord blood mesenchymal stem cells proved to be promising candidates for cell-based therapy in liver regeneration on an experimental level. Improved liver function was evident in the hepatocyte-treated group, and fibrosis control was more evident in the undifferentiated mesenchymal stem cell-treated group.

Expansion and maintenance of human embryonic stem cell–derived endothelial cells by TGFβ inhibition is Id1 dependent

PubMed Central

James, Daylon; Nam, Hyung-song; Seandel, Marco; Nolan, Daniel; Janovitz, Tyler; Tomishima, Mark; Studer, Lorenz; Lee, Gabsang; Lyden, David; Benezra, Robert; Zaninovic, Nikica; Rosenwaks, Zev; Rabbany, Sina Y; Rafii, Shahin

2010-01-01

Previous efforts to differentiate human embryonic stem cells (hESCs) into endothelial cells have not achieved sustained expansion and stability of vascular cells. To define vasculogenic developmental pathways and enhance differentiation, we used an endothelial cell–specific VE-cadherin promoter driving green fluorescent protein (GFP) (hVPr-GFP) to screen for factors that promote vascular commitment. In phase 1 of our method, inhibition of transforming growth factor (TGF)β at day 7 of differentiation increases hVPr-GFP+ cells by tenfold. In phase 2, TGFβ inhibition maintains the proliferation and vascular identity of purified endothelial cells, resulting in a net 36-fold expansion of endothelial cells in homogenous monolayers, which exhibited a transcriptional profile of Id1highVEGFR2highVE-cadherin+ ephrinB2+. Using an Id1-YFP hESC reporter line, we showed that TGFβ inhibition sustains Id1 expression in hESC-derived endothelial cells and that Id1 is required for increased proliferation and preservation of endothelial cell commitment. Our approach provides a serum-free method for differentiation and long-term maintenance of hESC-derived endothelial cells at a scale relevant to clinical application. PMID:20081865

Expression of the Pokemon proto-oncogene in nasopharyngeal carcinoma cell lines and tissues.

PubMed

Jiao, Wei; Liu, Fei; Tang, Feng-Zhu; Lan, Jiao; Xiao, Rui-Ping; Chen, Xing-Zhou; Ye, Hui-Lan; Cai, Yong-Lin

2013-01-01

To study the differentiated expression of the proto-oncogene Pokemon in nasopharyngeal carcinoma (NPC) cell lines and tissues, mRNA and protein expression levels of CNE1, CNE2, CNE3 and C666-1 were detected separately by reverse transcription polymerase chain reaction (RT-PCR), real-time PCR and Western-blotting. The immortalized nasopharyngeal epithelial cell line NP69 was used as a control. The Pokemon protein expression level in biopsy specimens from chronic rhinitis patients and undifferentiated non keratinizing NPC patients was determined by Western-blotting and arranged from high to low: C666-1>CNE1>CNE2> CNE3>NP69. The Pokemon mRNA expression level was also arranged from high to low: CNE1>CNE2>NP69>C666-1>CNE3. Pokemon expression of NP69 and C666-1 obviously varied from mRNA to protein. The Pokemon protein level of NPC biopsy specimens was obviously higher than in chronic rhinitis. The data suggest that high Pokemon protein expression is closely associated with undifferentiated non-keratinizing NPC and may provide useful information for NPC molecular target therapy.

Generation of a KLF15 homozygous knockout human embryonic stem cell line using paired CRISPR/Cas9n, and human cardiomyocytes derivation.

PubMed

Noack, Claudia; Haupt, Luis Peter; Zimmermann, Wolfram-Hubertus; Streckfuss-Bömeke, Katrin; Zelarayán, Laura Cecilia

2017-08-01

Krueppel-like factor 15 (KLF15) is abundantly expressed in liver, kidney, and muscle, including myocardium. In the adult heart KLF15 is important to maintain homeostasis and to repress hypertrophic remodeling. We generated a homozygous hESC KLF15 knockout (KO) line using paired CRISPR/Cas9n. KLF15-KO cells maintained full pluripotency and differentiation potential as well as genomic integrity. We demonstrated that KLF15-KO cells can be differentiated into morphologically normal cardiomyocytes turning them into a valuable tool for studying human KLF15-mediated mechanisms resulting in human cardiac dysfunction. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

PHB Associates with the HIRA Complex to Control an Epigenetic-Metabolic Circuit in Human ESCs.

PubMed

Zhu, Zhexin; Li, Chunliang; Zeng, Yanwu; Ding, Jianyi; Qu, Zepeng; Gu, Junjie; Ge, Laixiang; Tang, Fan; Huang, Xin; Zhou, Chenlin; Wang, Ping; Zheng, Deyou; Jin, Ying

2017-02-02

The chromatin landscape and cellular metabolism both contribute to cell fate determination, but their interplay remains poorly understood. Using genome-wide siRNA screening, we have identified prohibitin (PHB) as an essential factor in self-renewal of human embryonic stem cells (hESCs). Mechanistically, PHB forms protein complexes with HIRA, a histone H3.3 chaperone, and stabilizes the protein levels of HIRA complex components. Like PHB, HIRA is required for hESC self-renewal. PHB and HIRA act together to control global deposition of histone H3.3 and gene expression in hESCs. Of particular note, PHB and HIRA regulate the chromatin architecture at the promoters of isocitrate dehydrogenase genes to promote transcription and, thus, production of α-ketoglutarate, a key metabolite in the regulation of ESC fate. Our study shows that PHB has an unexpected nuclear role in hESCs that is required for self-renewal and that it acts with HIRA in chromatin organization to link epigenetic organization to a metabolic circuit. Copyright © 2017 Elsevier Inc. All rights reserved.

Inhibition of IKK/NF-κB Signaling Enhances Differentiation of Mesenchymal Stromal Cells from Human Embryonic Stem Cells.

PubMed

Deng, Peng; Zhou, Chenchen; Alvarez, Ruth; Hong, Christine; Wang, Cun-Yu

2016-04-12

Embryonic stem cell-derived mesenchymal stromal cells (MSCs; also known as mesenchymal stem cells) represent a promising source for bone regenerative medicine. Despite remarkable advances in stem cell biology, the molecular mechanism regulating differentiation of human embryonic stem cells (hESCs) into MSCs remains poorly understood. Here, we report that inhibition of IκB kinase (IKK)/nuclear factor kappa B (NF-κB) signaling enhances differentiation of hESCs into MSCs by expediting the loss of pluripotent markers and increasing the expression of MSC surface markers. In addition, a significantly higher quantity of MSCs was produced from hESCs with IKK/NF-κB suppression. These isolated MSCs displayed evident multipotency with capacity to terminally differentiate into osteoblasts, chondrocytes, and adipocytes in vitro and to form bone in vivo. Collectively, our data provide important insights into the role of NF-κB in mesenchymal lineage specification during hESC differentiation, suggesting that IKK inhibitors could be utilized as an adjuvant in generating MSCs for cell-mediated therapies. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Use of RUNX2 Expression to Identify Osteogenic Progenitor Cells Derived from Human Embryonic Stem Cells

PubMed Central

Zou, Li; Kidwai, Fahad K.; Kopher, Ross A.; Motl, Jason; Kellum, Cory A.; Westendorf, Jennifer J.; Kaufman, Dan S.

2015-01-01

Summary We generated a RUNX2-yellow fluorescent protein (YFP) reporter system to study osteogenic development from human embryonic stem cells (hESCs). Our studies demonstrate the fidelity of YFP expression with expression of RUNX2 and other osteogenic genes in hESC-derived osteoprogenitor cells, as well as the osteogenic specificity of YFP signal. In vitro studies confirm that the hESC-derived YFP+ cells have similar osteogenic phenotypes to osteoprogenitor cells generated from bone-marrow mesenchymal stem cells. In vivo studies demonstrate the hESC-derived YFP+ cells can repair a calvarial defect in immunodeficient mice. Using the engineered hESCs, we monitored the osteogenic development and explored the roles of osteogenic supplements BMP2 and FGF9 in osteogenic differentiation of these hESCs in vitro. Taken together, this reporter system provides a novel system to monitor the osteogenic differentiation of hESCs and becomes useful to identify soluble agents and cell signaling pathways that mediate early stages of human bone development. PMID:25680477

FGF signaling via MAPK is required early and improves Activin A-induced definitive endoderm formation from human embryonic stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sui, Lina, E-mail: linasui@vub.ac.be; Mfopou, Josue K.; Geens, Mieke

2012-09-28

Highlights: Black-Right-Pointing-Pointer Deep study the FGF signaling role during DE specification in the context of hESCs. Black-Right-Pointing-Pointer DE differentiation from hESCs has an early dependence on FGF signaling. Black-Right-Pointing-Pointer A serum-free DE protocol is developed based on the findings. Black-Right-Pointing-Pointer The DE cells showed potential to differentiate into pancreatic progenitor cells. -- Abstract: Considering their unlimited proliferation and pluripotency properties, human embryonic stem cells (hESCs) constitute a promising resource applicable for cell replacement therapy. To facilitate this clinical translation, it is critical to study and understand the early stage of hESCs differentiation wherein germ layers are defined. In this study,more » we examined the role of FGF signaling in Activin A-induced definitive endoderm (DE) differentiation in the absence of supplemented animal serum. We found that activated FGF/MAPK signaling is required at the early time point of Activin A-induced DE formation. In addition, FGF activation increased the number of DE cells compared to Activin A alone. These DE cells could further differentiate into PDX1 and NKX6.1 positive pancreatic progenitors in vitro. We conclude that Activin A combined with FGF/MAPK signaling efficiently induce DE cells in the absence of serum. These findings improve our understanding of human endoderm formation, and constitute a step forward in the generation of clinical grade hESCs progenies for cell therapy.« less

Viral single-strand DNA induces p53-dependent apoptosis in human embryonic stem cells.

PubMed

Hirsch, Matthew L; Fagan, B Matthew; Dumitru, Raluca; Bower, Jacquelyn J; Yadav, Swati; Porteus, Matthew H; Pevny, Larysa H; Samulski, R Jude

2011-01-01

Human embryonic stem cells (hESCs) are primed for rapid apoptosis following mild forms of genotoxic stress. A natural form of such cellular stress occurs in response to recombinant adeno-associated virus (rAAV) single-strand DNA genomes, which exploit the host DNA damage response for replication and genome persistence. Herein, we discovered a unique DNA damage response induced by rAAV transduction specific to pluripotent hESCs. Within hours following rAAV transduction, host DNA damage signaling was elicited as measured by increased gamma-H2AX, ser15-p53 phosphorylation, and subsequent p53-dependent transcriptional activation. Nucleotide incorporation assays demonstrated that rAAV transduced cells accumulated in early S-phase followed by the induction of apoptosis. This lethal signaling sequalae required p53 in a manner independent of transcriptional induction of Puma, Bax and Bcl-2 and was not evident in cells differentiated towards a neural lineage. Consistent with a lethal DNA damage response induced upon rAAV transduction of hESCs, empty AAV protein capsids demonstrated no toxicity. In contrast, DNA microinjections demonstrated that the minimal AAV origin of replication and, in particular, a 40 nucleotide G-rich tetrad repeat sequence, was sufficient for hESC apoptosis. Our data support a model in which rAAV transduction of hESCs induces a p53-dependent lethal response that is elicited by a telomeric sequence within the AAV origin of replication.

A Regulatory Network Involving β‐Catenin, e‐Cadherin, PI3k/Akt, and Slug Balances Self‐Renewal and Differentiation of Human Pluripotent Stem Cells In Response to Wnt Signaling

PubMed Central

Huang, Tyng‐Shyan; Li, Li; Moalim‐Nour, Lilian; Jia, Deyong; Bai, Jian; Yao, Zemin; Bennett, Steffany A. L.; Figeys, Daniel

2015-01-01

Abstract The mechanisms underlying disparate roles of the canonical Wnt signaling pathway in maintaining self‐renewal or inducing differentiation and lineage specification in embryonic stem cells (ESCs) are not clear. In this study, we provide the first demonstration that self‐renewal versus differentiation of human ESCs (hESCs) in response to Wnt signaling is predominantly determined by a two‐layer regulatory circuit involving β‐catenin, E‐cadherin, PI3K/Akt, and Slug in a time‐dependent manner. Short‐term upregulation of β‐catenin does not lead to the activation of T‐cell factor (TCF)‐eGFP Wnt reporter in hESCs. Instead, it enhances E‐cadherin expression on the cell membrane, thereby enhancing hESC self‐renewal through E‐cadherin‐associated PI3K/Akt signaling. Conversely, long‐term Wnt activation or loss of E‐cadherin intracellular β‐catenin binding domain induces TCF‐eGFP activity and promotes hESC differentiation through β‐catenin‐induced upregulation of Slug. Enhanced expression of Slug leads to a further reduction of E‐cadherin that serves as a β‐catenin “sink” sequestering free cytoplasmic β‐catenin. The formation of such a framework reinforces hESCs to switch from a state of temporal self‐renewal associated with short‐term Wnt/β‐catenin activation to definitive differentiation. Stem Cells 2015;33:1419–1433 PMID:25538040

A Regulatory Network Involving β-Catenin, e-Cadherin, PI3k/Akt, and Slug Balances Self-Renewal and Differentiation of Human Pluripotent Stem Cells In Response to Wnt Signaling.

PubMed

Huang, Tyng-Shyan; Li, Li; Moalim-Nour, Lilian; Jia, Deyong; Bai, Jian; Yao, Zemin; Bennett, Steffany A L; Figeys, Daniel; Wang, Lisheng

2015-05-01

The mechanisms underlying disparate roles of the canonical Wnt signaling pathway in maintaining self-renewal or inducing differentiation and lineage specification in embryonic stem cells (ESCs) are not clear. In this study, we provide the first demonstration that self-renewal versus differentiation of human ESCs (hESCs) in response to Wnt signaling is predominantly determined by a two-layer regulatory circuit involving β-catenin, E-cadherin, PI3K/Akt, and Slug in a time-dependent manner. Short-term upregulation of β-catenin does not lead to the activation of T-cell factor (TCF)-eGFP Wnt reporter in hESCs. Instead, it enhances E-cadherin expression on the cell membrane, thereby enhancing hESC self-renewal through E-cadherin-associated PI3K/Akt signaling. Conversely, long-term Wnt activation or loss of E-cadherin intracellular β-catenin binding domain induces TCF-eGFP activity and promotes hESC differentiation through β-catenin-induced upregulation of Slug. Enhanced expression of Slug leads to a further reduction of E-cadherin that serves as a β-catenin "sink" sequestering free cytoplasmic β-catenin. The formation of such a framework reinforces hESCs to switch from a state of temporal self-renewal associated with short-term Wnt/β-catenin activation to definitive differentiation. Stem Cells 2015;33:1419-1433. © 2015 AlphaMed Press.

Potential for pharmacological manipulation of human embryonic stem cells

PubMed Central

Atkinson, Stuart P; Lako, Majlinda; Armstrong, Lyle

2013-01-01

The therapeutic potential of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) is vast, allowing disease modelling, drug discovery and testing and perhaps most importantly regenerative therapies. However, problems abound; techniques for cultivating self-renewing hESCs tend to give a heterogeneous population of self-renewing and partially differentiated cells and general include animal-derived products that can be cost-prohibitive for large-scale production, and effective lineage-specific differentiation protocols also still remain relatively undefined and are inefficient at producing large amounts of cells for therapeutic use. Furthermore, the mechanisms and signalling pathways that mediate pluripotency and differentiation are still to be fully appreciated. However, over the recent years, the development/discovery of a range of effective small molecule inhibitors/activators has had a huge impact in hESC biology. Large-scale screening techniques, coupled with greater knowledge of the pathways involved, have generated pharmacological agents that can boost hESC pluripotency/self-renewal and survival and has greatly increased the efficiency of various differentiation protocols, while also aiding the delineation of several important signalling pathways. Within this review, we hope to describe the current uses of small molecule inhibitors/activators in hESC biology and their potential uses in the future. LINKED ARTICLES This article is part of a themed section on Regenerative Medicine and Pharmacology: A Look to the Future. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.169.issue-2 PMID:22515554

Rho/ROCK pathway is essential to the expansion, differentiation, and morphological rearrangements of human neural stem/progenitor cells induced by lysophosphatidic acid.

PubMed

Frisca, Frisca; Crombie, Duncan E; Dottori, Mirella; Goldshmit, Yona; Pébay, Alice

2013-05-01

We previously reported that lysophosphatidic acid (LPA) inhibits the neuronal differentiation of human embryonic stem cells (hESC). We extended these studies by analyzing LPA's effects on the expansion of neural stem/progenitor cells (NS/PC) derived from hESCs and human induced pluripotent stem cells (iPSC), and we assessed whether data obtained on the neural differentiation of hESCs were relevant to iPSCs. We showed that hESCs and iPSCs exhibited comparable mRNA expression profiles of LPA receptors and producing enzymes upon neural differentiation. We demonstrated that LPA inhibited the expansion of NS/PCs of both origins, mainly by increased apoptosis in a Rho/Rho-associated kinase (ROCK)-dependent mechanism. Furthermore, LPA inhibited the neuronal differentiation of iPSCs. Lastly, LPA induced neurite retraction of NS/PC-derived early neurons through Rho/ROCK, which was accompanied by myosin light chain (MLC) phosphorylation. Our data demonstrate the consistency of LPA effects across various sources of human NS/PCs, rendering hESCs and iPSCs valuable models for studying lysophospholipid signaling in human neural cells. Our data also highlight the importance of the Rho/ROCK pathway in human NS/PCs. As LPA levels are increased in the central nervous system (CNS) following injury, LPA-mediated effects on NS/PCs and early neurons could contribute to the poor neurogenesis observed in the CNS following injury.

Temporal Impact of Substrate Mechanics on Differentiation of Human Embryonic Stem Cells to Cardiomyocytes

PubMed Central

Hazeltine, Laurie B.; Badur, Mehmet G.; Lian, Xiaojun; Das, Amritava; Han, Wenqing; Palecek, Sean P.

2014-01-01

A significant clinical need exists to differentiate human pluripotent stem cells (hPSCs) into cardiomyocytes, enabling tissue modeling for in vitro discovery of new drugs or cell-based therapies for heart repair in vivo. Chemical and mechanical microenvironmental factors are known to impact efficiency of stem cell differentiation, but cardiac differentiation protocols in hPSCs are typically performed on rigid tissue culture polystyrene (TCPS) surfaces which do not present a physiological mechanical setting. To investigate the temporal effects of mechanics on cardiac differentiation, we cultured human embryonic stem cells (hESCs) and their derivatives on polyacrylamide hydrogel substrates with a physiologically relevant range of stiffnesses. In directed differentiation and embryoid body culture systems, differentiation of hESCs to cardiac Troponin T-expressing (cTnT+) cardiomyocytes peaked on hydrogels of intermediate stiffness. Brachyury expression also peaked on intermediate stiffness hydrogels at day 1 of directed differentiation, suggesting that stiffness impacted the initial differentiation trajectory of hESCs to mesendoderm. To investigate the impact of substrate mechanics during cardiac specification of mesodermal progenitors, we initiated directed cardiomyocyte differentiation on TCPS and transferred cells to hydrogels at the Nkx2.5/Isl1+ cardiac progenitor cell stage. No differences in cardiomyocyte purity with stiffness were observed on day 15. These experiments indicate that differentiation of hESCs is sensitive to substrate mechanics at early stages of mesodermal induction, and proper application of substrate mechanics can increase the propensity of hESCs to differentiate to cardiomyocytes. PMID:24200714

Fibrinogen Induces RUNX2 Activity and Osteogenic Development from Human Pluripotent Stem Cells

PubMed Central

Kidwai, Fahad; Edwards, Jessica; Zou, Li; Kaufman, Dan S.

2016-01-01

Pluripotent stem cells, both human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC), provide an important resource to produce specialized cells such as osteogenic cells for therapeutic applications such as repair or replacement of injured, diseased or damaged bone. hESCs and iPSCs can also be used to better define basic cellular and genetic mechanisms that regulate the earliest stages of human bone development. However, current strategies to mediate osteogenic differentiation of hESC and iPSC are typically limited by the use of xenogeneic components such as fetal bovine serum (FBS) that make defining specific agents that mediate human osteogenesis difficult. Runt-related transcription factor 2 (RUNX2) is a key regulator required for osteogenic differentiation. Here, we used a RUNX2-YFP reporter system to characterize the novel ability of fibrinogen to mediate human osteogenic development from hESC and iPSC in defined (serum-free) conditions. These studies demonstrate that fibrinogen mediates significant osteo-induction potential. Specifically, fibrinogen binds to the surface integrin (α9β1) to mediate RUNX2 gene expression through the SMAD1/5/8 signaling pathway. Additional studies characterize the fibrinogen-induced hESC/iPSC-derived osteogenic cells to demonstrate these osteogenic cells retain the capacity to express typical mature osteoblastic markers. Together, these studies define a novel fibrinogen-α9β1-SMAD1/5/8-RUNX2 signaling axis can efficiently induce osteogenic differentiation from hESCs and iPSCs. PMID:27331788

Effects of all-trans-retinoic acid on human SH-SY5Y neuroblastoma as in vitro model in neurotoxicity research.

PubMed

Cheung, Yuen-Ting; Lau, Way Kwok-Wai; Yu, Man-Shan; Lai, Cora Sau-Wan; Yeung, Sze-Chun; So, Kwok-Fai; Chang, Raymond Chuen-Chung

2009-01-01

Human neuroblastoma SH-SY5Y is a dopaminergic neuronal cell line which has been used as an in vitro model for neurotoxicity experiments. Although the neuroblastoma is usually differentiated by all-trans-retinoic acid (RA), both RA-differentiated and undifferentiated SH-SY5Y cells have been used in neuroscience research. However, the changes in neuronal properties triggered by RA as well as the subsequent responsiveness to neurotoxins have not been comprehensively studied. Therefore, we aim to re-evaluate the differentiation property of RA on this cell line. We hypothesize that modulation of signaling pathways and neuronal properties during RA-mediated differentiation in SH-SY5Y cells can affect their susceptibility to neurotoxins. The differentiation property of RA was confirmed by showing an extensive outgrowth of neurites, increased expressions of neuronal nuclei, neuron specific enolase, synaptophysin and synaptic associated protein-97, and decreased expression of inhibitor of differentiation-1. While undifferentiated SH-SY5Y cells were susceptible to 6-OHDA and MPP+, RA-differentiation conferred SH-SY5Y cells higher tolerance, potentially by up-regulating survival signaling, including Akt pathway as inhibition of Akt removed RA-induced neuroprotection against 6-OHDA. As a result, the real toxicity cannot be revealed in RA-differentiated cells. Therefore, undifferentiated SH-SY5Y is more appropriate for studying neurotoxicity or neuroprotection in experimental Parkinson's disease research.

The neuronal differentiation process involves a series of antioxidant proteins.

PubMed

Oh, J-E; Karlmark Raja, K; Shin, J-H; Hengstschläger, M; Pollak, A; Lubec, G

2005-11-01

Involvement of individual antioxidant proteins (AOXP) and antioxidants in the differentiation process has been already reported. A systematic search strategy for detecting differentially regulated AOXP in neuronal differentiation, however, has not been published so far. The aim of this study was to provide an analytical tool identifying AOXP and to generate a differentiation-related AOXP expressional pattern. The undifferentiated N1E-115 neuroblastoma cell line was switched into a neuronal phenotype by DMSO treatment and used for proteomic experiments: We used two-dimensional gel electrophoresis followed by unambiguous mass spectrometrical (MALDI-TOF-TOF) identification of proteins to generate a map of AOXP. 16 AOXP were unambiguously determined in both cell lines; catalase, thioredoxin domain-containing protein 4 and hypothetical glutaredoxin/glutathione S-transferase C terminus-containing protein were detectable in the undifferentiated cells only. Five AOXP were observed in both, undifferentiated and differentiated cells and thioredoxin, thioredoxin-like protein p19, thioredoxin reductase 1, superoxide dismutases (Mn and Cu-Zn), glutathione synthetase, glutathione S-transferase P1 and Mu1 were detected in differentiated cells exclusively. Herein a differential expressional pattern is presented that reveals so far unpublished antioxidant principles involved in neuronal differentiation by a protein chemical approach, unambiguously identifying AOXP. This finding not only shows concomitant determination of AOXP but also serves as an analytical tool and forms the basis for design of future studies addressing AOXP and differentiation per se.

Reprogramming somatic cells into iPS cells activates LINE-1 retroelement mobility

PubMed Central

Wissing, Silke; Muñoz-Lopez, Martin; Macia, Angela; Yang, Zhiyuan; Montano, Mauricio; Collins, William; Garcia-Perez, Jose Luis; Moran, John V.; Greene, Warner C.

2012-01-01

Long interspersed element-1 (LINE-1 or L1) retrotransposons account for nearly 17% of human genomic DNA and represent a major evolutionary force that has reshaped the structure and function of the human genome. However, questions remain concerning both the frequency and the developmental timing of L1 retrotransposition in vivo and whether the mobility of these retroelements commonly results in insertional and post-insertional mechanisms of genomic injury. Cells exhibiting high rates of L1 retrotransposition might be especially at risk for such injury. We assessed L1 mRNA expression and L1 retrotransposition in two biologically relevant cell types, human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), as well as in control parental human dermal fibroblasts (HDFs). Full-length L1 mRNA and the L1 open reading frame 1-encoded protein (ORF1p) were readily detected in hESCs and iPSCs, but not in HDFs. Sequencing analysis proved the expression of human-specific L1 element mRNAs in iPSCs. Bisulfite sequencing revealed that the increased L1 expression observed in iPSCs correlates with an overall decrease in CpG methylation in the L1 promoter region. Finally, retrotransposition of an engineered human L1 element was ∼10-fold more efficient in iPSCs than in parental HDFs. These findings indicate that somatic cell reprogramming is associated with marked increases in L1 expression and perhaps increases in endogenous L1 retrotransposition, which could potentially impact the genomic integrity of the resultant iPSCs. PMID:21989055

Expand and Regularize Federal Funding for Human Pluripotent Stem Cell Research

ERIC Educational Resources Information Center

Owen-Smith, Jason; Scott, Christopher Thomas; McCormick, Jennifer B.

2012-01-01

Human embryonic stem cell (hESC) research has sparked incredible scientific and public excitement, as well as significant controversy. hESCs are pluripotent, which means, in theory, that they can be differentiated into any type of cell found in the human body. Thus, they evoke great enthusiasm about potential clinical applications. They are…

Stem cell research ethics: consensus statement on emerging issues.

PubMed

Caulfield, Timothy; Ogbogu, Ubaka; Nelson, Erin; Einsiedel, Edna; Knoppers, Bartha; McDonald, Michael; Brunger, Fern; Downey, Robin; Fernando, Kanchana; Galipeau, Jacques; Geransar, Rose; Griener, Glenn; Grenier, Glenn; Hyun, Insoo; Isasi, Rosario; Kardel, Melanie; Knowles, Lori; Kucic, Terrence; Lotjonen, Salla; Lyall, Drew; Magnus, David; Mathews, Debra J H; Nisbet, Matthew; Nisker, Jeffrey; Pare, Guillaume; Pattinson, Shaun; Pullman, Daryl; Rudnicki, Michael; Williams-Jones, Bryn; Zimmerman, Susan

2007-10-01

This article is a consensus statement by an international interdisciplinary group of academic experts and Canadian policy-makers on emerging ethical, legal and social issues in human embryonic stem cells (hESC) research in Canada. The process of researching consensus included consultations with key stakeholders in hESC research (regulations, stem cell researchers, and research ethics experts), preparation and distribution of background papers, and an international workshop held in Montreal in February 2007 to discuss the papers and debate recommendations. The recommendations provided in the consensus statement focus on issues of immediate relevance to Canadian policy-makers, including informed consent to hESC research, the use of fresh embryos in research, management of conflicts of interest, and the relevance of public opinion research to policy-making.

Scaffolding for Three-Dimensional Embryonic Vasculogenesis

NASA Astrophysics Data System (ADS)

Kraehenbuehl, Thomas P.; Aday, Sezin; Ferreira, Lino S.

Biomaterial scaffolds have great potential to support efficient vascular differentiation of embryonic stem cells. Vascular cell fate-specific biochemical and biophysical cues have been identified and incorporated into three-dimensional (3D) biomaterials to efficiently direct embryonic vasculogenesis. The resulting vascular-like tissue can be used for regenerative medicine applications, further elucidation of biophysical and biochemical cues governing vasculogenesis, and drug discovery. In this chapter, we give an overview on the following: (1) developmental cues for directed differentiation of human embryonic stem cells (hESCs) into vascular cells, (2) 3D vascular differentiation in embryoid bodies (EBs), (3) preparation of 3D scaffolds for the vascular differentiation of hESCs, and (4) the most significant studies combining scaffolding and hESCs for development of vascular-like tissue.

Children's Developing Conceptions of the Mind and Brain.

ERIC Educational Resources Information Center

Johnson, Carl Nils; Wellman, Henry M.

1982-01-01

The development of concepts of both the mind and brain is examined in subjects from preschool age through adulthood. While young children begin with undifferentiated conceptions of the mind and brain, in subsequent developments these concepts are differentiated along ontological and functional lines. (Author/RH)

Differentiation of neuroblastoma cell line N1E-115 involves several signaling cascades.

PubMed

Oh, Ji-eun; Karlmark, Karlin Raja; Shin, Joo-ho; Pollak, Arnold; Freilinger, Angelika; Hengstschläger, Markus; Lubec, Gert

2005-03-01

No systematic searches for differential expression of signaling proteins (SP) in undifferentiated vs. differentiated cell lineages were published and herein we used protein profiling for this purpose. The NIE-115 cell line was cultivated and an aliquot was differentiated with dimethylsulfoxide (DMSO), that is known to lead to a neuronal phenotype. Cell lysates were prepared, run on two-dimensional gel electrophoresis followed by MALDI-TOF-TOF identification of proteins and maps of identified SPs were generated. Seven SPs were comparable, 27 SPs: GTP-binding/Ras-related proteins, kinases, growth factors, calcium binding proteins, phosphatase-related proteins were observed in differentiated NIE-115 cells and eight SPs of the groups mentioned above were observed in undifferentiated cells only. Switching-on/off of several individual SPs from different signaling cascades during the differentiation process is a key to understand mechanisms involved. The findings reported herein are challenging in vitro and in vivo studies to confirm a functional role for deranged SPs.

A model of early human embryonic stem cell differentiation reveals inter- and intracellular changes on transition to squamous epithelium.

PubMed

Galat, Vasiliy; Malchenko, Sergey; Galat, Yekaterina; Ishkin, Alex; Nikolsky, Yuri; Kosak, Steven T; Soares, Bento Marcelo; Iannaccone, Philip; Crispino, John D; Hendrix, Mary J C

2012-05-20

The molecular events leading to human embryonic stem cell (hESC) differentiation are the subject of considerable scrutiny. Here, we characterize an in vitro model that permits analysis of the earliest steps in the transition of hESC colonies to squamous epithelium on basic fibroblast growth factor withdrawal. A set of markers (GSC, CK18, Gata4, Eomes, and Sox17) point to a mesendodermal nature of the epithelial cells with subsequent commitment to definitive endoderm (Sox17, Cdx2, nestin, and Islet1). We assayed alterations in the transcriptome in parallel with the distribution of immunohistochemical markers. Our results indicate that the alterations of tight junctions in pluripotent culture precede the beginning of differentiation. We defined this cell population as "specified," as it is committed toward differentiation. The transitional zone between "specified" pluripotent and differentiated cells displays significant up-regulation of keratin-18 (CK18) along with a decrease in the functional activity of gap junctions and the down-regulation of 2 gap junction proteins, connexin 43 (Cx43) and connexin 45 (Cx45), which is coincidental with substantial elevation of intracellular Ca2+ levels. These findings reveal a set of cellular changes that may represent the earliest markers of in vitro hESC transition to an epithelial phenotype, before the induction of gene expression networks that guide hESC differentiation. Moreover, we hypothesize that these events may be common during the primary steps of hESC commitment to functionally varied epithelial tissue derivatives of different embryological origins.

A Model of Early Human Embryonic Stem Cell Differentiation Reveals Inter- and Intracellular Changes on Transition to Squamous Epithelium

PubMed Central

Malchenko, Sergey; Galat, Yekaterina; Ishkin, Alex; Nikolsky, Yuri; Kosak, Steven T.; Soares, Bento Marcelo; Iannaccone, Philip; Crispino, John D.; Hendrix, Mary J.C.

2012-01-01

The molecular events leading to human embryonic stem cell (hESC) differentiation are the subject of considerable scrutiny. Here, we characterize an in vitro model that permits analysis of the earliest steps in the transition of hESC colonies to squamous epithelium on basic fibroblast growth factor withdrawal. A set of markers (GSC, CK18, Gata4, Eomes, and Sox17) point to a mesendodermal nature of the epithelial cells with subsequent commitment to definitive endoderm (Sox17, Cdx2, nestin, and Islet1). We assayed alterations in the transcriptome in parallel with the distribution of immunohistochemical markers. Our results indicate that the alterations of tight junctions in pluripotent culture precede the beginning of differentiation. We defined this cell population as “specified,” as it is committed toward differentiation. The transitional zone between “specified” pluripotent and differentiated cells displays significant up-regulation of keratin-18 (CK18) along with a decrease in the functional activity of gap junctions and the down-regulation of 2 gap junction proteins, connexin 43 (Cx43) and connexin 45 (Cx45), which is coincidental with substantial elevation of intracellular Ca2+ levels. These findings reveal a set of cellular changes that may represent the earliest markers of in vitro hESC transition to an epithelial phenotype, before the induction of gene expression networks that guide hESC differentiation. Moreover, we hypothesize that these events may be common during the primary steps of hESC commitment to functionally varied epithelial tissue derivatives of different embryological origins. PMID:21861759

Aromatase inhibitor regulates let-7 expression and let-7f induced cell migration in endometrial cells from women with endometriosis

PubMed Central

Cho, SiHyun; Mutlu, Levent; Zhou, Yuping; Taylor, Hugh S.

2018-01-01

Objectives To evaluate associations between aromatase inhibitor (AI) treatment and let-7 family microRNA expression in endometriosis. Design In vitro study using Ishikawa cells and human endometrial stromal cells (HESC) obtained from patients with endometriosis Setting University research center. Patients Women undergoing laparoscopic surgery for endometriosis Interventions HESCs and Ishikawa cells treated with various letrozol concentrations and transfected with a mimic of let-7 subtypes of interest Main Outcome Measures microRNAs let7a-f and aromatase expression were evaluated. Migration potential after transfection with a let-7f mimic were analyzed. Results After letrozole treatment for 48 hours, all let-7 subtypes showed a trend toward increased expression in a dose dependent manner in Ishikawa cells, and significant differences were found in let-7b and let-7f between controls and the 20 μmol/L treated groups. Further, let-7f showed significant differences between control and 1.0 μmol/L treatment group, a typical therapeutic level, in HESCs. Transfection of a let-7f mimic decreased aromatase expression in both Ishikawa cells and HESC, and led to a significant decrease in number of migrating cells in both cell types. Conclusions AI treatment significantly increased expression of let-7f in Ishikawa cells and HESCs from patient with endometriosis; increased lef-7f expression effectively reduced the migration of endometrial cells. Modulation of miRNAs involved in the pathogenesis of endometriosis may have therapeutic potential for endometriosis. PMID:27320036

Temporal impact of substrate mechanics on differentiation of human embryonic stem cells to cardiomyocytes.

PubMed

Hazeltine, Laurie B; Badur, Mehmet G; Lian, Xiaojun; Das, Amritava; Han, Wenqing; Palecek, Sean P

2014-02-01

A significant clinical need exists to differentiate human pluripotent stem cells (hPSCs) into cardiomyocytes, enabling tissue modeling for in vitro discovery of new drugs or cell-based therapies for heart repair in vivo. Chemical and mechanical microenvironmental factors are known to impact the efficiency of stem cell differentiation, but cardiac differentiation protocols in hPSCs are typically performed on rigid tissue culture polystyrene (TCPS) surfaces, which do not present a physiological mechanical setting. To investigate the temporal effects of mechanics on cardiac differentiation, we cultured human embryonic stem cells (hESCs) and their derivatives on polyacrylamide hydrogel substrates with a physiologically relevant range of stiffnesses. In directed differentiation and embryoid body culture systems, differentiation of hESCs to cardiac troponin T-expressing (cTnT+) cardiomyocytes peaked on hydrogels of intermediate stiffness. Brachyury expression also peaked on intermediate stiffness hydrogels at day 1 of directed differentiation, suggesting that stiffness impacted the initial differentiation trajectory of hESCs to mesendoderm. To investigate the impact of substrate mechanics during cardiac specification of mesodermal progenitors, we initiated directed cardiomyocyte differentiation on TCPS and transferred cells to hydrogels at the Nkx2.5/Isl1+ cardiac progenitor cell stage. No differences in cardiomyocyte purity with stiffness were observed on day 15. These experiments indicate that differentiation of hESCs is sensitive to substrate mechanics at early stages of mesodermal induction, and proper application of substrate mechanics can increase the propensity of hESCs to differentiate to cardiomyocytes. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

A homozygous Keap1-knockout human embryonic stem cell line generated using CRISPR/Cas9 mediates gene targeting.

PubMed

Kim, So-Jung; Habib, Omer; Kim, Jin-Soo; Han, Hyo-Won; Koo, Soo Kyung; Kim, Jung-Hyun

2017-03-01

Kelch-like ECH-associated protein 1 (keap1) is a cysteine-rich protein that interacts with transcription factor Nrf2 in a redox-sensitive manner, leading to the degradation of Nrf2 (Kim et al., 2014a). Disruption of Keap1 results in the induction of Nrf2-related signaling pathways involving the expression of a set of anti-oxidant and anti-inflammatory genes. We generated biallelic mutants of the Keap1 gene using a CRISPR-Cas9 genome editing method in the H9 human embryonic stem cell (hESC). The Keap1 homozygous-knockout H9 cell line retained normal morphology, gene expression, and in vivo differentiation potential. Copyright © 2016 The Author(s). Published by Elsevier B.V. All rights reserved.

Transforming Growth Factor β1 (TGFβ1) and Progesterone Regulate Matrix Metalloproteinases (MMP) in Human Endometrial Stromal Cells

PubMed Central

Itoh, Hiroko; Kishore, Annavarapu Hari; Lindqvist, Annika; Rogers, David E.

2012-01-01

Context: Menstruation is preceded by progesterone withdrawal and endometrial matrix remodeling predominantly through induction of matrix metalloproteinases (MMP) and recruitment of invading neutrophils. Design: Using endometrial tissues from women during various phases of the menstrual cycle, we found that MMP2, MMP9, and MMP11 were up-regulated in the late secretory phase/premenstrual phase. Because TGFβ-responsive genes were also up-regulated in endometrium during this time, we tested the hypothesis that TGFβ1 and progesterone regulate expression of MMP in human endometrial stromal cells (HESC). Results: Treatment of HESC with TGFβ1 resulted in marked increases in MMP2 and MMP11 mRNA and pro- and active MMP2 activity. Progesterone inhibited TGFβ1-induced stimulation of MMP2 and MMP11 through its nuclear hormone receptors. Interestingly, TGFβ1 also decreased progesterone receptor (PR)-A and PR-B in HESC with a more pronounced effect on PR-A. Conclusions: These data support the hypothesis that TGFβ1 has endogenous anti-progestational effects in HESC and that the opposing effects of progesterone and TGFβ1 are important in regulation of matrix integrity in human endometrium. PMID:22466340

Human stem cell neuronal differentiation on silk-carbon nanotube composite

NASA Astrophysics Data System (ADS)

Chen, Chi-Shuo; Soni, Sushant; Le, Catherine; Biasca, Matthew; Farr, Erik; Chen, Eric Y.-T.; Chin, Wei-Chun

2012-02-01

Human embryonic stem cells [hESCs] are able to differentiate into specific lineages corresponding to regulated spatial and temporal signals. This unique attribute holds great promise for regenerative medicine and cell-based therapy for many human diseases such as spinal cord injury [SCI] and multiple sclerosis [MS]. Carbon nanotubes [CNTs] have been successfully used to promote neuronal differentiation, and silk has been widely applied in tissue engineering. This study aims to build silk-CNT composite scaffolds for improved neuron differentiation efficiency from hESCs. Two neuronal markers (β-III tubulin and nestin) were utilized to determine the hESC neuronal lineage differentiation. In addition, axonal lengths were measured to evaluate the progress of neuronal development. The results demonstrated that cells on silk-CNT scaffolds have a higher β-III tubulin and nestin expression, suggesting augmented neuronal differentiation. In addition, longer axons with higher density were found to associate with silk-CNT scaffolds. Our silk-CNT-based composite scaffolds can promote neuronal differentiation of hESCs. The silk-CNT composite scaffolds developed here can serve as efficient supporting matrices for stem cell-derived neuronal transplants, offering a promising opportunity for nerve repair treatments for SCI and MS patients.

A simple and economical route to generate functional hepatocyte-like cells from hESCs and their application in evaluating alcohol induced liver damage.

PubMed

Pal, Rajarshi; Mamidi, Murali Krishna; Das, Anjan Kumar; Gupta, Pawan Kumar; Bhonde, Ramesh

2012-01-01

The in vitro derived hepatocytes from human embryonic stem cells (hESC) is a promising tool to acquire improved knowledge of the cellular and molecular events underlying early human liver development under physiological and pathological conditions. Here we report a simple two-step protocol employing conditioned medium (CM) from human hepatocellular carcinoma cell line, HepG2 to generate functional hepatocyte-like cells from hESC. Immunocytochemistry, flow cytometry, quantitative RT-PCR, and biochemical analyses revealed that the endodermal progenitors appeared as pockets in culture, and the cascade of genes associated with the formation of definitive endoderm (HNF-3β, SOX-17, DLX-5, CXCR4) was consistent and in concurrence with the up-regulation of the markers for hepatic progenitors [alpha-feto protein (AFP), HNF-4α, CK-19, albumin, alpha-1-antitrypsin (AAT)], followed by maturation into functional hepatocytes [tyrosine transferase (TAT), tryptophan-2, 3-dioxygenase (TDO), glucose 6-phosphate (G6P), CYP3A4, CYP7A1]. We witnessed that the gene expression profile during this differentiation process recapitulated in vivo liver development demonstrating a gradual down-regulation of extra embryonic endodermal markers (SOX-7, HNF-1β, SNAIL-1, LAMININ-1, CDX2), and the generated hepatic cells performed multiple liver functions. Since prenatal alcohol exposure is known to provoke irreversible abnormalities in the fetal cells and developing tissues, we exposed in vitro generated hepatocytes to ethanol (EtOH) and found that EtOH treatment not only impairs the survival and proliferation, but also induces apoptosis and perturbs differentiation of progenitor cells into hepatocytes. This disruption was accompanied by alterations in the expression of genes and proteins involved in hepatogenesis. Our results provide new insights into the wider range of destruction caused by alcohol on the dynamic process of liver organogenesis. Copyright © 2011 Wiley Periodicals, Inc.

Resveratrol Ameliorates the Maturation Process of β-Cell-Like Cells Obtained from an Optimized Differentiation Protocol of Human Embryonic Stem Cells

PubMed Central

Pezzolla, Daniela; López-Beas, Javier; Lachaud, Christian C.; Domínguez-Rodríguez, Alejandro; Smani, Tarik; Hmadcha, Abdelkrim; Soria, Bernat

2015-01-01

Human embryonic stem cells (hESCs) retain the extraordinary capacity to differentiate into different cell types of an adult organism, including pancreatic β-cells. For this particular lineage, although a lot of effort has been made in the last ten years to achieve an efficient and reproducible differentiation protocol, it was not until recently that this aim was roughly accomplished. Besides, several studies evidenced the impact of resveratrol (RSV) on insulin secretion, even though the mechanism by which this polyphenol potentiates glucose-stimulated insulin secretion (GSIS) is still not clear. The aim of this study was to optimize an efficient differentiation protocol that mimics in vivo pancreatic organogenesis and to investigate whether RSV may improve the final maturation step to obtain functional insulin-secreting cells. Our results indicate that treatment of hESCs (HS-181) with activin-A induced definitive endoderm differentiation as detected by the expression of SOX17 and FOXA2. Addition of retinoic acid (RA), Noggin and Cyclopamine promoted pancreatic differentiation as indicated by the expression of the early pancreatic progenitor markers ISL1, NGN3 and PDX1. Moreover, during maturation in suspension culture, differentiating cells assembled in islet-like clusters, which expressed specific endocrine markers such as PDX1, SST, GCG and INS. Similar results were confirmed with the human induced Pluripotent Stem Cell (hiPSC) line MSUH-001. Finally, differentiation protocols incorporating RSV treatment yielded numerous insulin-positive cells, induced significantly higher PDX1 expression and were able to transiently normalize glycaemia when transplanted in streptozotocin (STZ) induced diabetic mice thus promoting its survival. In conclusion, our strategy allows the efficient differentiation of hESCs into pancreatic endoderm capable of generating β-cell-like cells and demonstrates that RSV improves the maturation process. PMID:25774684

Protein-based human iPS cells efficiently generate functional dopamine neurons and can treat a rat model of Parkinson disease.

PubMed

Rhee, Yong-Hee; Ko, Ji-Yun; Chang, Mi-Yoon; Yi, Sang-Hoon; Kim, Dohoon; Kim, Chun-Hyung; Shim, Jae-Won; Jo, A-Young; Kim, Byung-Woo; Lee, Hyunsu; Lee, Suk-Ho; Suh, Wonhee; Park, Chang-Hwan; Koh, Hyun-Chul; Lee, Yong-Sung; Lanza, Robert; Kim, Kwang-Soo; Lee, Sang-Hun

2011-06-01

Parkinson disease (PD) involves the selective loss of midbrain dopamine (mDA) neurons and is a possible target disease for stem cell-based therapy. Human induced pluripotent stem cells (hiPSCs) are a potentially unlimited source of patient-specific cells for transplantation. However, it is critical to evaluate the safety of hiPSCs generated by different reprogramming methods. Here, we compared multiple hiPSC lines derived by virus- and protein-based reprogramming to human ES cells (hESCs). Neuronal precursor cells (NPCs) and dopamine (DA) neurons delivered from lentivirus-based hiPSCs exhibited residual expression of exogenous reprogramming genes, but those cells derived from retrovirus- and protein-based hiPSCs did not. Furthermore, NPCs derived from virus-based hiPSCs exhibited early senescence and apoptotic cell death during passaging, which was preceded by abrupt induction of p53. In contrast, NPCs derived from hESCs and protein-based hiPSCs were highly expandable without senescence. DA neurons derived from protein-based hiPSCs exhibited gene expression, physiological, and electrophysiological properties similar to those of mDA neurons. Transplantation of these cells into rats with striatal lesions, a model of PD, significantly rescued motor deficits. These data support the clinical potential of protein-based hiPSCs for personalized cell therapy of PD.

Induced pluripotent stem cell-derived cardiomyocytes for cardiovascular disease modeling and drug screening.

PubMed

Sharma, Arun; Wu, Joseph C; Wu, Sean M

2013-12-24

Human induced pluripotent stem cells (hiPSCs) have emerged as a novel tool for drug discovery and therapy in cardiovascular medicine. hiPSCs are functionally similar to human embryonic stem cells (hESCs) and can be derived autologously without the ethical challenges associated with hESCs. Given the limited regenerative capacity of the human heart following myocardial injury, cardiomyocytes derived from hiPSCs (hiPSC-CMs) have garnered significant attention from basic and translational scientists as a promising cell source for replacement therapy. However, ongoing issues such as cell immaturity, scale of production, inter-line variability, and cell purity will need to be resolved before human clinical trials can begin. Meanwhile, the use of hiPSCs to explore cellular mechanisms of cardiovascular diseases in vitro has proven to be extremely valuable. For example, hiPSC-CMs have been shown to recapitulate disease phenotypes from patients with monogenic cardiovascular disorders. Furthermore, patient-derived hiPSC-CMs are now providing new insights regarding drug efficacy and toxicity. This review will highlight recent advances in utilizing hiPSC-CMs for cardiac disease modeling in vitro and as a platform for drug validation. The advantages and disadvantages of using hiPSC-CMs for drug screening purposes will be explored as well.

An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells.

PubMed

Guo, Jianying; Ma, Dacheng; Huang, Rujin; Ming, Jia; Ye, Min; Kee, Kehkooi; Xie, Zhen; Na, Jie

2017-05-01

Human pluripotent stem cells (hPSCs) are an important system to study early human development, model human diseases, and develop cell replacement therapies. However, genetic manipulation of hPSCs is challenging and a method to simultaneously activate multiple genomic sites in a controllable manner is sorely needed. Here, we constructed a CRISPR-ON system to efficiently upregulate endogenous genes in hPSCs. A doxycycline (Dox) inducible dCas9-VP64-p65-Rta (dCas9-VPR) transcription activator and a reverse Tet transactivator (rtTA) expression cassette were knocked into the two alleles of the AAVS1 locus to generate an iVPR hESC line. We showed that the dCas9-VPR level could be precisely and reversibly controlled by the addition and withdrawal of Dox. Upon transfection of multiplexed gRNA plasmid targeting the NANOG promoter and Dox induction, we were able to control NANOG gene expression from its endogenous locus. Interestingly, an elevated NANOG level promoted naïve pluripotent gene expression, enhanced cell survival and clonogenicity, and enabled hESCs to integrate with the inner cell mass (ICM) of mouse blastocysts in vitro. Thus, iVPR cells provide a convenient platform for gene function studies as well as high-throughput screens in hPSCs.

Establishment and proteomic characterization of a novel cell line, NCC-UPS2-C1, derived from a patient with undifferentiated pleomorphic sarcoma.

PubMed

Oyama, Rieko; Kito, Fusako; Sakumoto, Marimu; Shiozawa, Kumiko; Toki, Shunichi; Yoshida, Akihiko; Kawai, Akira; Kondo, Tadashi

2018-03-01

Undifferentiated pleomorphic sarcoma (UPS) is an aggressive mesenchymal malignancy requiring novel therapeutic approaches to improve clinical outcome. Patient-derived cancer cell lines are an essential tool for investigating molecular mechanisms underlying cancer initiation and development; however, there is a lack of patient-derived cell lines of UPS available for research. The objective of this study was to develop a patient-derived cell model of UPS. A cell line designated NCC-UPS2-C1 was established from the primary tumor tissue of an 84-yr-old female patient with UPS. The short tandem repeat pattern of NCC-UPS2-C1 cells was identical to that of the original tumor and distinct from that of any other cell lines deposited in public cell banks. NCC-UPS2-C1 cells were maintained as a monolayer culture for over 80 passages during 30 mo and exhibited spindle-like morphology, continuous growth, and ability for spheroid formation and invasion. Proteomic profiling using mass spectrometry and functional treemap analysis revealed that the original tumor and the derived NCC-UPS2-C1 cells had similar but distinct protein expression patterns. Our results indicate that a novel UPS cell line was successfully established and could be used to study UPS development and effects of anti-cancer drugs. However, the revealed difference between proteomes of the original tumor and NCC-UPS2-C1 cells should be further investigated to determine the appropriate applications of this cell line in UPS research.

PreImplantation factor (PIF*) promotes embryotrophic and neuroprotective decidual genes: effect negated by epidermal growth factor.

PubMed

Duzyj, Christina M; Paidas, Michael J; Jebailey, Lellean; Huang, Jing Shun; Barnea, Eytan R

2014-01-01

Intimate embryo-maternal interaction is paramount for pregnancy success post-implantation. The embryo follows a specific developmental timeline starting with neural system, dependent on endogenous and decidual factors. Beyond altered genetics/epigenetics, post-natal diseases may initiate at prenatal/neonatal, post-natal period, or through a continuum. Preimplantation factor (PIF) secreted by viable embryos promotes implantation and trophoblast invasion. Synthetic PIF reverses neuroinflammation in non-pregnant models. PIF targets embryo proteins that protect against oxidative stress and protein misfolding. We report of PIF's embryotrophic role and potential to prevent developmental disorders by regulating uterine milieu at implantation and first trimester. PIF's effect on human implantation (human endometrial stromal cells (HESC)) and first-trimester decidua cultures (FTDC) was examined, by global gene expression (Affymetrix), disease-biomarkers ranking (GeneGo), neuro-specific genes (Ingenuity) and proteins (mass-spectrometry). PIF co-cultured epidermal growth factor (EGF) in both HESC and FTDC (Affymetrix) was evaluated. In HESC, PIF promotes neural differentiation and transmission genes (TLX2, EPHA10) while inhibiting retinoic acid receptor gene, which arrests growth. PIF promotes axon guidance and downregulates EGF-dependent neuroregulin signaling. In FTDC, PIF promotes bone morphogenetic protein pathway (SMAD1, 53-fold) and axonal guidance genes (EPH5) while inhibiting PPP2R2C, negative cell-growth regulator, involved in Alzheimer's and amyotrophic lateral sclerosis. In HESC, PIF affects angiotensin via beta-arrestin, transforming growth factor-beta (TGF-β), notch, BMP, and wingless-int (WNT) signaling pathways that promote neurogenesis involved in childhood neurodevelopmental diseases-autism and also affected epithelial-mesenchymal transition involved in neuromuscular disorders. In FTDC, PIF upregulates neural development and hormone signaling, while downregulating genes protecting against xenobiotic response leading to connective tissue disorders. In both HESC and FTDC, PIF affects neural development and transmission pathways. In HESC interactome, PIF promotes FUS gene, which controls genome integrity, while in FTDC, PIF upregulates STAT3 critical transcription signal. EGF abolished PIF's effect on HESC, decreasing metalloproteinase and prolactin receptor genes, thereby interfering with decidualization, while in FTDC, EGF co-cultured with PIF reduced ZHX2, gene that regulates neural AFP secretion. PIF promotes decidual trophic genes and proteins to regulate neural development. By regulating the uterine milieu, PIF may decrease embryo vulnerability to post-natal neurodevelopmental disorders. Examination of PIF-based intervention strategies used during embryogenesis to improve pregnancy prognosis and reduce post-natal vulnerability is clearly in order.

PreImplantation factor (PIF*) promotes embryotrophic and neuroprotective decidual genes: effect negated by epidermal growth factor

PubMed Central

2014-01-01

Background Intimate embryo-maternal interaction is paramount for pregnancy success post-implantation. The embryo follows a specific developmental timeline starting with neural system, dependent on endogenous and decidual factors. Beyond altered genetics/epigenetics, post-natal diseases may initiate at prenatal/neonatal, post-natal period, or through a continuum. Preimplantation factor (PIF) secreted by viable embryos promotes implantation and trophoblast invasion. Synthetic PIF reverses neuroinflammation in non-pregnant models. PIF targets embryo proteins that protect against oxidative stress and protein misfolding. We report of PIF’s embryotrophic role and potential to prevent developmental disorders by regulating uterine milieu at implantation and first trimester. Methods PIF’s effect on human implantation (human endometrial stromal cells (HESC)) and first-trimester decidua cultures (FTDC) was examined, by global gene expression (Affymetrix), disease-biomarkers ranking (GeneGo), neuro-specific genes (Ingenuity) and proteins (mass-spectrometry). PIF co-cultured epidermal growth factor (EGF) in both HESC and FTDC (Affymetrix) was evaluated. Results In HESC, PIF promotes neural differentiation and transmission genes (TLX2, EPHA10) while inhibiting retinoic acid receptor gene, which arrests growth. PIF promotes axon guidance and downregulates EGF-dependent neuroregulin signaling. In FTDC, PIF promotes bone morphogenetic protein pathway (SMAD1, 53-fold) and axonal guidance genes (EPH5) while inhibiting PPP2R2C, negative cell-growth regulator, involved in Alzheimer’s and amyotrophic lateral sclerosis. In HESC, PIF affects angiotensin via beta-arrestin, transforming growth factor-beta (TGF-β), notch, BMP, and wingless-int (WNT) signaling pathways that promote neurogenesis involved in childhood neurodevelopmental diseases—autism and also affected epithelial-mesenchymal transition involved in neuromuscular disorders. In FTDC, PIF upregulates neural development and hormone signaling, while downregulating genes protecting against xenobiotic response leading to connective tissue disorders. In both HESC and FTDC, PIF affects neural development and transmission pathways. In HESC interactome, PIF promotes FUS gene, which controls genome integrity, while in FTDC, PIF upregulates STAT3 critical transcription signal. EGF abolished PIF’s effect on HESC, decreasing metalloproteinase and prolactin receptor genes, thereby interfering with decidualization, while in FTDC, EGF co-cultured with PIF reduced ZHX2, gene that regulates neural AFP secretion. Conclusions PIF promotes decidual trophic genes and proteins to regulate neural development. By regulating the uterine milieu, PIF may decrease embryo vulnerability to post-natal neurodevelopmental disorders. Examination of PIF-based intervention strategies used during embryogenesis to improve pregnancy prognosis and reduce post-natal vulnerability is clearly in order. PMID:26085845

Ultrastructural characteristics of three undifferentiated mouse embryonic stem cell lines and their differentiated three-dimensional derivatives: a comparative study.

PubMed

Alharbi, Suzan; Elsafadi, Mona; Mobarak, Mohammed; Alrwili, Ali; Vishnubalaji, Radhakrishnan; Manikandan, Muthurangan; Al-Qudsi, Fatma; Karim, Saleh; Al-Nabaheen, May; Aldahmash, Abdullah; Mahmood, Amer

2014-04-01

The fine structures of mouse embryonic stem cells (mESCs) grown as colonies and differentiated in three-dimensional (3D) culture as embryoid bodies (EBs) were analyzed by transmission electron microscopy. Undifferentiated mESCs expressed markers that proved their pluripotency. Differentiated EBs expressed different differentiation marker proteins from the three germ layers. The ultrastructure of mESCs revealed the presence of microvilli on the cell surfaces, large and deep infolded nuclei, low cytoplasm-to-nuclear ratios, frequent lipid droplets, nonprominent Golgi apparatus, and smooth endoplasmic reticulum. In addition, we found prominent juvenile mitochondria and free ribosomes-rich cytoplasm in mESCs. Ultrastructure of the differentiated mESCs as EBs showed different cell arrangements, which indicate the different stages of EB development and differentiation. The morphologies of BALB/c and 129 W9.5 EBs were very similar at day 4, whereas C57BL/6 EBs were distinct from the others at day 4. This finding suggested that differentiation of EBs from different cell lines occurs in the same pattern but not at the same rate. Conversely, the ultrastructure results of BALB/c and 129 W9.5 ESCs revealed differentiating features, such as the dilated profile of a rough endoplasmic reticulum. In addition, we found low expression levels of undifferentiated markers on the outer cells of BALB/c and 129 W9.5 mESC colonies, which suggests a faster differentiation potential.

Pluripotency of adult stem cells derived from human and rat pancreas

NASA Astrophysics Data System (ADS)

Kruse, C.; Birth, M.; Rohwedel, J.; Assmuth, K.; Goepel, A.; Wedel, T.

Adult stem cells are undifferentiated cells found within fully developed tissues or organs of an adult individuum. Until recently, these cells have been considered to bear less self-renewal ability and differentiation potency compared to embryonic stem cells. In recent studies an undifferentiated cell type was found in primary cultures of isolated acini from exocrine pancreas termed pancreatic stellate cells. Here we show that pancreatic stellate-like cells have the capacity of extended self-renewal and are able to differentiate spontaneously into cell types of all three germ layers expressing markers for smooth muscle cells, neurons, glial cells, epithelial cells, chondrocytes and secretory cells (insulin, amylase). Differentiation and subsequent formation of three-dimensional cellular aggregates (organoid bodies) were induced by merely culturing pancreatic stellate-like cells in hanging drops. These cells were developed into stable, long-term, in vitro cultures of both primary undifferentiated cell lines as well as organoid cultures. Thus, evidence is given that cell lineages of endodermal, mesodermal, and ectodermal origin arise spontaneously from a single adult undifferentiated cell type. Based on the present findings it is assumed that pancreatic stellate-like cells are a new class of lineage uncommitted pluripotent adult stem cells with a remarkable self-renewal ability and differentiation potency. The data emphasize the versatility of adult stem cells and may lead to a reappraisal of their use for the treatment of inherited disorders or acquired degenerative diseases.

Growing Stem Cells: The Impact of Federal Funding Policy on the U.S. Scientific Frontier

ERIC Educational Resources Information Center

Furman, Jeffrey L.; Murray, Fiona; Stern, Scott

2012-01-01

This paper articulates a citation-based approach to science policy evaluation and employs that approach to investigate the impact of the United States' 2001 policy regarding the federal funding of human embryonic stem cell (hESC) research. We evaluate the impact of the policy on the level of U.S. hESC research, the U.S. position at the knowledge…

Chemically defined serum-free conditions for cartilage regeneration from human embryonic stem cells.

PubMed

Yang, Dandan; Chen, Shubin; Gao, Changzhao; Liu, Xiaobo; Zhou, Yulai; Liu, Pengfei; Cai, Jinglei

2016-11-01

The aim of this study was to improve a method that induce cartilage differentiation of human embryoid stem cells (hESCs) in vitro, and test the effect of in vivo environments on the further maturation of hESCs derived cells. Embryoid bodies (EBs) formed from hESCs, with serum-free KSR-based medium and mesodermal specification related factors, CHIR, and Noggin for first 8days. Then cells were digested and cultured as micropellets in serum-free KSR-based chondrogenic medium that was supplemented with PDGF-BB, TGF β3, BMP4 in sequence for 24days. The morphology, FACS, histological staining as well as the expression of chondrogenic specific genes were detected in each stage, and further in vivo experiments, cell injections and tissue transplantations, further verified the formation of chondrocytes. We were able to obtain chondrocyte/cartilage from hESCs using serum-free KSR-based conditioned medium. qPCR analysis showed that expression of the chondroprogenitor genes and the chondrocyte/cartilage matrix genes. Morphology analysis demonstrated we got PG+COL2+COL1-particles. It indicated we obtained hyaline cartilage-like particles. 32-Day differential cells were injected subcutaneous. Staining results showed grafts developed further mature in vivo. But when transplanted in subrenal capsule, their effect was not good as in subcutaneous. Microenvironment might affect the cartilage formation. The results of this study provide an absolute serum-free and efficient approach for generation of hESC-derived chondrocytes, and cells will become further maturation in vivo. It provides evidence and technology for the hypothesis that hESCs may be a promising therapy for the treatment of cartilage disease. Copyright © 2016 Elsevier Inc. All rights reserved.

Heat shock instructs hESCs to exit from the self-renewal program through negative regulation of OCT4 by SAPK/JNK and HSF1 pathway.

PubMed

Byun, Kyunghee; Kim, Taek-Kyun; Oh, Jeehyun; Bayarsaikhan, Enkhjargal; Kim, Daesik; Lee, Min Young; Pack, Chan-Gi; Hwang, Daehee; Lee, Bonghee

2013-11-01

Environmental factors affect self-renewal of stem cells by modulating the components of self-renewal networks. Heat shock, an environmental factor, induces heat shock factors (HSFs), which up-regulate stress response-related genes. However, the link of heat shock to self-renewal of stem cells has not been elucidated yet. Here, we present the direct link of heat shock to a core stem cell regulator, OCT4, in the self-renewal network through SAPK/JNK and HSF1 pathway. We first showed that heat shock initiated differentiation of human embryonic stem cells (hESCs). Gene expression analysis revealed that heat shock increased the expression of many genes involved in cellular processes related to differentiation of stem cells. We then examined the effects of HSFs induced by heat shock on core self-renewal factors. Among HSFs, heat shock induced mainly HSF1 in hESCs. The HSF1 repressed the expression of OCT4, leading to the differentiation of hESCs and the above differentiation-related gene expression change. We further examined the effects of the upstream MAP (mitogen-activated protein) kinases of HSF1 on the repression of OCT4 expression by HSF1. Among the MAP kinases, SAPK/JNK controlled predominantly the repression of the OCT4 expression by HSF1. The direct link of heat shock to the core self-renewal regulator through SAPK/JNK and HSF1 provides a fundamental basis for understanding the effect of heat and other stresses involving activation of HSF1 on the self-renewal program and further controlling differentiation of hESCs in a broad spectrum of stem cell applications using these stresses. © 2013.

CAPN 7 promotes the migration and invasion of human endometrial stromal cell by regulating matrix metalloproteinase 2 activity.

PubMed

Liu, Hongyu; Jiang, Yue; Jin, Xiaoyan; Zhu, Lihua; Shen, Xiaoyue; Zhang, Qun; Wang, Bin; Wang, Junxia; Hu, Yali; Yan, Guijun; Sun, Haixiang

2013-07-15

Matrix metalloproteinase 2 (MMP-2) has been reported to be an important regulator of cell migration and invasion through degradation of the extracellular matrix (ECM) in many diseases, such as cancer and endometriosis. Here, we found calcium-activated neutral protease 7 (CAPN 7) expression was markedly upregulated in the eutopic endometrium and endometrial stromal cells of women diagnosed with endometriosis. Our studies were carried out to detect the effects of CAPN 7 on human endometrial stromal cell (hESC) migration and invasion. Western blotting and quantitative real-time PCR were used to detect the expression of CAPN 7 in endometriosis patients and normal fertile women. Scratch-wound-healing and invasion chamber assay were used to investigate the role of CAPN 7 in hESC migration and invasion. Western blotting, quantitative real-time PCR and zymography were carried out to detect the effect of CAPN 7 on the expressions and activity of MMP-2. CAPN 7 was markedly up-regulated in endometriosis, thereby promoting the migration and invasion of hESC. CAPN 7 overexpression led to increased expression of MMP-2 and tissue inhibitor of metalloproteinases 2 (TIMP-2); CAPN 7 knockdown reversed these changes. CAPN 7 increased MMP-2 activity by increasing the ratio of MMP-2 to TIMP-2. We also found that OA-Hy (an MMP-2 inhibitor) decreased the effects of CAPN 7 overexpression on hESC migration and invasion by approximately 50% and 55%, respectively. Additionally, a coimmunoprecipitation assay demonstrated that CAPN 7 interacted with activator protein 2α (AP-2α): an important transcription factor of MMP-2. CAPN 7 promotes hESC migration and invasion by increasing the activity of MMP-2 via an increased ratio of MMP-2 to TIMP-2.

Biochemical and morphological effects of hypoxic environment on human embryonic stem cells in long-term culture and differentiating embryoid bodies.

PubMed

Lim, Hee-Joung; Han, Jiyou; Woo, Dong-Hun; Kim, Sung-Eun; Kim, Suel-Kee; Kang, Hee-Gyoo; Kim, Jong-Hoon

2011-02-01

The mammalian reproductive tract is known to contain 1.5-5.3% oxygen (O(2)), but human embryonic stem cells (hESCs) derived from preimplantation embryos are typically cultured under 21% O(2) tension. The aim of this study was to investigate the effects of O(2) tension on the long-term culture of hESCs and on cell-fate determination during early differentiation. hESCs and embryoid bodies (EBs) were grown under different O(2) tensions (3, 12, and 21% O(2)). The expression of markers associated with pluripotency, embryonic germ layers, and hypoxia was analyzed using RTPCR, immunostaining, and Western blotting. Proliferation, apoptosis, and chromosomal aberrations were examined using BrdU incorporation, caspase-3 immunostaining, and karyotype analysis, respectively. Structural and morphological changes of EBs under different O(2) tensions were comparatively examined using azan- and hematoxylineosin staining, and scanning and transmission electron microscopy. Mild hypoxia (12% O(2)) increased the number of cells expressing Oct4/Nanog and reduced BrdU incorporation and aneuploidy. The percentage of cells positive for active caspase-3, which was high during normoxia (21% O(2)), gradually decreased when hESCs were continuously cultured under mild hypoxia. EBs subjected to hypoxia (3% O(2)) exhibited well-differentiated microvilli on their surface, secreted high levels of collagen, and showed enhanced differentiation into primitive endoderm. These changes were associated with increased expression of Foxa2, Sox17, AFP, and GATA4 on the EB periphery. Our data suggest that mild hypoxia facilitates the slow mitotic division of hESCs in long-term culture and reduces the frequency of chromosomal abnormalities and apoptosis. In addition, hypoxia promotes the differentiation of EBs into extraembryonic endoderm.

Leucine-rich Repeat Neuronal Protein 1 Regulates Differentiation of Embryonic Stem Cells by Posttranslational Modifications of Pluripotency Factors.

PubMed

Liao, Chien Huang; Wang, Ya-Hui; Chang, Wei-Wei; Yang, Bei-Chia; Wu, Tsai-Jung; Liu, Wei-Li; Yu, Alice L; Yu, John

2018-06-11

Stem cell surface markers may facilitate a better understanding of stem cell biology through molecular function studies or serve as tools to monitor the differentiation status and behavior of stem cells in culture or tissue. Thus, it is important to identify additional, novel stem cell markers. We used glycoproteomics to discover surface glycoproteins on human embryonic stem cells (hESCs) that may be useful stem cell markers. We found that a surface glycoprotein, leucine-rich repeat neuronal protein 1 (LRRN1), is expressed abundantly on the surface of hESCs prior to differentiation into embryoid bodies (EBs). Silencing of LRRN1 with short hairpin RNA (shLRRN1) in hESCs resulted in decreased capacity of self-renewal, and skewed differentiation toward endoderm/mesoderm lineages in vitro and in vivo. Meanwhile, the protein expression levels of the pluripotency factors OCT4, NANOG and SOX2 were reduced. Interestingly, the mRNA levels of these pluripotency factors were not affected in LRRN1 silenced cells, but protein half-lives were substantially shortened. Furthermore, we found LRRN1 silencing led to nuclear export and proteasomal degradation of all three pluripotency factors. In addition, the effects on nuclear export were mediated by AKT phosphorylation. These results suggest that LRRN1 plays an important role in maintaining the protein stability of pluripotency factors through AKT phosphorylation, thus maintaining hESC self-renewal capacity and pluripotency. Overall, we found that LRRN1 contributes to pluripotency of hESC by preventing translocation of OCT4, NANOG and SOX2 from nucleus to cytoplasm, thereby lessening their post-translational modification and degradation. This article is protected by copyright. All rights reserved. © 2018 AlphaMed Press.

Changing Nuclear Landscape and Unique PML Structures During Early Epigenetic Transitions of Human Embryonic Stem Cells

PubMed Central

Butler, John T.; Hall, Lisa L.; Smith, Kelly P.; Lawrence, Jeanne B.

2010-01-01

The complex nuclear structure of somatic cells is important to epigenomic regulation, yet little is known about nuclear organization of human embryonic stem cells (hESC). Here we surveyed several nuclear structures in pluripotent and transitioning hESC. Observations of centromeres, telomeres, SC35 speckles, Cajal Bodies, lamin A/C and emerin, nuclear shape and size demonstrate a very different “nuclear landscape” in hESC. This landscape is remodeled during a brief transitional window, concomitant with or just prior to differentiation onset. Notably, hESC initially contain abundant signal for spliceosome assembly factor, SC35, but lack discrete SC35 domains; these form as cells begin to specialize, likely reflecting cell-type specific genomic organization. Concomitantly, nuclear size increases and shape changes as lamin A/C and emerin incorporate into the lamina. During this brief window, hESC exhibit dramatically different PML-defined structures, which in somatic cells are linked to gene regulation and cancer. Unlike the numerous, spherical somatic PML bodies, hES cells often display ~1–3 large PML structures of two morphological types: long linear “rods” or elaborate “rosettes”, which lack substantial SUMO-1, Daxx, and Sp100.These occur primarily between Day 0–2 of differentiation and become rare thereafter. PML rods may be “taut” between other structures, such as centromeres, but clearly show some relationship with the lamina, where PML often abuts or fills a “gap” in early lamin A/C staining. Findings demonstrate that pluripotent hES cells have a markedly different overall nuclear architecture, remodeling of which is linked to early epigenomic programming and involves formation of unique PML-defined structures. PMID:19449340

Quantitative Dynamics of Proteome, Acetylome, and Succinylome during Stem-Cell Differentiation into Hepatocyte-like Cells.

PubMed

Liu, Zekun; Zhang, Qing-Bin; Bu, Chen; Wang, Dawei; Yu, Kai; Gan, Zhixue; Chang, Jianfeng; Cheng, Zhongyi; Liu, Zexian

2018-06-21

Stem-cell differentiation is a complex biological process controlled by a series of functional protein clusters and signaling transductions, especially metabolism-related pathways. Although previous studies have quantified the proteome and phosphoproteome for stem-cell differentiation, the investigation of acylation-mediated regulation is still absent. In this study, we quantitatively profiled the proteome, acetylome, and succinylome in pluripotent human embryonic stem cells (hESCs) and differentiated hepatocyte-like cells (HLCs). In total, 3843 proteins, 185 acetylation sites in 103 proteins, and 602 succinylation sites in 391 proteins were quantified. The quantitative proteome showed that in differentiated HLCs the TGF-β, JAK-STAT, and RAS signaling pathways were activated, whereas ECM-related processes such as sulfates and leucine degradation were depressed. Interestingly, it was observed that the acetylation and succinylation were more intensive in hESCs, whereas protein processing in endoplasmic reticulum and the carbon metabolic pathways were especially highly succinylated. Because the metabolism patterns in pluripotent hESCs and the differentiated HLCs were different, we proposed that the dynamic acylations, especially succinylation, might regulate the Warburg-like effect and TCA cycle during differentiation. Taken together, we systematically profiled the protein and acylation levels of regulation in pluripotent hESCs and differentiated HLCs, and the results indicated the important roles of acylation in pluripotency maintenance and differentiation.

The Role of ARX in Human Pancreatic Endocrine Specification

PubMed Central

Gage, Blair K.; Asadi, Ali; Baker, Robert K.; Webber, Travis D.; Wang, Rennian; Itoh, Masayuki; Hayashi, Masaharu; Miyata, Rie; Akashi, Takumi; Kieffer, Timothy J.

2015-01-01

The in vitro differentiation of human embryonic stem cells (hESCs) offers a model system to explore human development. Humans with mutations in the transcription factor Aristaless Related Homeobox (ARX) often suffer from the syndrome X-linked lissencephaly with ambiguous genitalia (XLAG), affecting many cell types including those of the pancreas. Indeed, XLAG pancreatic islets lack glucagon and pancreatic polypeptide-positive cells but retain somatostatin, insulin, and ghrelin-positive cells. To further examine the role of ARX in human pancreatic endocrine development, we utilized genomic editing in hESCs to generate deletions in ARX. ARX knockout hESCs retained pancreatic differentiation capacity and ARX knockout endocrine cells were biased toward somatostatin-positive cells (94% of endocrine cells) with reduced pancreatic polypeptide (rarely detected), glucagon (90% reduced) and insulin-positive (65% reduced) lineages. ARX knockout somatostatin-positive cells shared expression patterns with human fetal and adult δ-cells. Differentiated ARX knockout cells upregulated PAX4, NKX2.2, ISL1, HHEX, PCSK1, PCSK2 expression while downregulating PAX6 and IRX2. Re-expression of ARX in ARX knockout pancreatic progenitors reduced HHEX and increased PAX6 and insulin expression following differentiation. Taken together these data suggest that ARX plays a key role in pancreatic endocrine fate specification of pancreatic polypeptide, somatostatin, glucagon and insulin positive cells from hESCs. PMID:26633894

The Role of ARX in Human Pancreatic Endocrine Specification.

PubMed

Gage, Blair K; Asadi, Ali; Baker, Robert K; Webber, Travis D; Wang, Rennian; Itoh, Masayuki; Hayashi, Masaharu; Miyata, Rie; Akashi, Takumi; Kieffer, Timothy J

2015-01-01

The in vitro differentiation of human embryonic stem cells (hESCs) offers a model system to explore human development. Humans with mutations in the transcription factor Aristaless Related Homeobox (ARX) often suffer from the syndrome X-linked lissencephaly with ambiguous genitalia (XLAG), affecting many cell types including those of the pancreas. Indeed, XLAG pancreatic islets lack glucagon and pancreatic polypeptide-positive cells but retain somatostatin, insulin, and ghrelin-positive cells. To further examine the role of ARX in human pancreatic endocrine development, we utilized genomic editing in hESCs to generate deletions in ARX. ARX knockout hESCs retained pancreatic differentiation capacity and ARX knockout endocrine cells were biased toward somatostatin-positive cells (94% of endocrine cells) with reduced pancreatic polypeptide (rarely detected), glucagon (90% reduced) and insulin-positive (65% reduced) lineages. ARX knockout somatostatin-positive cells shared expression patterns with human fetal and adult δ-cells. Differentiated ARX knockout cells upregulated PAX4, NKX2.2, ISL1, HHEX, PCSK1, PCSK2 expression while downregulating PAX6 and IRX2. Re-expression of ARX in ARX knockout pancreatic progenitors reduced HHEX and increased PAX6 and insulin expression following differentiation. Taken together these data suggest that ARX plays a key role in pancreatic endocrine fate specification of pancreatic polypeptide, somatostatin, glucagon and insulin positive cells from hESCs.

Comparative study of human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) as a treatment for retinal dystrophies

PubMed Central

Riera, Marina; Fontrodona, Laura; Albert, Silvia; Ramirez, Diana Mora; Seriola, Anna; Salas, Anna; Muñoz, Yolanda; Ramos, David; Villegas-Perez, Maria Paz; Zapata, Miguel Angel; Raya, Angel; Ruberte, Jesus; Veiga, Anna; Garcia-Arumi, Jose

2016-01-01

Retinal dystrophies (RD) are major causes of familial blindness and are characterized by progressive dysfunction of photoreceptor and/or retinal pigment epithelium (RPE) cells. In this study, we aimed to evaluate and compare the therapeutic effects of two pluripotent stem cell (PSC)-based therapies. We differentiated RPE from human embryonic stem cells (hESCs) or human-induced pluripotent stem cells (hiPSCs) and transplanted them into the subretinal space of the Royal College of Surgeons (RCS) rat. Once differentiated, cells from either source of PSC resembled mature RPE in their morphology and gene expression profile. Following transplantation, both hESC- and hiPSC-derived cells maintained the expression of specific RPE markers, lost their proliferative capacity, established tight junctions, and were able to perform phagocytosis of photoreceptor outer segments. Remarkably, grafted areas showed increased numbers of photoreceptor nuclei and outer segment disk membranes. Regardless of the cell source, human transplants protected retina from cell apoptosis, glial stress and accumulation of autofluorescence, and responded better to light stimuli. Altogether, our results show that hESC- and hiPSC-derived cells survived, migrated, integrated, and functioned as RPE in the RCS rat retina, providing preclinical evidence that either PSC source could be of potential benefit for treating RD. PMID:27006969

Transplantation of Human Embryonic Stem Cells in Patients with Multiple Sclerosis and Lyme Disease.

PubMed

Shroff, Geeta

2016-12-13

BACKGROUND Multiple sclerosis (MS) is an inflammatory and neurodegenerative disease in which the myelin sheath of nerve cells is damaged. It can cause delayed neurologic symptoms similar to those seen in Lyme disease (LD) patients. Thymus derived T-cells (myelin reactive) migrate to the blood brain barrier and stimulate an inflammatory cascade in the central nervous system. Cell based therapies play an important role in treating neurological diseases such as MS and LD. CASE REPORT Human embryonic stem cell (hESC) therapy was used to treat two patients with both MS and LD. The hESCs were administered via different routes including intramuscular, intravenous, and supplemental routes (e.g., deep spinal, caudal, intercostal through eye drops) to regenerate the injured cells. Both the patients showed remarkable improvement in their functional skills, overall stamina, cognitive abilities, and muscle strength. Furthermore, the improvement in the patients' conditions were assessed by magnetic resonance tractography and single photon emission computed tomography (SPECT). CONCLUSIONS Therapy with hESCs might emerge as an effective and safe treatment for patients with both MS and LD. Well-designed clinical trials and follow-up studies are needed to prove the long-term efficacy and safety of hESC therapy in the treatment of patients with MS and LD.

Extraction of Blebs in Human Embryonic Stem Cell Videos.

PubMed

Guan, Benjamin X; Bhanu, Bir; Talbot, Prue; Weng, Nikki Jo-Hao

2016-01-01

Blebbing is an important biological indicator in determining the health of human embryonic stem cells (hESC). Especially, areas of a bleb sequence in a video are often used to distinguish two cell blebbing behaviors in hESC: dynamic and apoptotic blebbings. This paper analyzes various segmentation methods for bleb extraction in hESC videos and introduces a bio-inspired score function to improve the performance in bleb extraction. Full bleb formation consists of bleb expansion and retraction. Blebs change their size and image properties dynamically in both processes and between frames. Therefore, adaptive parameters are needed for each segmentation method. A score function derived from the change of bleb area and orientation between consecutive frames is proposed which provides adaptive parameters for bleb extraction in videos. In comparison to manual analysis, the proposed method provides an automated fast and accurate approach for bleb sequence extraction.

SP600125 has a remarkable anticancer potential against undifferentiated thyroid cancer through selective action on ROCK and p53 pathways.

PubMed

Grassi, Elisa Stellaria; Vezzoli, Valeria; Negri, Irene; Lábadi, Árpád; Fugazzola, Laura; Vitale, Giovanni; Persani, Luca

2015-11-03

Thyroid cancer is the most common endocrine malignancy with increasing incidence worldwide.The majority of thyroid cancer cases are well differentiated with favorable outcome. However, undifferentiated thyroid cancers are one of the most lethal human malignancies because of their invasiveness, metastatization and refractoriness even to the most recently developed therapies.In this study we show for the first time a significant hyperactivation of ROCK/HDAC6 pathway in thyroid cancer tissues, and its negative correlation with p53 DNA binding ability.We demonstrate that a small compound, SP600125 (SP), is able to induce cell death selectively in undifferentiated thyroid cancer cell lines by specifically acting on the pathogenic pathways of cancer development. In detail, SP acts on the ROCK/HDAC6 pathway involved in dedifferentiation and invasiveness of undifferentiated human cancers, by restoring its physiological activity level. As main consequence, cancer cell migration is inhibited and, at the same time, cell death is induced through the mitotic catastrophe. Moreover, SP exerts a preferential action on the mutant p53 by increasing its DNA binding ability. In TP53-mutant cells that survive mitotic catastrophe this process results in p21 induction and eventually lead to premature senescence. In conclusion, SP has been proved to be able to simultaneously block cell replication and migration, the two main processes involved in cancer development and dissemination, making it an ideal candidate for developing new drugs against anaplastic thyroid cancer.

Variability of human pluripotent stem cell lines.

PubMed

Ortmann, Daniel; Vallier, Ludovic

2017-10-01

Human pluripotent stem cells derived from embryos (human Embryonic Stem Cells or hESCs) or generated by direct reprogramming of somatic cells (human Induced Pluripotent Stem Cells or hiPSCs) can proliferate almost indefinitely in vitro while maintaining the capacity to differentiate into a broad diversity of cell types. These two properties (self-renewal and pluripotency) confers human pluripotent stem cells a unique interest for clinical applications since they could allow the production of infinite quantities of cells for disease modelling, drug screening and cell based therapy. However, recent studies have clearly established that human pluripotent stem cell lines can display variable capacity to differentiate into specific lineages. Consequently, the development of universal protocols of differentiation which could work efficiently with any human pluripotent cell line is complicated substantially. As a consequence, each protocol needs to be adapted to every cell line thereby limiting large scale applications and precluding personalised therapies. Here, we summarise our knowledge concerning the origin of this variability and describe potential solutions currently available to bypass this major challenge. Copyright © 2017 Elsevier Ltd. All rights reserved.

Combination of heterologous fibrin sealant and bioengineered human embryonic stem cells to improve regeneration following autogenous sciatic nerve grafting repair.

PubMed

Mozafari, Roghayeh; Kyrylenko, Sergiy; Castro, Mateus Vidigal; Ferreira, Rui Seabra; Barraviera, Benedito; Oliveira, Alexandre Leite Rodrigues

2018-01-01

Peripheral nerve injury is a worldwide clinical problem, and the preferred surgical method for treating it is the end-to-end neurorrhaphy. When it is not possible due to a large nerve gap, autologous nerve grafting is used. However, these surgical techniques result in nerve regeneration at highly variable degrees. It is thus very important to seek complementary techniques to improve motor and sensory recovery. One promising approach could be cell therapy. Transplantation therapy with human embryonic stem cells (hESCs) is appealing because these cells are pluripotent and can differentiate into specialized cell types and have self-renewal ability. Therefore, the main objective of this study was to find conditions under which functional recovery is improved after sciatic nerve neurorrhaphy. We assumed that hESC, either alone or in combination with heterologous fibrin sealant scaffold, could be used to support regeneration in a mouse model of sciatic nerve injury and repair via autografting with end-to-end neurorrhaphy. Five millimeters of the sciatic nerve of C57BL/6 J mice were transected off and rotated 180 degrees to simulate an injury, and then stumps were sutured. Next, we applied heterologous fibrin sealant and/or human embryonic stem cells genetically altered to overexpress fibroblast growth factor 2 (FGF2) at the site of the injury. The study was designed to include six experimental groups comprising neurorrhaphy (N), neurorrhaphy + heterologous fibrin sealant (N + F), neurorrhaphy + heterologous fibrin sealant + doxycycline (N + F + D), neurorrhaphy + heterologous fibrin sealant + wild-type hESC (N + F + W), neurorrhaphy + heterologous fibrin sealant + hESC off (N + F + T), and neurorrhaphy + heterologous fibrin sealant + hESC on via doxycycline (N + F + D + T). We evaluated the recovery rate using Catwalk and von Frey functional recovery tests, as well as immunohistochemistry analysis. The experiments indicated that sensory function improved when transgenic hESCs were used. The regeneration of sensory fibers indeed led to increased reflexes, upon stimulation of the paw ipsilateral to the lesion, as seen by von-Frey evaluation, which was supported by immunohistochemistry. Overall, the present data demonstrated that transgenic embryonic stem cells, engineered to overexpress FGF-2 in an inducible fashion, could be employed to support regeneration aiming at the recovery of both motor and sensory functions.

Myocardial commitment from human pluripotent stem cells: Rapid production of human heart grafts.

PubMed

Garreta, Elena; de Oñate, Lorena; Fernández-Santos, M Eugenia; Oria, Roger; Tarantino, Carolina; Climent, Andreu M; Marco, Andrés; Samitier, Mireia; Martínez, Elena; Valls-Margarit, Maria; Matesanz, Rafael; Taylor, Doris A; Fernández-Avilés, Francisco; Izpisua Belmonte, Juan Carlos; Montserrat, Nuria

2016-08-01

Genome editing on human pluripotent stem cells (hPSCs) together with the development of protocols for organ decellularization opens the door to the generation of autologous bioartificial hearts. Here we sought to generate for the first time a fluorescent reporter human embryonic stem cell (hESC) line by means of Transcription activator-like effector nucleases (TALENs) to efficiently produce cardiomyocyte-like cells (CLCs) from hPSCs and repopulate decellularized human heart ventricles for heart engineering. In our hands, targeting myosin heavy chain locus (MYH6) with mCherry fluorescent reporter by TALEN technology in hESCs did not alter major pluripotent-related features, and allowed for the definition of a robust protocol for CLCs production also from human induced pluripotent stem cells (hiPSCs) in 14 days. hPSCs-derived CLCs (hPSCs-CLCs) were next used to recellularize acellular cardiac scaffolds. Electrophysiological responses encountered when hPSCs-CLCs were cultured on ventricular decellularized extracellular matrix (vdECM) correlated with significant increases in the levels of expression of different ion channels determinant for calcium homeostasis and heart contractile function. Overall, the approach described here allows for the rapid generation of human cardiac grafts from hPSCs, in a total of 24 days, providing a suitable platform for cardiac engineering and disease modeling in the human setting. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Improvement of Cell Survival During Human Pluripotent Stem Cell Definitive Endoderm Differentiation

PubMed Central

Wang, Han; Luo, Xie; Yao, Li; Lehman, Donna M.

2015-01-01

Definitive endoderm (DE) is a vital precursor for internal organs such as liver and pancreas. Efficient protocol to differentiate human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs) to DE is essential for regenerative medicine and for modeling diseases; yet, poor cell survival during DE differentiation remains unsolved. In this study, our use of B27 supplement in modified differentiation protocols has led to a substantial improvement. We used an SOX17-enhanced green fluorescent protein (eGFP) reporter hESC line to compare and modify established DE differentiation protocols. Both total live cell numbers and the percentages of eGFP-positive cells were used to assess differentiation efficiency. Among tested protocols, three modified protocols with serum-free B27 supplement were developed to generate a high number of DE cells. Massive cell death was avoided during DE differentiation and the percentage of DE cells remained high. When the resulting DE cells were further differentiated toward the pancreatic lineage, the expression of pancreatic-specific markers was significantly increased. Similar high DE differentiation efficiency was observed in H1 hESCs and iPSCs through the modified protocols. In B27 components, bovine serum albumin was found to facilitate DE differentiation and cell survival. Using our modified DE differentiation protocols, satisfactory quantities of quality DE can be produced as primary material for further endoderm lineage differentiation. PMID:26132288

Identification of Transposable Elements Contributing to Tissue-Specific Expression of Long Non-Coding RNAs

PubMed Central

Chishima, Takafumi; Iwakiri, Junichi

2018-01-01

It has been recently suggested that transposable elements (TEs) are re-used as functional elements of long non-coding RNAs (lncRNAs). This is supported by some examples such as the human endogenous retrovirus subfamily H (HERVH) elements contained within lncRNAs and expressed specifically in human embryonic stem cells (hESCs), as required to maintain hESC identity. There are at least two unanswered questions about all lncRNAs. How many TEs are re-used within lncRNAs? Are there any other TEs that affect tissue specificity of lncRNA expression? To answer these questions, we comprehensively identify TEs that are significantly related to tissue-specific expression levels of lncRNAs. We downloaded lncRNA expression data corresponding to normal human tissue from the Expression Atlas and transformed the data into tissue specificity estimates. Then, Fisher’s exact tests were performed to verify whether the presence or absence of TE-derived sequences influences the tissue specificity of lncRNA expression. Many TE–tissue pairs associated with tissue-specific expression of lncRNAs were detected, indicating that multiple TE families can be re-used as functional domains or regulatory sequences of lncRNAs. In particular, we found that the antisense promoter region of L1PA2, a LINE-1 subfamily, appears to act as a promoter for lncRNAs with placenta-specific expression. PMID:29315213

CAPN 7 promotes the migration and invasion of human endometrial stromal cell by regulating matrix metalloproteinase 2 activity

PubMed Central

2013-01-01

Background Matrix metalloproteinase 2 (MMP-2) has been reported to be an important regulator of cell migration and invasion through degradation of the extracellular matrix (ECM) in many diseases, such as cancer and endometriosis. Here, we found calcium-activated neutral protease 7 (CAPN 7) expression was markedly upregulated in the eutopic endometrium and endometrial stromal cells of women diagnosed with endometriosis. Our studies were carried out to detect the effects of CAPN 7 on human endometrial stromal cell (hESC) migration and invasion. Methods Western blotting and quantitative real-time PCR were used to detect the expression of CAPN 7 in endometriosis patients and normal fertile women. Scratch-wound-healing and invasion chamber assay were used to investigate the role of CAPN 7 in hESC migration and invasion. Western blotting, quantitative real-time PCR and zymography were carried out to detect the effect of CAPN 7 on the expressions and activity of MMP-2. Results CAPN 7 was markedly up-regulated in endometriosis, thereby promoting the migration and invasion of hESC. CAPN 7 overexpression led to increased expression of MMP-2 and tissue inhibitor of metalloproteinases 2 (TIMP-2); CAPN 7 knockdown reversed these changes. CAPN 7 increased MMP-2 activity by increasing the ratio of MMP-2 to TIMP-2. We also found that OA-Hy (an MMP-2 inhibitor) decreased the effects of CAPN 7 overexpression on hESC migration and invasion by approximately 50% and 55%, respectively. Additionally, a coimmunoprecipitation assay demonstrated that CAPN 7 interacted with activator protein 2α (AP-2α): an important transcription factor of MMP-2. Conclusions CAPN 7 promotes hESC migration and invasion by increasing the activity of MMP-2 via an increased ratio of MMP-2 to TIMP-2. PMID:23855590

Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) blocks differentiation and maintains the expression of pluripotency markers in human embryonic stem cells.

PubMed

Burton, Peter; Adams, David R; Abraham, Achamma; Allcock, Robert W; Jiang, Zhong; McCahill, Angela; Gilmour, Jane; McAbney, John; Kaupisch, Alexandra; Kane, Nicole M; Baillie, George S; Baker, Andrew H; Milligan, Graeme; Houslay, Miles D; Mountford, Joanne C

2010-12-15

hESCs (human embryonic stem cells) have enormous potential for use in pharmaceutical development and therapeutics; however, to realize this potential, there is a requirement for simple and reproducible cell culture methods that provide adequate numbers of cells of suitable quality. We have discovered a novel way of blocking the spontaneous differentiation of hESCs in the absence of exogenous cytokines by supplementing feeder-free conditions with EHNA [erythro-9-(2-hydroxy-3-nonyl)adenine], an established inhibitor of ADA (adenosine deaminase) and cyclic nucleotide PDE2 (phosphodiesterase 2). hESCs maintained in feeder-free conditions with EHNA for more than ten passages showed no reduction in hESC-associated markers including NANOG, POU5F1 (POU domain class 5 transcription factor 1, also known as Oct-4) and SSEA4 (stage-specific embryonic antigen 4) compared with cells maintained in feeder-free conditions containing bFGF (basic fibroblast growth factor). Spontaneous differentiation was reversibly suppressed by the addition of EHNA, but, upon removing EHNA, hESC populations underwent efficient spontaneous, multi-lineage and directed differentiation. EHNA also acts as a strong blocker of directed neuronal differentiation. Chemically distinct inhibitors of ADA and PDE2 lacked the capacity of EHNA to suppress hESC differentiation, suggesting that the effect is not driven by inhibition of either ADA or PDE2. Preliminary structure-activity relationship analysis found the differentiation-blocking properties of EHNA to reside in a pharmacophore comprising a close adenine mimetic with an extended hydrophobic substituent in the 8- or 9-position. We conclude that EHNA and simple 9-alkyladenines can block directed neuronal and spontaneous differentiation in the absence of exogenous cytokine addition, and may provide a useful replacement for bFGF in large-scale or cGMP-compliant processes.

Isolation, culture, and imaging of human fetal pancreatic cell clusters.

PubMed

Lopez, Ana D; Kayali, Ayse G; Hayek, Alberto; King, Charles C

2014-05-18

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation(1-9). However in vitro, genesis of insulin producing cells from human fetal ICCs is low(10); results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust(11-17). A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro(11-22), far fewer exist for ICCs(10,23,24). Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue(6). Although the examples presented here focus upon the pancreatic endoderm proliferation and β cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

Efficient stage-specific differentiation of human pluripotent stem cells toward retinal photoreceptor cells.

PubMed

Mellough, Carla B; Sernagor, Evelyne; Moreno-Gimeno, Inmaculada; Steel, David H W; Lako, Majlinda

2012-04-01

Recent successes in the stem cell field have identified some of the key chemical and biological cues which drive photoreceptor derivation from human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC); however, the efficiency of this process is variable. We have designed a three-step photoreceptor differentiation protocol combining previously published methods that direct the differentiation of hESC and hiPSC toward a retinal lineage, which we further modified with additional supplements selected on the basis of reports from the eye field and retinal development. We report that hESC and hiPSC differentiating under our regimen over a 60 day period sequentially acquire markers associated with neural, retinal field, retinal pigmented epithelium and photoreceptor cells, including mature photoreceptor markers OPN1SW and RHODOPSIN with a higher efficiency than previously reported. In addition, we report the ability of hESC and hiPSC cultures to generate neural and retinal phenotypes under minimal culture conditions, which may be linked to their ability to endogenously upregulate the expression of a range of factors important for retinal cell type specification. However, cultures that were differentiated with full supplementation under our photoreceptor-induction regimen achieve this within a significantly shorter time frame and show a substantial increase in the expression of photoreceptor-specific markers in comparison to cultures differentiated under minimal conditions. Interestingly, cultures supplemented only with B27 and/or N2 displayed comparable differentiation efficiency to those under full supplementation, indicating a key role for B27 and N2 during the differentiation process. Furthermore, our data highlight an important role for Dkk1 and Noggin in enhancing the differentiation of hESC and hiPSC toward retinal progenitor cells and photoreceptor precursors during the early stages of differentiation, while suggesting that further maturation of these cells into photoreceptors may not require additional factors and can ensue under minimal culture conditions. Copyright © 2012 AlphaMed Press.

The processing of asparagine-linked oligosaccharides in HT-29 cells is a function of their state of enterocytic differentiation. An accumulation of Man9,8-GlcNAc2-Asn species is indicative of an impaired N-glycan trimming in undifferentiated cells.

PubMed

Ogier-Denis, E; Codogno, P; Chantret, I; Trugnan, G

1988-05-05

Studies on the regulation of the enterocytic differentiation of the human colon cancer cell line HT-29, which is differentiated in the absence (Glc-) but not in the presence of glucose (Glc+), have recently shown that the post-translational processing of sucrase-isomaltase and particularly its glycosylation vary as a function of cell differentiation (Trugnan G., Rousset, M., Chantret, I., Barbat, A., and Zweibaum, A. (1987) J. Cell Biol. 104, 1199-1205). Other studies indicate that in undifferentiated HT-29 Glc+ cells there is an accumulation of UDP-N-acetylhexosamine, which is involved in the glycosylation process (Wice, B. M., Trugnan, G., Pinto, M., Rousset, M., Chevalier, G., Dussaulx, E., Lacroix, B., and Zweibaum, A. (1985) J. Biol. Chem. 260, 139-146). The purpose of the present work is to investigate whether an overall alteration of protein glycosylation is associated with the inability of HT-29 cells to differentiate. At least three alterations are detected: (i) after a 10-min pulse, the incorporation of D-[2-3H]mannose in undifferentiated cells is severely reduced, compared to differentiated cells. (ii) After a 24-h period of labeling with D-[2-3H]mannose, undifferentiated cells accumulate more than 60% of the radioactivity in the high mannose glycopeptides, whereas differentiated HT-29 Glc- cells accumulate only 38%. (iii) The analysis of the high mannose oligosaccharides transferred "en bloc" from the lipid precursor shows that Man9,8-GlcNAc2 species accumulate in undifferentiated cells, whereas no such accumulation can be detected in differentiated cells. This glycosylation pattern is consistent with an impairment of the trimming of high mannose into complex glycans. It is concluded that N-glycan processing is correlated with the state of enterocytic differentiation of HT-29 cells.

Brüstle v. Greenpeace: Implications for Commercialisation of Translational Stem Cell Research.

PubMed

Mansnérus, Juli

2015-04-01

The lack of consensus on a common definition of the term 'embryo' has resulted in legal uncertainty affecting the permissibility of human embryonic stem cell (hESC) research and the commercialisation prospects and patenting of inventions of hESC origin in the EU. The Brüstle v. Greenpeace case, which by providing a very broad definition of a human embryo restricts the patentability of hESC-based inventions, aims at harmonising the patenting practices regarding interpretation of Article 6.2.c of Directive 98/44/ EC. It fills the gaps in national laws by providing binding interpretation guidelines for national courts. As currently no marketing authorisations have been granted to hESC-based products, implications of this judgment for translational hESC research together with other barriers to commercialisation of such research need to be analysed. In addition, whether the main obstacles relate to patenting restrictions or whether something else in the innovation system is impeding the market entry of these innovative products is discussed.

Genome-wide DNA methylation drives human embryonic stem cell erythropoiesis by remodeling gene expression dynamics.

PubMed

Liu, Zhijing; Feng, Qiang; Sun, Pengpeng; Lu, Yan; Yang, Minlan; Zhang, Xiaowei; Jin, Xiangshu; Li, Yulin; Lu, Shi-Jiang; Quan, Chengshi

2017-12-01

To investigate the role of DNA methylation during erythrocyte production by human embryonic stem cells (hESCs). We employed an erythroid differentiation model from hESCs, and then tracked the genome-wide DNA methylation maps and gene expression patterns through an Infinium HumanMethylation450K BeadChip and an Ilumina Human HT-12 v4 Expression Beadchip, respectively. A negative correlation between DNA methylation and gene expression was substantially enriched during the later differentiation stage and was present in both the promoter and the gene body. Moreover, erythropoietic genes with differentially methylated CpG sites that were primarily enriched in nonisland regions were upregulated, and demethylation of their gene bodies was associated with the presence of enhancers and DNase I hypersensitive sites. Finally, the components of JAK-STAT-NF-κB signaling were DNA hypomethylated and upregulated, which targets the key genes for erythropoiesis. Erythroid lineage commitment by hESCs requires genome-wide DNA methylation modifications to remodel gene expression dynamics.

Knockdown of Fanconi anemia genes in human embryonic stem cells reveals early developmental defects in the hematopoietic lineage.

PubMed

Tulpule, Asmin; Lensch, M William; Miller, Justine D; Austin, Karyn; D'Andrea, Alan; Schlaeger, Thorsten M; Shimamura, Akiko; Daley, George Q

2010-04-29

Fanconi anemia (FA) is a genetically heterogeneous, autosomal recessive disorder characterized by pediatric bone marrow failure and congenital anomalies. The effect of FA gene deficiency on hematopoietic development in utero remains poorly described as mouse models of FA do not develop hematopoietic failure and such studies cannot be performed on patients. We have created a human-specific in vitro system to study early hematopoietic development in FA using a lentiviral RNA interference (RNAi) strategy in human embryonic stem cells (hESCs). We show that knockdown of FANCA and FANCD2 in hESCs leads to a reduction in hematopoietic fates and progenitor numbers that can be rescued by FA gene complementation. Our data indicate that hematopoiesis is impaired in FA from the earliest stages of development, suggesting that deficiencies in embryonic hematopoiesis may underlie the progression to bone marrow failure in FA. This work illustrates how hESCs can provide unique insights into human development and further our understanding of genetic disease.

A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells

PubMed Central

Nakagawa, Masato; Taniguchi, Yukimasa; Senda, Sho; Takizawa, Nanako; Ichisaka, Tomoko; Asano, Kanako; Morizane, Asuka; Doi, Daisuke; Takahashi, Jun; Nishizawa, Masatoshi; Yoshida, Yoshinori; Toyoda, Taro; Osafune, Kenji; Sekiguchi, Kiyotoshi; Yamanaka, Shinya

2014-01-01

In order to apply human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) to regenerative medicine, the cells should be produced under restricted conditions conforming to GMP guidelines. Since the conventional culture system has some issues that need to be addressed to achieve this goal, we developed a novel culture system. We found that recombinant laminin-511 E8 fragments are useful matrices for maintaining hESCs and hiPSCs when used in combination with a completely xeno-free (Xf) medium, StemFit™. Using this system, hESCs and hiPSCs can be easily and stably passaged by dissociating the cells into single cells for long periods, without any karyotype abnormalities. Human iPSCs could be generated under feeder-free (Ff) and Xf culture systems from human primary fibroblasts and blood cells, and they possessed differentiation abilities. These results indicate that hiPSCs can be generated and maintained under this novel Ff and Xf culture system. PMID:24399248

Distinct epigenomic landscapes of pluripotent and lineage-committed human cells.

PubMed

Hawkins, R David; Hon, Gary C; Lee, Leonard K; Ngo, Queminh; Lister, Ryan; Pelizzola, Mattia; Edsall, Lee E; Kuan, Samantha; Luu, Ying; Klugman, Sarit; Antosiewicz-Bourget, Jessica; Ye, Zhen; Espinoza, Celso; Agarwahl, Saurabh; Shen, Li; Ruotti, Victor; Wang, Wei; Stewart, Ron; Thomson, James A; Ecker, Joseph R; Ren, Bing

2010-05-07

Human embryonic stem cells (hESCs) share an identical genome with lineage-committed cells, yet possess the remarkable properties of self-renewal and pluripotency. The diverse cellular properties in different cells have been attributed to their distinct epigenomes, but how much epigenomes differ remains unclear. Here, we report that epigenomic landscapes in hESCs and lineage-committed cells are drastically different. By comparing the chromatin-modification profiles and DNA methylomes in hESCs and primary fibroblasts, we find that nearly one-third of the genome differs in chromatin structure. Most changes arise from dramatic redistributions of repressive H3K9me3 and H3K27me3 marks, which form blocks that significantly expand in fibroblasts. A large number of potential regulatory sequences also exhibit a high degree of dynamics in chromatin modifications and DNA methylation. Additionally, we observe novel, context-dependent relationships between DNA methylation and chromatin modifications. Our results provide new insights into epigenetic mechanisms underlying properties of pluripotency and cell fate commitment.

Nature vs. nurture: gold perpetuates "stemness".

PubMed

Paul, Willi; Sharma, Chandra P; Deb, Kaushik Dilip

2011-01-01

Adult tissues contain quiescent reservoirs of multipotent somatic stem cells and pluripotent embryonic-like stem cells (ELSCs). Credited with regenerative properties gold is used across both -contemporary and -ancient medicines. Here, we show that gold exerted these effects by enhancing the pool of pluripotent ELSC while improving their stemness. We used hESCs as an in-vitro model to understand if gold could enhance self-renewal and pluripotency. Swarna-bhasma (SB), an ancient Indian gold microparticulate (41.1 nm), preparation, reduced spontaneous-differentiation, improved self-renewal, pluripotency and proliferation of hESCs. Colloidal gold-nanoparticles (GNP) (15.59 nm) were tested to confirm that the observations were attributable to nanoparticulate-gold. SB and GNP exposure: maintained -stemness, -karyotypic stability, enhanced pluripotency till day-12, increased average colony-sizes, and reduced the number of autonomously-derived differentiated FGFR1 positive fibroblast-niche-cells/colony. Particulate-gold induced upregulation of FGFR1 and IGF2 expression, and decrease in IGF1 secretion indicates IGF1/2 mediated support for enhanced pluripotency and self-renewal in hESCs.

Pluripotent stem cell-derived natural killer cells for cancer therapy

PubMed Central

Knorr, David A.; Kaufman, Dan S.

2010-01-01

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) provide an accessible, genetically tractable and homogenous starting cell populations to efficiently study human blood cell development. These cell populations provide platforms to develop new cell-based therapies to treat both malignant and non-malignant hematological diseases. Our group has previously demonstrated the ability of hESC-derived hematopoietic precursors to produce functional natural killer (NK) cells as well as an explanation of the underlying mechanism responsible for inefficient development of T and B cells from hESCs. hESCs and iPSCs, which can be reliably engineered in vitro, provide an important new model system to study human lymphocyte development and produce enhanced cell-based therapies with potential to serve as a “universal” source of anti-tumor lymphocytes for novel clinical therapies. This review will focus on the application of hESC-derived NK cells with currently used and novel therapeutics for clinical trials, current barriers to translation, and future applications through genetic engineering approaches. PMID:20801411

Targeting the Gdnf Gene in peritubular myoid cells disrupts undifferentiated spermatogonial cell development

PubMed Central

Chen, Liang-Yu; Willis, William D.; Eddy, Edward M.

2016-01-01

Spermatogonial stem cells (SSCs) are a subpopulation of undifferentiated spermatogonia located in a niche at the base of the seminiferous epithelium delimited by Sertoli cells and peritubular myoid (PM) cells. SSCs self-renew or differentiate into spermatogonia that proliferate to give rise to spermatocytes and maintain spermatogenesis. Glial cell line-derived neurotrophic factor (GDNF) is essential for this process. Sertoli cells produce GDNF and other growth factors and are commonly thought to be responsible for regulating SSC development, but limited attention has been paid to the role of PM cells in this process. A conditional knockout (cKO) of the androgen receptor gene in PM cells resulted in male infertility. We found that testosterone (T) induces GDNF expression in mouse PM cells in vitro and neonatal spermatogonia (including SSCs) co-cultured with T-treated PM cells were able to colonize testes of germ cell-depleted mice after transplantation. This strongly suggested that T-regulated production of GDNF by PM cells is required for spermatogonial development, but PM cells might produce other factors in vitro that are responsible. In this study, we tested the hypothesis that production of GDNF by PM cells is essential for spermatogonial development by generating mice with a cKO of the Gdnf gene in PM cells. The cKO males sired up to two litters but became infertile due to collapse of spermatogenesis and loss of undifferentiated spermatogonia. These studies show for the first time, to our knowledge, that the production of GDNF by PM cells is essential for undifferentiated spermatogonial cell development in vivo. PMID:26831079

Recent Developments in β-Cell Differentiation of Pluripotent Stem Cells Induced by Small and Large Molecules

PubMed Central

Kumar, S. Suresh; Alarfaj, Abdullah A.; Munusamy, Murugan A.; Singh, A. J. A. Ranjith; Peng, I-Chia; Priya, Sivan Padma; Hamat, Rukman Awang; Higuchi, Akon

2014-01-01

Human pluripotent stem cells, including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), hold promise as novel therapeutic tools for diabetes treatment because of their self-renewal capacity and ability to differentiate into beta (β)-cells. Small and large molecules play important roles in each stage of β-cell differentiation from both hESCs and hiPSCs. The small and large molecules that are described in this review have significantly advanced efforts to cure diabetic disease. Lately, effective protocols have been implemented to induce hESCs and human mesenchymal stem cells (hMSCs) to differentiate into functional β-cells. Several small molecules, proteins, and growth factors promote pancreatic differentiation from hESCs and hMSCs. These small molecules (e.g., cyclopamine, wortmannin, retinoic acid, and sodium butyrate) and large molecules (e.g. activin A, betacellulin, bone morphogentic protein (BMP4), epidermal growth factor (EGF), fibroblast growth factor (FGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), noggin, transforming growth factor (TGF-α), and WNT3A) are thought to contribute from the initial stages of definitive endoderm formation to the final stages of maturation of functional endocrine cells. We discuss the importance of such small and large molecules in uniquely optimized protocols of β-cell differentiation from stem cells. A global understanding of various small and large molecules and their functions will help to establish an efficient protocol for β-cell differentiation. PMID:25526563

Human Embryonic and Induced Pluripotent Stem Cells Express TRAIL Receptors and Can Be Sensitized to TRAIL-Induced Apoptosis

PubMed Central

Vinarsky, Vladimir; Krivanek, Jan; Rankel, Liina; Nahacka, Zuzana; Barta, Tomas; Jaros, Josef; Andera, Ladislav

2013-01-01

Death ligands and their tumor necrosis factor receptor (TNFR) family receptors are the best-characterized and most efficient inducers of apoptotic signaling in somatic cells. In this study, we analyzed whether these prototypic activators of apoptosis are also expressed and able to be activated in human pluripotent stem cells. We examined human embryonic stem cells (hESC) and human-induced pluripotent stem cells (hiPSC) and found that both cell types express primarily TNF-related apoptosis-inducing ligand (TRAIL) receptors and TNFR1, but very low levels of Fas/CD95. We also found that although hESC and hiPSC contain all the proteins required for efficient induction and progression of extrinsic apoptotic signaling, they are resistant to TRAIL-induced apoptosis. However, both hESC and hiPSC can be sensitized to TRAIL-induced apoptosis by co-treatment with protein synthesis inhibitors such as the anti-leukemia drug homoharringtonine (HHT). HHT treatment led to suppression of cellular FLICE inhibitory protein (cFLIP) and Mcl-1 expression and, in combination with TRAIL, enhanced processing of caspase-8 and full activation of caspase-3. cFLIP likely represents an important regulatory node, as its shRNA-mediated down-regulation significantly sensitized hESC to TRAIL-induced apoptosis. Thus, we provide the first evidence that, irrespective of their origin, human pluripotent stem cells express canonical components of the extrinsic apoptotic system and on stress can activate death receptor-mediated apoptosis. PMID:23806100

Functional high-resolution time-course expression analysis of human embryonic stem cells undergoing cardiac induction.

PubMed

Piccini, Ilaria; Araúzo-Bravo, Marcos; Seebohm, Guiscard; Greber, Boris

2016-12-01

Cardiac induction of human embryonic stem cells (hESCs) is a process bearing increasing medical relevance, yet it is poorly understood from a developmental biology perspective. Anticipated technological progress in deriving stably expandable cardiac precursor cells or in advancing cardiac subtype specification protocols will likely require deeper insights into this fascinating system. Recent improvements in controlling hESC differentiation now enable a near-homogeneous induction of the cardiac lineage. This is based on an optimized initial stimulation of mesoderm-inducing signaling pathways such as Activin and/or FGF, BMP, and WNT, followed by WNT inhibition as a secondary requirement. Here, we describe a comprehensive data set based on varying hESC differentiation conditions in a systematic manner and recording high-resolution differentiation time-courses analyzed by genome-wide expression profiling (GEO accession number GSE67154). As a baseline, hESCs were differentiated into cardiomyocytes under optimal conditions. Moreover, in additional time-series, individual signaling factors were withdrawn from the initial stimulation cocktail to reveal their specific roles via comparison to the standard condition. Hence, this data set presents a rich resource for hypothesis generation in studying human cardiac induction, as we reveal numbers of known as well as uncharacterized genes prominently marking distinct intermediate stages in the process. These data will also be useful for identifying putative cardiac master regulators in the human system as well as for characterizing expandable cardiac stem cells.

The orphan nuclear receptor Nur77 regulates decidual prolactin expression in human endometrial stromal cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jiang, Yue; Hu, Yali; Zhao, Jing

2011-01-14

Research highlights: {yields} Decidually produced PRL plays a key role during pregnancy. {yields} Overexpression of Nur77 increased PRL mRNA expression and enhanced decidual PRL promoter activity. {yields} Knockdown of Nur77 decreased decidual PRL secretion induced by 8-Br-cAMP and MPA. {yields} Nur77 is a novel transcription factor that plays an active role in decidual prolactin expression. -- Abstract: Prolactin (PRL) is synthesized and released by several extrapituitary tissues, including decidualized stromal cells. Despite the important role of decidual PRL during pregnancy, little is understood about the factors involved in the proper regulation of decidual PRL expression. Here we present evidence thatmore » the transcription factor Nur77 plays an active role in decidual prolactin expression in human endometrial stromal cells (hESCs). Nur77 mRNA expression in hESCs was significantly increased after decidualization stimulated by 8-Br-cAMP and medroxyprogesterone acetate (MPA). Adenovirus-mediated overexpression of Nur77 in hESCs markedly increased PRL mRNA expression and enhanced decidual PRL promoter (dPRL/-332Luc) activity in a concentration-dependent manner. Furthermore, knockdown of Nur77 in hESCs significantly decreased decidual PRL promoter activation and substantially attenuated PRL mRNA expression and PRL secretion (P < 0.01) induced by 8-Br-cAMP and MPA. These results demonstrate that Nur77 is a novel transcription factor that contributes significantly to the regulation of prolactin gene expression in human endometrial stromal cells.« less

Undifferentiated Connective Tissue Disease

MedlinePlus

... Home Conditions Undifferentiated Connective Tissue Disease (UCTD) Undifferentiated Connective Tissue Disease (UCTD) Make an Appointment Find a Doctor ... by Barbara Goldstein, MD (February 01, 2016) Undifferentiated connective tissue disease (UCTD) is a systemic autoimmune disease. This ...

Discovery of Novel Isoforms of Huntingtin Reveals a New Hominid-Specific Exon

PubMed Central

Popowski, Melissa; Haremaki, Tomomi; Croft, Gist F.; Deglincerti, Alessia; Brivanlou, Ali H.

2015-01-01

Huntington’s disease (HD) is a devastating neurological disorder that is caused by an expansion of the poly-Q tract in exon 1 of the Huntingtin gene (HTT). HTT is an evolutionarily conserved and ubiquitously expressed protein that has been linked to a variety of functions including transcriptional regulation, mitochondrial function, and vesicle transport. This large protein has numerous caspase and calpain cleavage sites and can be decorated with several post-translational modifications such as phosphorylations, acetylations, sumoylations, and palmitoylations. However, the exact function of HTT and the role played by its modifications in the cell are still not well understood. Scrutiny of HTT function has been focused on a single, full length mRNA. In this study, we report the discovery of 5 novel HTT mRNA splice isoforms that are expressed in normal and HTT-expanded human embryonic stem cell (hESC) lines as well as in cortical neurons differentiated from hESCs. Interestingly, none of the novel isoforms generates a truncated protein. Instead, 4 of the 5 new isoforms specifically eliminate domains and modifications to generate smaller HTT proteins. The fifth novel isoform incorporates a previously unreported additional exon, dubbed 41b, which is hominid-specific and introduces a potential phosphorylation site in the protein. The discovery of this hominid-specific isoform may shed light on human-specific pathogenic mechanisms of HTT, which could not be investigated with current mouse models of the disease. PMID:26010866

The embryo research debate in Brazil: from the National Congress to the Federal Supreme Court.

PubMed

Cesarino, Letícia; Luna, Naara

2011-04-01

New forms of life produced by biomedical research, such as human embryonic stem cells (hESC), have been the object of public debate beyond the scientific fields involved. This article brings to light the case of Brazil, where recently passed federal legislation has authorized research with in vitro human embryos. It focuses on the legislative debate in the Brazilian National Congress between 2003 and 2005 on the Biosafety Bill of Law, which cleared for hESC research a certain share of supernumerary and unviable human embryos frozen in the country's assisted reproduction clinics. The passing of this Bill triggered other public reactions, chiefly a Direct Action of Unconstitutionality in Brazil's Federal Supreme Court. This study adopts an anthropological perspective for describing and analyzing the chief arguments in both debates, in terms of how the notion of 'life' was deployed and negotiated by contending parties. If, on the one hand, the definition of life appeared firmly attached to a conception of both the in vitro embryo and the fetus as a human person, on the other a movement towards breaking down life along utilitarian lines was found when the potential beneficiaries of stem cell therapy came into the equation. In all cases, however, notions of life were negotiated from a hybrid continuum of (biological) facts and (religious, moral and juridical) values, and resonated in different ways with the idea of the individual as privileged mode of constructing personhood in the context of modern nation states.

Lentiviral Vectors and Protocols for Creation of Stable hESC Lines for Fluorescent Tracking and Drug Resistance Selection of Cardiomyocytes

PubMed Central

Kita-Matsuo, Hiroko; Barcova, Maria; Prigozhina, Natalie; Salomonis, Nathan; Wei, Karen; Jacot, Jeffrey G.; Nelson, Brandon; Spiering, Sean; Haverslag, René; Kim, Changsung; Talantova, Maria; Bajpai, Ruchi; Calzolari, Diego; Terskikh, Alexey; McCulloch, Andrew D.; Price, Jeffrey H.; Conklin, Bruce R.; Chen, H. S. Vincent; Mercola, Mark

2009-01-01

Background Developmental, physiological and tissue engineering studies critical to the development of successful myocardial regeneration therapies require new ways to effectively visualize and isolate large numbers of fluorescently labeled, functional cardiomyocytes. Methodology/Principal Findings Here we describe methods for the clonal expansion of engineered hESCs and make available a suite of lentiviral vectors for that combine Blasticidin, Neomycin and Puromycin resistance based drug selection of pure populations of stem cells and cardiomyocytes with ubiquitous or lineage-specific promoters that direct expression of fluorescent proteins to visualize and track cardiomyocytes and their progenitors. The phospho-glycerate kinase (PGK) promoter was used to ubiquitously direct expression of histone-2B fused eGFP and mCherry proteins to the nucleus to monitor DNA content and enable tracking of cell migration and lineage. Vectors with T/Brachyury and α-myosin heavy chain (αMHC) promoters targeted fluorescent or drug-resistance proteins to early mesoderm and cardiomyocytes. The drug selection protocol yielded 96% pure cardiomyocytes that could be cultured for over 4 months. Puromycin-selected cardiomyocytes exhibited a gene expression profile similar to that of adult human cardiomyocytes and generated force and action potentials consistent with normal fetal cardiomyocytes, documenting these parameters in hESC-derived cardiomyocytes and validating that the selected cells retained normal differentiation and function. Conclusion/Significance The protocols, vectors and gene expression data comprise tools to enhance cardiomyocyte production for large-scale applications. PMID:19352491

Method for evaluation of human induced pluripotent stem cell quality using image analysis based on the biological morphology of cells.

PubMed

Wakui, Takashi; Matsumoto, Tsuyoshi; Matsubara, Kenta; Kawasaki, Tomoyuki; Yamaguchi, Hiroshi; Akutsu, Hidenori

2017-10-01

We propose an image analysis method for quality evaluation of human pluripotent stem cells based on biologically interpretable features. It is important to maintain the undifferentiated state of induced pluripotent stem cells (iPSCs) while culturing the cells during propagation. Cell culture experts visually select good quality cells exhibiting the morphological features characteristic of undifferentiated cells. Experts have empirically determined that these features comprise prominent and abundant nucleoli, less intercellular spacing, and fewer differentiating cellular nuclei. We quantified these features based on experts' visual inspection of phase contrast images of iPSCs and found that these features are effective for evaluating iPSC quality. We then developed an iPSC quality evaluation method using an image analysis technique. The method allowed accurate classification, equivalent to visual inspection by experts, of three iPSC cell lines.

Dynamic Proteomic Analysis of Pancreatic Mesenchyme Reveals Novel Factors That Enhance Human Embryonic Stem Cell to Pancreatic Cell Differentiation.

PubMed

Russ, Holger A; Landsman, Limor; Moss, Christopher L; Higdon, Roger; Greer, Renee L; Kaihara, Kelly; Salamon, Randy; Kolker, Eugene; Hebrok, Matthias

2016-01-01

Current approaches in human embryonic stem cell (hESC) to pancreatic beta cell differentiation have largely been based on knowledge gained from developmental studies of the epithelial pancreas, while the potential roles of other supporting tissue compartments have not been fully explored. One such tissue is the pancreatic mesenchyme that supports epithelial organogenesis throughout embryogenesis. We hypothesized that detailed characterization of the pancreatic mesenchyme might result in the identification of novel factors not used in current differentiation protocols. Supplementing existing hESC differentiation conditions with such factors might create a more comprehensive simulation of normal development in cell culture. To validate our hypothesis, we took advantage of a novel transgenic mouse model to isolate the pancreatic mesenchyme at distinct embryonic and postnatal stages for subsequent proteomic analysis. Refined sample preparation and analysis conditions across four embryonic and prenatal time points resulted in the identification of 21,498 peptides with high-confidence mapping to 1,502 proteins. Expression analysis of pancreata confirmed the presence of three potentially important factors in cell differentiation: Galectin-1 (LGALS1), Neuroplastin (NPTN), and the Laminin α-2 subunit (LAMA2). Two of the three factors (LGALS1 and LAMA2) increased expression of pancreatic progenitor transcript levels in a published hESC to beta cell differentiation protocol. In addition, LAMA2 partially blocks cell culture induced beta cell dedifferentiation. Summarily, we provide evidence that proteomic analysis of supporting tissues such as the pancreatic mesenchyme allows for the identification of potentially important factors guiding hESC to pancreas differentiation.

Dynamic Proteomic Analysis of Pancreatic Mesenchyme Reveals Novel Factors That Enhance Human Embryonic Stem Cell to Pancreatic Cell Differentiation

PubMed Central

Russ, Holger A.; Landsman, Limor; Moss, Christopher L.; Higdon, Roger; Greer, Renee L.; Kaihara, Kelly; Salamon, Randy; Kolker, Eugene; Hebrok, Matthias

2016-01-01

Current approaches in human embryonic stem cell (hESC) to pancreatic beta cell differentiation have largely been based on knowledge gained from developmental studies of the epithelial pancreas, while the potential roles of other supporting tissue compartments have not been fully explored. One such tissue is the pancreatic mesenchyme that supports epithelial organogenesis throughout embryogenesis. We hypothesized that detailed characterization of the pancreatic mesenchyme might result in the identification of novel factors not used in current differentiation protocols. Supplementing existing hESC differentiation conditions with such factors might create a more comprehensive simulation of normal development in cell culture. To validate our hypothesis, we took advantage of a novel transgenic mouse model to isolate the pancreatic mesenchyme at distinct embryonic and postnatal stages for subsequent proteomic analysis. Refined sample preparation and analysis conditions across four embryonic and prenatal time points resulted in the identification of 21,498 peptides with high-confidence mapping to 1,502 proteins. Expression analysis of pancreata confirmed the presence of three potentially important factors in cell differentiation: Galectin-1 (LGALS1), Neuroplastin (NPTN), and the Laminin α-2 subunit (LAMA2). Two of the three factors (LGALS1 and LAMA2) increased expression of pancreatic progenitor transcript levels in a published hESC to beta cell differentiation protocol. In addition, LAMA2 partially blocks cell culture induced beta cell dedifferentiation. Summarily, we provide evidence that proteomic analysis of supporting tissues such as the pancreatic mesenchyme allows for the identification of potentially important factors guiding hESC to pancreas differentiation. PMID:26681951

Hepatic Differentiation and Maturation of Human Embryonic Stem Cells Cultured in a Perfused Three-Dimensional Bioreactor

PubMed Central

Synnergren, Jane; Jensen, Janne; Björquist, Petter; Ingelman-Sundberg, Magnus

2013-01-01

Drug-induced liver injury is a serious and frequently occurring adverse drug reaction in the clinics and is hard to predict during preclinical studies. Today, primary hepatocytes are the most frequently used cell model for drug discovery and prediction of toxicity. However, their use is marred by high donor variability regarding drug metabolism and toxicity, and instable expression levels of liver-specific genes such as cytochromes P450. An in vitro model system based on human embryonic stem cells (hESC), with their unique properties of pluripotency and self-renewal, has potential to provide a stable and unlimited supply of human hepatocytes. Much effort has been made to direct hESC toward the hepatic lineage, mostly using 2-dimensional (2D) cultures. Although the results are encouraging, these cells lack important functionality. Here, we investigate if hepatic differentiation of hESC can be improved by using a 3-dimensional (3D) bioreactor system. Human ESCs were differentiated toward the hepatic lineage using the same cells in either the 3D or 2D system. A global transcriptional analysis identified important differences between the 2 differentiation regimes, and we identified 10 pathways, highly related to liver functions, which were significantly upregulated in cells differentiated in the bioreactor compared to 2D control cultures. The enhanced hepatic differentiation observed in the bioreactor system was also supported by immunocytochemistry. Taken together, our results suggest that hepatic differentiation of hESC is improved when using this 3D bioreactor technology as compared to 2D culture systems. PMID:22970843

Hepatic differentiation and maturation of human embryonic stem cells cultured in a perfused three-dimensional bioreactor.

PubMed

Sivertsson, Louise; Synnergren, Jane; Jensen, Janne; Björquist, Petter; Ingelman-Sundberg, Magnus

2013-02-15

Drug-induced liver injury is a serious and frequently occurring adverse drug reaction in the clinics and is hard to predict during preclinical studies. Today, primary hepatocytes are the most frequently used cell model for drug discovery and prediction of toxicity. However, their use is marred by high donor variability regarding drug metabolism and toxicity, and instable expression levels of liver-specific genes such as cytochromes P450. An in vitro model system based on human embryonic stem cells (hESC), with their unique properties of pluripotency and self-renewal, has potential to provide a stable and unlimited supply of human hepatocytes. Much effort has been made to direct hESC toward the hepatic lineage, mostly using 2-dimensional (2D) cultures. Although the results are encouraging, these cells lack important functionality. Here, we investigate if hepatic differentiation of hESC can be improved by using a 3-dimensional (3D) bioreactor system. Human ESCs were differentiated toward the hepatic lineage using the same cells in either the 3D or 2D system. A global transcriptional analysis identified important differences between the 2 differentiation regimes, and we identified 10 pathways, highly related to liver functions, which were significantly upregulated in cells differentiated in the bioreactor compared to 2D control cultures. The enhanced hepatic differentiation observed in the bioreactor system was also supported by immunocytochemistry. Taken together, our results suggest that hepatic differentiation of hESC is improved when using this 3D bioreactor technology as compared to 2D culture systems.

Comparison of Nutech Functional Score with European Stroke Scale for Patients with Cerebrovascular Accident Treated with Human Embryonic Stem Cells: NFS for CVA Patients Treated with hESCs.

PubMed

Shroff, Geeta

2017-06-01

Stem cell therapy is a promising modality for treatment of patients with chronic cerebrovascular accident (CVA) in whom treatment other than physiotherapy or occupational therapy does not address the repair or recovery of the lost function. In this study, the author aimed at evaluating CVA patients treated with human embryonic stem cell (hESC) therapy and comparing their study outcomes with globally accepted European Stroke Scale (ESS) to that with novel scoring system, Nutech functional score (NFS), a 21-point positional and directional scoring system for assessing patients with CVA. Patients diagnosed with CVA were assessed with NFS and ESS before and after hESC therapy. NFS assessed the patients in the direction of 1-5 (bad to good), where 5 was considered as the highest possible grade (HPG). The findings were obtained for the patients who scored HPG, and had shown improvement by at least one grade. Overall, 66.7% of patients scored HPG level on the NFS scale and about 62.5% of the patients scored HPG according to the ESS scale. Approximately, 52.2% patients showed an improvement of 100% (by at least one grade) on NFS scale. None of the patients showed 100% improvement in the alteration of the score by at least one grade when scored with ESS. NFS and ESS scores show that a large population of CVA patients was benefitted with hESC therapy. NFS was found to give more convincing results than ESS, and overcomes the shortcomings of ESS.

Transplantation of Human Embryonic Stem Cells in Patients with Multiple Sclerosis and Lyme Disease

PubMed Central

Shroff, Geeta

2016-01-01

Case series Patient: Male, 42 • Female, 30 Final Diagnosis: Human embryonic stem cells showed good therapeutic potential for treatment of multiple sclerosis with lyme disease Symptoms: Fatigue • weakness in limbs Medication: — Clinical Procedure: Human embryonic stem cells transplantation Specialty: Transplantology Objective: Rare disease Background: Multiple sclerosis (MS) is an inflammatory and neurodegenerative disease in which the myelin sheath of nerve cells is damaged. It can cause delayed neurologic symptoms similar to those seen in Lyme disease (LD) patients. Thymus derived T-cells (myelin reactive) migrate to the blood brain barrier and stimulate an inflammatory cascade in the central nervous system. Cell based therapies play an important role in treating neurological diseases such as MS and LD. Case Report: Human embryonic stem cell (hESC) therapy was used to treat two patients with both MS and LD. The hESCs were administered via different routes including intramuscular, intravenous, and supplemental routes (e.g., deep spinal, caudal, intercostal through eye drops) to regenerate the injured cells. Both the patients showed remarkable improvement in their functional skills, overall stamina, cognitive abilities, and muscle strength. Furthermore, the improvement in the patients’ conditions were assessed by magnetic resonance tractography and single photon emission computed tomography (SPECT). Conclusions: Therapy with hESCs might emerge as an effective and safe treatment for patients with both MS and LD. Well-designed clinical trials and follow-up studies are needed to prove the long-term efficacy and safety of hESC therapy in the treatment of patients with MS and LD. PMID:27956736

miR-373 is regulated by TGFβ signaling and promotes mesendoderm differentiation in human Embryonic Stem Cells

PubMed Central

Rosa, Alessandro; Papaioannou, Marilena D.; Krzyspiak, Joanna E.; Brivanlou, Ali H.

2014-01-01

MicroRNAs (miRNAs) belonging to the evolutionary conserved miR-302 family play important functions in Embryonic Stem Cells (ESCs). The expression of some members, such as the human miR-302 and mouse miR-290 clusters, is regulated by ESC core transcription factors. However, whether miRNAs act downstream of signaling pathways involved in human ESC pluripotency remains unknown. The maintenance of pluripotency in hESCs is under the control of the TGFβ pathway. Here, we show that inhibition of the Activin/Nodal branch of this pathway affects the expression of a subset of miRNAs in hESCs. Among them, we found miR-373, a member of the miR-302 family. Proper levels of miR-373 are crucial for the maintenance of hESC pluripotency, since its overexpression leads to differentiation towards the mesendodermal lineage. Among miR-373 predicted targets, involved in TGFβ signaling, we validated the Nodal inhibitor Lefty. Our work suggests a crucial role for the interplay between miRNAs and signaling pathways in ESCs. PMID:24709321

Laser-induced fusion of human embryonic stem cells with optical tweezers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen Shuxun; Wang Xiaolin; Sun Dong

2013-07-15

We report a study on the laser-induced fusion of human embryonic stem cells (hESCs) at the single-cell level. Cells were manipulated by optical tweezers and fused under irradiation with pulsed UV laser at 355 nm. Successful fusion was indicated by green fluorescence protein transfer. The influence of laser pulse energy on the fusion efficiency was investigated. The fused products were viable as gauged by live cell staining. Successful fusion of hESCs with somatic cells was also demonstrated. The reported fusion outcome may facilitate studies of cell differentiation, maturation, and reprogramming.

Undifferentiated negative affect and impulsivity in borderline personality and depressive disorders: A momentary perspective.

PubMed

Tomko, Rachel L; Lane, Sean P; Pronove, Lisa M; Treloar, Hayley R; Brown, Whitney C; Solhan, Marika B; Wood, Phillip K; Trull, Timothy J

2015-08-01

Individuals with borderline personality disorder (BPD) often report experiencing several negative emotions simultaneously, an indicator of "undifferentiated" negative affect. The current study examined the relationship between undifferentiated negative affect and impulsivity. Participants with a current BPD (n = 67) or depressive disorder (DD; n = 38) diagnosis carried an electronic diary for 28 days, reporting on emotions and impulsivity when randomly prompted (up to 6 times per day). Undifferentiated negative affect was quantified using momentary intraclass correlation coefficients, which indicated how consistently negative emotion items were rated across fear, hostility, and sadness subscales. Undifferentiated negative affect at the occasion-level, day-level, and across 28 days was used to predict occasion-level impulsivity. Multilevel modeling was used to test the hypothesis that undifferentiated negative emotion would be a significant predictor of momentary impulsivity above and beyond levels of overall negative affect. Undifferentiated negative affect at the occasion and day levels were significant predictors of occasion-level impulsivity, but undifferentiated negative affect across the 28-day study period was only marginally significant. Results did not differ depending on BPD or DD status, though individuals with BPD did report significantly greater momentary impulsivity and undifferentiated negative affect. Undifferentiated negative affect may increase risk for impulsivity among individuals with BPD and depressive disorders, and the current data suggest that this process can be relatively immediate as well as cumulative over the course of a day. This research supports the consideration of undifferentiated negative affect as a transdiagnostic construct, but one that may be particularly relevant for those with BPD. (c) 2015 APA, all rights reserved).

Human Decidua-Derived Mesenchymal Cells Are a Promising Source for the Generation and Cell Banking of Human Induced Pluripotent Stem Cells

PubMed Central

Shofuda, Tomoko; Kanematsu, Daisuke; Fukusumi, Hayato; Yamamoto, Atsuyo; Bamba, Yohei; Yoshitatsu, Sumiko; Suemizu, Hiroshi; Nakamura, Masato; Sugimoto, Yoshikazu; Furue, Miho Kusuda; Kohara, Arihiro; Akamatsu, Wado; Okada, Yohei; Okano, Hideyuki; Yamasaki, Mami; Kanemura, Yonehiro

2013-01-01

Placental tissue is a biomaterial with remarkable potential for use in regenerative medicine. It has a three-layer structure derived from the fetus (amnion and chorion) and the mother (decidua), and it contains huge numbers of cells. Moreover, placental tissue can be collected without any physical danger to the donor and can be matched with a variety of HLA types. The decidua-derived mesenchymal cells (DMCs) are highly proliferative fibroblast-like cells that express a similar pattern of CD antigens as bone marrow-derived mesenchymal cells (BM-MSCs). Here we demonstrated that induced pluripotent stem (iPS) cells could be efficiently generated from DMCs by retroviral transfer of reprogramming factor genes. DMC-hiPS cells showed equivalent characteristics to human embryonic stem cells (hESCs) in colony morphology, global gene expression profile (including human pluripotent stem cell markers), DNA methylation status of the OCT3/4 and NANOG promoters, and ability to differentiate into components of the three germ layers in vitro and in vivo. The RNA expression of XIST and the methylation status of its promoter region suggested that DMC-iPSCs, when maintained undifferentiated and pluripotent, had three distinct states: (1) complete X-chromosome reactivation, (2) one inactive X-chromosome, or (3) an epigenetic aberration. Because DMCs are derived from the maternal portion of the placenta, they can be collected with the full consent of the adult donor and have considerable ethical advantages for cell banking and the subsequent generation of human iPS cells for regenerative applications. PMID:26858858

Roles of Estrogen Receptor-α and the Coactivator MED1 During Human Endometrial Decidualization

PubMed Central

Kaya Okur, Hatice S.; Das, Amrita; Taylor, Robert N.; Bagchi, Indrani C.

2016-01-01

The steroid hormones 17β-estradiol and progesterone are critical regulators of endometrial stromal cell differentiation, known as decidualization, which is a prerequisite for successful establishment of pregnancy. The present study using primary human endometrial stromal cells (HESCs) addressed the role of estrogen receptor-α (ESR1) in decidualization. Knockdown of ESR1 transcripts by RNA interference led to a marked reduction in decidualization of HESCs. Gene expression profiling at an early stage of decidualization indicated that ESR1 negatively regulates several cell cycle regulatory factors, thereby suppressing the proliferation of HESCs as these cells enter the differentiation program. ESR1 also controls the expression of WNT4, FOXO1, and progesterone receptor (PGR), well-known mediators of decidualization. Whereas ESR1 knockdown strongly inhibited the expression of FOXO1 and WNT4 transcripts within 24 hours of the initiation of decidualization, PGR expression remained unaffected at this early time point. Our study also revealed a major role of cAMP signaling in influencing the function of ESR1 during decidualization. Using a proteomic approach, we discovered that the cAMP-dependent protein kinase A (PKA) phosphorylates Mediator 1 (MED1), a subunit of the mediator coactivator complex, during HESC differentiation. Using immunoprecipitation, we demonstrated that PKA-phosphorylated MED1 interacts with ESR1. The PKA-dependent phosphorylation of MED1 was also correlated with its enhanced recruitment to estrogen-responsive elements in the WNT4 gene. Knockdown of MED1 transcripts impaired the expression of ESR1-induced WNT4 and FOXO1 transcripts and blocked decidualization. Based on these findings, we conclude that modulation of ESR1-MED1 interactions by cAMP signaling plays a critical role in human decidualization. PMID:26849466

Pluripotent and Multipotent Stem Cells Display Distinct Hypoxic miRNA Expression Profiles

PubMed Central

Agrawal, Rahul; Dale, Tina P.; Al-Zubaidi, Mohammed A.; Benny Malgulwar, Prit; Forsyth, Nicholas R.; Kulshreshtha, Ritu

2016-01-01

MicroRNAs are reported to have a crucial role in the regulation of self-renewal and differentiation of stem cells. Hypoxia has been identified as a key biophysical element of the stem cell culture milieu however, the link between hypoxia and miRNA expression in stem cells remains poorly understood. We therefore explored miRNA expression in hypoxic human embryonic and mesenchymal stem cells (hESCs and hMSCs). A total of 50 and 76 miRNAs were differentially regulated by hypoxia (2% O2) in hESCs and hMSCs, respectively, with a negligible overlap of only three miRNAs. We found coordinate regulation of precursor and mature miRNAs under hypoxia suggesting their regulation mainly at transcriptional level. Hypoxia response elements were located upstream of 97% of upregulated hypoxia regulated miRNAs (HRMs) suggesting hypoxia-inducible-factor (HIF) driven transcription. HIF binding to the candidate cis-elements of specific miRNAs under hypoxia was confirmed by Chromatin immunoprecipitation coupled with qPCR. Role analysis of a subset of upregulated HRMs identified linkage to reported inhibition of differentiation while a downregulated subset of HRMs had a putative role in the promotion of differentiation. MiRNA-target prediction correlation with published hypoxic hESC and hMSC gene expression profiles revealed HRM target genes enriched in the cytokine:cytokine receptor, HIF signalling and pathways in cancer. Overall, our study reveals, novel and distinct hypoxia-driven miRNA signatures in hESCs and hMSCs with the potential for application in optimised culture and differentiation models for both therapeutic application and improved understanding of stem cell biology. PMID:27783707

In Silico Analysis Identifies a Novel Role for Androgens in the Regulation of Human Endometrial Apoptosis

PubMed Central

Marshall, Elaine; Lowrey, Jacqueline; MacPherson, Sheila; Maybin, Jacqueline A.; Collins, Frances; Critchley, Hilary O. D.

2011-01-01

Context: The endometrium is a multicellular, steroid-responsive tissue that undergoes dynamic remodeling every menstrual cycle in preparation for implantation and, in absence of pregnancy, menstruation. Androgen receptors are present in the endometrium. Objective: The objective of the study was to investigate the impact of androgens on human endometrial stromal cells (hESC). Design: Bioinformatics was used to identify an androgen-regulated gene set and processes associated with their function. Regulation of target genes and impact of androgens on cell function were validated using primary hESC. Setting: The study was conducted at the University Research Institute. Patients: Endometrium was collected from women with regular menses; tissues were used for recovery of cells, total mRNA, or protein and for immunohistochemistry. Results: A new endometrial androgen target gene set (n = 15) was identified. Bioinformatics revealed 12 of these genes interacted in one pathway and identified an association with control of cell survival. Dynamic androgen-dependent changes in expression of the gene set were detected in hESC with nine significantly down-regulated at 2 and/or 8 h. Treatment of hESC with dihydrotestosterone reduced staurosporine-induced apoptosis and cell migration/proliferation. Conclusions: Rigorous in silico analysis resulted in identification of a group of androgen-regulated genes expressed in human endometrium. Pathway analysis and functional assays suggest androgen-dependent changes in gene expression may have a significant impact on stromal cell proliferation, migration, and survival. These data provide the platform for further studies on the role of circulatory or local androgens in the regulation of endometrial function and identify androgens as candidates in the pathogenesis of common endometrial disorders including polycystic ovarian syndrome, cancer, and endometriosis. PMID:21865353

Transcriptome analysis reveals determinant stages controlling human embryonic stem cell commitment to neuronal cells.

PubMed

Li, Yuanyuan; Wang, Ran; Qiao, Nan; Peng, Guangdun; Zhang, Ke; Tang, Ke; Han, Jing-Dong J; Jing, Naihe

2017-12-01

Proper neural commitment is essential for ensuring the appropriate development of the human brain and for preventing neurodevelopmental diseases such as autism spectrum disorders, schizophrenia, and intellectual disorders. However, the molecular mechanisms underlying the neural commitment in humans remain elusive. Here, we report the establishment of a neural differentiation system based on human embryonic stem cells (hESCs) and on comprehensive RNA sequencing analysis of transcriptome dynamics during early hESC differentiation. Using weighted gene co-expression network analysis, we reveal that the hESC neurodevelopmental trajectory has five stages: pluripotency (day 0); differentiation initiation (days 2, 4, and 6); neural commitment (days 8-10); neural progenitor cell proliferation (days 12, 14, and 16); and neuronal differentiation (days 18, 20, and 22). These stages were characterized by unique module genes, which may recapitulate the early human cortical development. Moreover, a comparison of our RNA-sequencing data with several other transcriptome profiling datasets from mice and humans indicated that Module 3 associated with the day 8-10 stage is a critical window of fate switch from the pluripotency to the neural lineage. Interestingly, at this stage, no key extrinsic signals were activated. In contrast, using CRISPR/Cas9-mediated gene knockouts, we also found that intrinsic hub transcription factors, including the schizophrenia-associated SIX3 gene and septo-optic dysplasia-related HESX1 gene, are required to program hESC neural determination. Our results improve the understanding of the mechanism of neural commitment in the human brain and may help elucidate the etiology of human mental disorders and advance therapies for managing these conditions. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Early embryonic sensitivity to cyclophosphamide in cardiac differentiation from human embryonic stem cells.

PubMed

Zhu, Ming-Xia; Zhao, Jin-Yuan; Chen, Gui-An; Guan, Li

2011-09-01

hESCs (human embryonic stem cells) can differentiate into tissue derivatives of all three germ layers in vitro and mimic the development of the embryo in vivo. In this study, we have investigated the potential of an hESC-based assay for the detection of toxicity to cardiac differentiation in embryonic development. First of all, we developed the protocol of cardiac induction from hESCs according to our previous work and distinguished cardiac precursor cells and late mature cardiomyocytes from differentiated cells, demonstrated by the Q-PCR (quantitative real-time PCR), immunocytochemistry and flow cytometry analysis. In order to test whether CPA (cyclophosphamide) induces developmental and cellular toxicity in the human embryo, we exposed the differentiating cells from hESCs to CPA (a well-known proteratogen) at different stages. We have found that a high concentration of CPA could inhibit cardiac differentiation of hESCs. Two separate exposure intervals were used to determine the effects of CPA on cardiac precursor cells and late mature cardiomyocytes respectively. The cardiac precursor cells were sensitive to CPA in non-cytotoxic concentrations for the expression of the cardiac-specific mRNA markers Nkx2.5 (NK2 transcription factor related, locus 5), GATA-4 (GATA binding protein 4 transcription factor) and TNNT2 (troponin T type 2). Non-cytotoxic CPA concentrations did not affect the mRNA markers' expression in late mature cardiomyocytes, indicating that cardiac precursors were more sensitive to CPA than late cardiomyocytes in cardiogenesis. We set up the in vitro developmental toxicity test model so as to reduce the number of test animals and expenses without compromising the safety of consumers and patients. Furthermore, such in vitro methods may be possibly suited to test a large number of chemicals than the classical employed in vivo tests.

Raf-1 levels determine the migration rate of primary endometrial stromal cells of patients with endometriosis

PubMed Central

Yotova, Iveta; Quan, Ping; Gaba, Aulona; Leditznig, Nadja; Pateisky, Petra; Kurz, Christine; Tschugguel, Walter

2012-01-01

Endometriosis is a disease characterized by the localization of endometrial tissue outside the uterine cavity. The differences observed in migration of human endometrial stromal cells (hESC) obtained from patients with endometriosis versus healthy controls were proposed to correlate with the abnormal activation of Raf-1/ROCKII signalling pathway. To evaluate the mechanism by which Raf-1 regulates cytoskeleton reorganization and motility, we used primary eutopic (Eu-, n = 16) and ectopic (Ec-, n = 8; isolated from ovarian cysts) hESC of patients with endometriosis and endometriosis-free controls (Co-hESC, n = 14). Raf-1 siRNA knockdown in Co- and Eu-hESC resulted in contraction and decreased migration versus siRNA controls. This phenotype was reversed following the re-expression of Raf-1 in these cells. Lowest Raf-1 levels in Ec-hESC were associated with hyperactivated ROCKII and ezrin/radixin/moesin (E/R/M), impaired migration and a contracted phenotype similar to Raf-1 knockdown in Co- and Eu-hESC. We further show that the mechanism by which Raf-1 mediates migration in hESC includes direct myosin light chain phosphatase (MYPT1) phosphorylation and regulation of the levels of E/R/M, paxillin, MYPT1 and myosin light chain (MLC) phosphorylation indirectly via the hyperactivation of ROCKII kinase. Furthermore, we suggest that in contrast to Co-and Eu-hESC, where the cellular Raf-1 levels regulate the rate of migration, the low cellular Raf-1 content in Ec-hESC, might ensure their restricted migration by preserving the contracted cellular phenotype. In conclusion, our findings suggest that cellular levels of Raf-1 adjust the threshold of hESC migration in endometriosis. PMID:22225925

Quality Assurance in Stem Cell Banking: Emphasis on Embryonic and Induced Pluripotent Stem Cell Banking.

PubMed

Kallur, Therése; Blomberg, Pontus; Stenfelt, Sonya; Tryggvason, Kristian; Hovatta, Outi

2017-01-01

For quality assurance (QA) in stem cell banking, a planned system is needed to ensure that the banked products, stem cells, meet the standards required for research, clinical use, and commercial biotechnological applications. QA is process oriented, avoids, or minimizes unacceptable product defects, and particularly encompasses the management and operational systems of the bank, as well as the ethical and legal frameworks. Quality control (QC ) is product oriented and therefore ensures the stem cells of a bank are what they are expected to be. Testing is for controlling, not assuring, product quality, and is therefore a part of QC , not QA. Like QA, QC is essential for banking cells for quality research and translational application (Schwartz et al., Lancet 379:713-720, 2012). Human embryonic stem cells (hESCs), as cells derived from donated supernumerary embryos from in vitro fertilization (IVF) therapy, are different from other stem cell types in resulting from an embryo that has had two donors . This imposes important ethical and legal constraints on the utility of the cells, which, together with quite specific culture conditions, require special attention in the QA system. Importantly, although the origin and derivation of induced pluripotent stem cells (iPSCs ) differ from that of hESCs, many of the principles of QA for hESC banking are applicable to iPSC banking (Stacey et al., Cell Stem Cell 13:385-388, 2013). Furthermore, despite differences between the legal and regulatory frameworks for hESC and iPSC banking between different countries, the requirements for QA are being harmonized (Stacey et al., Cell Stem Cell 13:385-388, 2013; International Stem Cell Banking Initiative, Stem Cell Rev 5:301-314, 2009).

ARG1 Functions in the Physiological Adaptation of Undifferentiated Plant Cells to Spaceflight

NASA Astrophysics Data System (ADS)

Zupanska, Agata K.; Schultz, Eric R.; Yao, JiQiang; Sng, Natasha J.; Zhou, Mingqi; Callaham, Jordan B.; Ferl, Robert J.; Paul, Anna-Lisa

2017-11-01

Scientific access to spaceflight and especially the International Space Station has revealed that physiological adaptation to spaceflight is accompanied or enabled by changes in gene expression that significantly alter the transcriptome of cells in spaceflight. A wide range of experiments have shown that plant physiological adaptation to spaceflight involves gene expression changes that alter cell wall and other metabolisms. However, while transcriptome profiling aptly illuminates changes in gene expression that accompany spaceflight adaptation, mutation analysis is required to illuminate key elements required for that adaptation. Here we report how transcriptome profiling was used to gain insight into the spaceflight adaptation role of Altered response to gravity 1 (Arg1), a gene known to affect gravity responses in plants on Earth. The study compared expression profiles of cultured lines of Arabidopsis thaliana derived from wild-type (WT) cultivar Col-0 to profiles from a knock-out line deficient in the gene encoding ARG1 (ARG1 KO), both on the ground and in space. The cell lines were launched on SpaceX CRS-2 as part of the Cellular Expression Logic (CEL) experiment of the BRIC-17 spaceflight mission. The cultured cell lines were grown within 60 mm Petri plates in Petri Dish Fixation Units (PDFUs) that were housed within the Biological Research In Canisters (BRIC) hardware. Spaceflight samples were fixed on orbit. Differentially expressed genes were identified between the two environments (spaceflight and comparable ground controls) and the two genotypes (WT and ARG1 KO). Each genotype engaged unique genes during physiological adaptation to the spaceflight environment, with little overlap. Most of the genes altered in expression in spaceflight in WT cells were found to be Arg1-dependent, suggesting a major role for that gene in the physiological adaptation of undifferentiated cells to spaceflight.

Sodium and calcium currents in neuroblastoma x glioma hybrid cells before and after morphological differentiation by dibutyryl cyclic AMP.

PubMed

Bodewei, R; Hering, S; Schubert, B; Wollenberger, A

1985-04-01

Sodium and calcium inward currents (INa and ICa) were measured in neuroblastoma X glioma hybrid cells of clones 108CC5 and 108CC15 by a single suction pipette method for internal perfusion and voltage clamp. Morphologically undifferentiated, exponentially growing cells were compared with cells differentiated by cultivation with 1 mmol/l dibutyryl cyclic AMP. Outward currents were eliminated by perfusing the cells with a K+-free solution. Voltage dependence and ion selectivity as well as steady state inactivation characteristics of INa and ICa resembled those of differentiated mouse neuroblastoma cells, clone N1E-115 (Moolenaar and Spector 1978, 1979). These parameters were identical in undifferentiated and differentiated cells of both clones. After differentiation the average density of the peak sodium and calcium currents was increased two and four-fold, respectively, in both cell lines. Our data indicate that exponentially growing, morphologically undifferentiated 108CC5 and 108CC15 neuroblastoma X glioma hybrid cells possess functional Na+ and Ca2+ channels undistinguishable from those of non-proliferating cells of these clones differentiated morphologically by treatment with dibutyryl cyclic AMP. That Na+ and Ca2+ spikes were not detected by other authors in these cells prior to morphological differentiation by dibutyryl cyclic AMP may be attributed to the fact that at the low resting membrane potential measured the Na+ and Ca2+ channels are inactivated.

Characterization of Phenotypic and Transcriptional Differences in Human Pluripotent Stem Cells under 2D and 3D Culture Conditions.

PubMed

Kamei, Ken-Ichiro; Koyama, Yoshie; Tokunaga, Yumie; Mashimo, Yasumasa; Yoshioka, Momoko; Fockenberg, Christopher; Mosbergen, Rowland; Korn, Othmar; Wells, Christine; Chen, Yong

2016-11-01

Human pluripotent stem cells hold great promise for applications in drug discovery and regenerative medicine. Microfluidic technology is a promising approach for creating artificial microenvironments; however, although a proper 3D microenvironment is required to achieve robust control of cellular phenotypes, most current microfluidic devices provide only 2D cell culture and do not allow tuning of physical and chemical environmental cues simultaneously. Here, the authors report a 3D cellular microenvironment plate (3D-CEP), which consists of a microfluidic device filled with thermoresponsive poly(N-isopropylacrylamide)-β-poly(ethylene glycol) hydrogel (HG), which enables systematic tuning of both chemical and physical environmental cues as well as in situ cell monitoring. The authors show that H9 human embryonic stem cells (hESCs) and 253G1 human induced pluripotent stem cells in the HG/3D-CEP system maintain their pluripotent marker expression under HG/3D-CEP self-renewing conditions. Additionally, global gene expression analyses are used to elucidate small variations among different test environments. Interestingly, the authors find that treatment of H9 hESCs under HG/3D-CEP self-renewing conditions results in initiation of entry into the neural differentiation process by induction of PAX3 and OTX1 expression. The authors believe that this HG/3D-CEP system will serve as a versatile platform for developing targeted functional cell lines and facilitate advances in drug screening and regenerative medicine. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Familial Dysautonomia (FD) Human Embryonic Stem Cell Derived PNS Neurons Reveal that Synaptic Vesicular and Neuronal Transport Genes Are Directly or Indirectly Affected by IKBKAP Downregulation

PubMed Central

Kantor, Gal; Cheishvili, David; Even, Aviel; Birger, Anastasya; Turetsky, Tikva; Gil, Yaniv; Even-Ram, Sharona; Aizenman, Einat; Bashir, Nibal; Maayan, Channa; Razin, Aharon; Reubinoff, Benjamim E.; Weil, Miguel

2015-01-01

A splicing mutation in the IKBKAP gene causes Familial Dysautonomia (FD), affecting the IKAP protein expression levels and proper development and function of the peripheral nervous system (PNS). Here we found new molecular insights for the IKAP role and the impact of the FD mutation in the human PNS lineage by using a novel and unique human embryonic stem cell (hESC) line homozygous to the FD mutation originated by pre implantation genetic diagnosis (PGD) analysis. We found that IKBKAP downregulation during PNS differentiation affects normal migration in FD-hESC derived neural crest cells (NCC) while at later stages the PNS neurons show reduced intracellular colocalization between vesicular proteins and IKAP. Comparative wide transcriptome analysis of FD and WT hESC-derived neurons together with the analysis of human brains from FD and WT 12 weeks old embryos and experimental validation of the results confirmed that synaptic vesicular and neuronal transport genes are directly or indirectly affected by IKBKAP downregulation in FD neurons. Moreover we show that kinetin (a drug that corrects IKBKAP alternative splicing) promotes the recovery of IKAP expression and these IKAP functional associated genes identified in the study. Altogether, these results support the view that IKAP might be a vesicular like protein that might be involved in neuronal transport in hESC derived PNS neurons. This function seems to be mostly affected in FD-hESC derived PNS neurons probably reflecting some PNS neuronal dysfunction observed in FD. PMID:26437462

Role of Sida cordifolia L. leaves on biochemical and antioxidant profile during myocardial injury.

PubMed

Kubavat, J B; Asdaq, S M B

2009-07-06

The Sida cordifolia L. (Family: Malvaceae) is a widely allocated herb by folk tribes of Gujarat state of India for the treatment of coronary manifestations. However, no published data relevant to use of the plant is available. The aim of the present study was to evaluate the antioxidant and biochemical profile of hydroalcoholic extract of Sida cordifolia L. (HESC) leaves against myocardial infarction (MI) in rats. Albino rats were administered HESC (100 and 500 mg/kg) and propranolol (10 mg/kg) once daily orally for 30 days. At the end of treatment period, MI was induced by administering isoproterenol (ISO) or by subjecting heart to ischemia reperfusion injury (IRI). Endogenous biomarkers (LDH and CK-MB) and antioxidants (SOD and catalase) were estimated in serum/perfusate and heart tissue homogenate (HTH). The LDH and CK-MB activities were elevated in HTH and depleted in serum/perfusate of HESC and propranolol groups when compared to ISO/IRI control. Further, it was found that both doses significantly increased endogenous antioxidants in HTH. Moreover, biochemical findings were supported by histopathological observations. The result confirm, at least in part, for the use of Sida cordifolia in folk medicine to treat MI.

Epigenetic stability, adaptability, and reversibility in human embryonic stem cells

PubMed Central

Tompkins, Joshua D.; Hall, Christine; Chen, Vincent Chang-yi; Li, Arthur Xuejun; Wu, Xiwei; Hsu, David; Couture, Larry A.; Riggs, Arthur D.

2012-01-01

The stability of human embryonic stem cells (hESCs) is of critical importance for both experimental and clinical applications. We find that as an initial response to altered culture conditions, hESCs change their transcription profile for hundreds of genes and their DNA methylation profiles for several genes outside the core pluripotency network. After adaption to conditions of feeder-free defined and/or xeno-free culture systems, expression and DNA methylation profiles are quite stable for additional passaging. However, upon reversion to the original feeder-based culture conditions, numerous transcription changes are not reversible. Similarly, although the majority of DNA methylation changes are reversible, highlighting the plasticity of DNA methylation, a few are persistent. Collectively, this indicates these cells harbor a memory of culture history. For culture-induced DNA methylation changes, we also note an intriguing correlation: hypomethylation of regions 500–2440 bp upstream of promoters correlates with decreased expression, opposite to that commonly seen at promoter-proximal regions. Lastly, changes in regulation of G-coupled protein receptor pathways provide a partial explanation for many of the unique transcriptional changes observed during hESC adaptation and reverse adaptation. PMID:22802633

Human embryonic stem cell-derived NK cells acquire functional receptors and cytolytic activity.

PubMed

Woll, Petter S; Martin, Colin H; Miller, Jeffrey S; Kaufman, Dan S

2005-10-15

Human embryonic stem cells (hESCs) provide a unique resource to analyze early stages of human hematopoiesis. However, little is known about the ability to use hESCs to evaluate lymphocyte development. In the present study, we use a two-step culture method to demonstrate efficient generation of functional NK cells from hESCs. The CD56(+)CD45(+) hESC-derived lymphocytes express inhibitory and activating receptors typical of mature NK cells, including killer cell Ig-like receptors, natural cytotoxicity receptors, and CD16. Limiting dilution analysis suggests that these cells can be produced from hESC-derived hemopoietic progenitors at a clonal frequency similar to CD34(+) cells isolated from cord blood. The hESC-derived NK cells acquire the ability to lyse human tumor cells by both direct cell-mediated cytotoxicity and Ab-dependent cellular cytotoxicity. Additionally, activated hESC-derived NK cells up-regulate cytokine production. hESC-derived lymphoid progenitors provide a novel means to characterize specific cellular and molecular mechanisms that lead to development of specific human lymphocyte populations. These cells may also provide a source for innovative cellular immune therapies.

Roadblocks en route to the clinical application of induced pluripotent stem cells.

PubMed

Lowry, William E; Quan, William L

2010-03-01

Since the first studies of human embryonic stem cells (hESCs) and, more recently, human induced pluripotent stem cells (hiPSCs), the stem-cell field has been abuzz with the promise that these pluripotent populations will one day be a powerful therapeutic tool. Although it has been proposed that hiPSCs will supersede hESCs with respect to their research and/or clinical potential because of the ease of their derivation and the ability to create immunologically matched iPSCs for each individual patient, recent evidence suggests that iPSCs in fact have several underappreciated characteristics that might mean they are less suitable for clinical application. Continuing research is revealing the similarities, differences and deficiencies of various pluripotent stem-cell populations, and suggests that many years will pass before the clinical utility of hESCs and hiPSCs is realized. There are a plethora of ethical, logistical and technical roadblocks on the route to the clinical application of pluripotent stem cells, particularly of iPSCs. In this Essay, we discuss what we believe are important issues that should be considered when attempting to bring hiPSC-based technology to the clinic.

Phosphatidic acid induces decidualization by stimulating Akt-PP2A binding in human endometrial stromal cells.

PubMed

Lee, So Young; Lee, Yun Young; Choi, Joong Sub; Yoon, Mee-Sup; Han, Joong-Soo

2016-11-01

Decidualization of human endometrial stromal cells (hESCs) is crucial for successful uterine implantation and maintaining pregnancy. We previously reported that phospholipase D1 (PLD1) is required for cAMP-induced decidualization of hESCs. However, the mechanism by which phosphatidic acid (PA), the product of PLD1 action, might regulate decidualization is not known. We confirmed that PA induced decidualization of hESCs by observing morphological changes and measuring increased levels of decidualization markers such as IGFBP1 and prolactin transcripts (P < 0.05). Treatment with PA reduced phosphorylation of Akt and consequently that of FoxO1, which led to the increased IGFBP1 and prolactin mRNA levels (P < 0.05). Conversely, PLD1 knockdown rescued Akt phosphorylation. Binding of PP2A and Akt increased in response to cAMP or PA, suggesting that their binding is directly responsible for the inactivation of Akt during decidualization. Consistent with this observation, treatment with okadaic acid, a PP2A inhibitor, also inhibited cAMP-induced decidualization by blocking Akt dephosphorylation. © 2016 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

Naked regulators: moral pluralism, deliberative democracy and authoritative regulation of human embryonic stem cell (hESC) research.

PubMed

Parker, Malcolm

2009-02-01

Bioethical issues pose challenges for pluralist, democratic societies due to the need to arbitrate between incompatible views over fundamental beliefs. The legitimacy of public policy is increasingly seen to depend on taking public consultation seriously, and subsequently regulating contested activities such as therapeutic cloning and hESC research. In December 2006, the Australian Federal Parliament lifted the ban on therapeutic cloning, following recommendations of the Legislation Review Committee (Lockhart Committee), which recently reported on its approach and methods in this journal. This column analyses recent accounts of democratic deliberative processes, authoritative regulation and the committee's own account. Authoritative regulation turns out to be largely an appeasement strategy, directed towards the losers of the contest, in this case the opponents of therapeutic cloning and hESC research. This is because regulation fails to minimise harm as perceived by the losers, and fails to meaningfully limit what it is the winners wish to do. Moreover, regulation adds an unnecessary layer of red tape to the work of the winners. Committees of inquiry in bioethical matters should be more open about their processes and their normative recommendations, at the risk of eroding trust in parts of their processes.

Stem cells in cardiac repair.

PubMed

Henning, Robert J

2011-01-01

Myocardial infarction is the leading cause of death among people in industrialized nations. Although the heart has some ability to regenerate after infarction, myocardial restoration is inadequate. Consequently, investigators are currently exploring the use of human embryonic stem cells (hESCs), skeletal myoblasts and adult bone marrow stem cells to limit infarct size. hESCs are pluripotent cells that can regenerate myocardium in infarcted hearts, attenuate heart remodeling and contribute to left ventricle (LV) systolic force development. Since hESCs can form heart teratomas, investigators are differentiating hESCs toward cardiac progenitor cells prior to transplantation into hearts. Large quantities of hESCs cardiac progenitor cells, however, must be generated, immune rejection must be prevented and grafts must survive over the long term to significantly improve myocardial performance. Transplanted autologous skeletal myoblasts can survive in infarcted myocardium in small numbers, proliferate, differentiate into skeletal myofibers and increase the LV ejection fraction. These cells, however, do not form electromechanical connections with host cardiomyocytes. Consequently, electrical re-entry can occur and cause cardiac arrhythmias. Autologous bone marrow mononuclear cells contain hematopoietic and mesenchymal stem cells. In several meta-analyses, patients with coronary disease who received autologous bone marrow cells by intracoronary injection show significant 3.7% (range: 1.9-5.4%) increases in LV ejection fraction, decreases in LV end-systolic volume of -4.8 ml (range: -1.4 to -8.2 ml) and reductions in infarct size of 5.5% (-1.9 to -9.1%), without experiencing arrhythmias. Bone marrow cells appear to release biologically active factors that limit myocardial damage. Unfortunately, bone marrow cells from patients with chronic diseases propagate poorly and can die prematurely. Substantial challenges must be addressed and resolved to advance the use of stem cells in cardiac repair including identifying the optimal stem cell(s) that permit transplantation without requirements for host immune suppression; timing of stem cell transplantation that maximizes chemoattraction of stem cells to infarcts; and determining the optimal technique for injecting stem cells for cardiac repair. Techniques must be developed to enhance survival and propagation of stem cells in the myocardium. These studies will require close cooperation and interaction of scientists and clinicians. Cell-based cardiac repair in the 21st century will offer new hope for millions of patients worldwide with myocardial infarctions who, otherwise, would suffer from the relentless progression of heart disease to heart failure and death.

An endogenous 55 kDa TNF receptor mediates cell death in a neural cell line.

PubMed

Sipe, K J; Srisawasdi, D; Dantzer, R; Kelley, K W; Weyhenmeyer, J A

1996-06-01

Tumor necrosis factor-alpha (TNF) is associated with developmental and injury-related events in the central nervous system (CNS). In the present study, we have examined the role of TNF on neurons using the clonal murine neuroblastoma line, N1E-115 (N1E). N1E cells represent a well-defined model for studying neuronal development since they can be maintained as either undifferentiated, mitotically active neuroblasts or as differentiated, mature neurons. Northern and reverse transcription-polymerase chain reaction (RT-PCR) analyses revealed that both undifferentiated and differentiated N1Es express transcripts for the 55 kDa TNF receptor (TNFR), but not the 75 kDa TNFR. The biological activity of the expressed TNF receptor was demonstrated by a dose dependent cytotoxicity to either recombinant murine or human TNF when the cells were incubated with the transcriptional inhibitor actinomycin D. The lack of the 75 kDa receptor mRNA expression and the dose dependent response to rHuTNF, an agonist specific for the murine 55 kDa receptor, suggest that the TNF induced cytotoxicity is mediated through the 55 kDa receptor in both the undifferentiated and differentiated N1Es. Light microscopic observations, flow cytometric analysis of hypodiploid DNA, and electrophoretic analysis of nucleosomal DNA fragmentation of N1Es treated with actinomycin D and TNF revealed features characteristic of both necrotic and apoptotic cell death. These findings demonstrate that blast and mature N1E cells express the 55 kDa TNF receptor which is responsible for inducing both necrotic and apoptotic death in these cells. The observation that actinomycin D renders N1E cells susceptible to the cytotoxic effects of TNF indicates that a sensitization step, such as removal of an endogenous protective factor or viral-mediated inhibition of transcription, may be necessary for TNF cytotoxicity in neurons.

Oxygen Tension Modulates Differentiation and Primary Macrophage Functions in the Human Monocytic THP-1 Cell Line

PubMed Central

Grodzki, Ana Cristina G.; Giulivi, Cecilia; Lein, Pamela J.

2013-01-01

The human THP-1 cell line is widely used as an in vitro model system for studying macrophage differentiation and function. Conventional culture conditions for these cells consist of ambient oxygen pressure (∼20% v/v) and medium supplemented with the thiol 2-mercaptoethanol (2-ME) and serum. In consideration of the redox activities of O2 and 2-ME, and the extensive experimental evidence supporting a role for reactive oxygen species (ROS) in the differentiation and function of macrophages, we addressed the question of whether culturing THP-1 cells under a more physiologically relevant oxygen tension (5% O2) in the absence of 2-ME and serum would alter THP-1 cell physiology. Comparisons of cultures maintained in 18% O2 versus 5% O2 indicated that reducing oxygen tension had no effect on the proliferation of undifferentiated THP-1 cells. However, decreasing the oxygen tension to 5% O2 significantly increased the rate of phorbol ester-induced differentiation of THP-1 cells into macrophage-like cells as well as the metabolic activity of both undifferentiated and PMA-differentiated THP-1 cells. Removal of both 2-ME and serum from the medium decreased the proliferation of undifferentiated THP-1 cells but increased metabolic activity and the rate of differentiation under either oxygen tension. In differentiated THP-1 cells, lowering the oxygen tension to 5% O2 decreased phagocytic activity, the constitutive release of β-hexosaminidase and LPS-induced NF-κB activation but enhanced LPS-stimulated release of cytokines. Collectively, these data demonstrate that oxygen tension influences THP-1 cell differentiation and primary macrophage functions, and suggest that culturing these cells under tightly regulated oxygen tension in the absence of exogenous reducing agent and serum is likely to provide a physiologically relevant baseline from which to study the role of the local redox environment in regulating THP-1 cell physiology. PMID:23355903

Undifferentiated Negative Affect and Impulsivity in Borderline Personality and Depressive Disorders: A Momentary Perspective

PubMed Central

Pronove, Lisa M.; Treloar, Hayley R.; Brown, Whitney C.; Solhan, Marika B.; Wood, Phillip K.; Trull, Timothy J.

2015-01-01

Individuals with borderline personality disorder (BPD) often report experiencing several negative emotions simultaneously, an indicator of “undifferentiated” negative affect. The current study examined the relationship between undifferentiated negative affect and impulsivity. Participants with a current BPD (n = 67) or depressive disorder (DD; n = 38) diagnosis carried an electronic diary for 28 days, reporting on emotions and impulsivity when randomly prompted (up to 6 times per day). Undifferentiated negative affect was quantified using momentary intraclass correlation coefficients, which indicated how consistently negative emotion items were rated across fear, hostility, and sadness subscales. Undifferentiated negative affect at the occasion-level, day-level, and across 28 days was used to predict occasion-level impulsivity. Multilevel modeling was used to test the hypothesis that undifferentiated negative emotion would be a significant predictor of momentary impulsivity above and beyond levels of overall negative affect. Undifferentiated negative affect at the occasion and day levels were significant predictors of occasion-level impulsivity, but undifferentiated negative affect across the 28-day study period was only marginally significant. Results did not differ depending on BPD or DD status, though BPD individuals did report significantly greater momentary impulsivity and undifferentiated negative affect. Undifferentiated negative affect may increase risk for impulsivity among individuals with BPD and depressive disorders, and the current data suggest that this process can be relatively immediate as well as cumulative over the course of a day. This research supports the consideration of undifferentiated negative affect as a transdiagnostic construct, but one that may be particularly relevant for those with BPD. PMID:26147324

Niche players

PubMed Central

Seandel, Marco; Falciatori, Ilaria; Shmelkov, Sergey V.; Kim, Jiyeon; James, Daylon; Rafii, Shahin

2010-01-01

The undifferentiated spermatogonia of adult mouse testes are composed of both true stem cells and committed progenitors. It is unclear what normally prevents these adult germ cells from manifesting multipotency. The critical elements of the spermatogonial stem cell niche, while poorly understood, are thought to be composed of Sertoli cells with several other somatic cell types in close proximity. We recently discovered a novel orphan G-protein coupled receptor (GPR125) that is restricted to undifferentiated spermatogonia within the testis. GPR125 expression was maintained when the progenitor cells were extracted from the in vivo niche and propagated under growth conditions that recapitulate key elements of the niche. Such conditions preserved the ability of the cells to generate multipotent derivatives, known as multipotent adult spermatogonial derived progenitor cells (MASCs). Upon differentiation, the latter produced a variety tissues including functional endothelium, illustrating the potential applications of such cells. Thus, GPR125 represents a novel target for purifying adult stem and progenitors from tissues, with the goal of developing autologous multipotent cell lines. PMID:18256534

A unique cell-surface protein phenotype distinguishes human small-cell from non-small-cell lung cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baylin, S.B.; Gazdar, A.F.; Minna, J.D.

1982-08-01

Radioiodination (/sup 125/I) and two-dimensional polyacrylamide gel electrophoresis was used to determine that small-(oat) cell lung carcinoma (SCC)-a tumor with neuroedocrine features-possesses a surface protein pattern distinct from the other types of lung cancer cells (squamous, adeno-, and large-cell undifferentiated carcinoma). Twelve distinguishing proteins, 40 to 70 kilodaltons (kDal), characterized four separate lines of SCC; three of these, designated E (60 kDal; pI = 7.3), S (30 kDal; pI = 6.0), and U 57 kDal; pI = 5.6), may be unique SCC gene products and were identified only in (/sup 35/S)methionine labeling of SCC and not in non-SCC or humanmore » fibroblasts. Two lines of adeno-, one of squamous, and one of undifferentiated large-cell lung carcinoma exhibited similar surface protein patterns to one another. Nine distinguishing proteins (40 to 100 kDal) and at least five large proteins (>100 kDal) were unique to these lines. The surface protein phenotypes for SCC and non-SCC were distinct from those for human lymphoblastoid cells and fibroblasts. However, the neuroendocrine features of SCC were further substantiated because 6 of the 12 distinguishing SCC surface proteins, including E and U, were identified on human neuroblastoma cells. The proteins identified should (i) help define differentiation steps for normal and neoplastic bronchial epithelial cells, (ii) prove useful in better classifying lung cancers, and (iii) be instrumental in tracing formation of neuroendocrine cells.« less

The cytokine-dependent MUTZ-3 cell line as an in vitro model for the screening of contact sensitizers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Azam, Philippe; Peiffer, Jean-Luc; Chamousset, Delphine

2006-04-01

Langerhans cells (LC) are key mediators of contact allergenicity in the skin. However, no in vitro methods exist which are based on the activation process of LC to predict the sensitization potential of chemicals. In this study, we have evaluated the performances of MUTZ-3, a cytokine-dependent human monocytic cell line, in its response to sensitizers. First, we compared undifferentiated MUTZ-3 cells with several standard human cells such as THP-1, KG-1, HL-60, K-562, and U-937 in their response to the strong sensitizer DNCB and the irritant SDS by monitoring the expression levels of HLA-DR, CD54, and CD86 by flow cytometry. Onlymore » MUTZ-3 and THP-1 cells show a strong and specific response to sensitizer, while other cell lines showed very variable responses. Then, we tested MUTZ-3 cells against a wider panel of sensitizers and irritants on a broader spectrum of cell surface markers (HLA-DR, CD40, CD54, CD80, CD86, B7-H1, B7-H2, B7-DC). Of these markers, CD86 proved to be the most reliable since it detected all sensitizers, including benzocaine, a classical false negative in local lymph node assay (LLNA) but not irritants. We confirmed the MUTZ-3 response to DNCB by real-time PCR analysis. Taken together, our data suggest that undifferentiated MUTZ-3 cells may represent a valuable in vitro model for the screening of potential sensitizers.« less

[Role of CD2-associated protein in podocyte differentiation.].

PubMed

Jiang, Hua-Jun; Chang, Ying; Zhu, Zhong-Hua; Liu, Jian-She; Deng, An-Guo; Zhang, Chun

2008-02-25

To study the cellular changes and the potential role of CD2-associated protein (CD2AP) in podocyte differentiation, conditionally immortalized murine podocyte cell line was cultured in RPMI 1640 medium under permissive condition at 33 °C. After transfection with CD2AP small interfering RNA (siRNA) the cells were shifted to non-permissive condition at 37 °C. Simultaneously, untransfected cells were taken as differentiation control. The podocyte proliferation rate was determined by MTT method. The expressions of CD2AP, WT1, synaptopodin and nephrin mRNAs were examined by RT-PCR. CD2AP, WT1 and nephrin protein expressions were examined by Western blot. The distribution of CD2AP, nephrin, F-actin and tubulin in differentiated and undifferentiated podocytes was detected by laser scanning confocal microscopy. The results showed: (1) CD2AP, WT1 and nephrin were stably expressed in differentiated and undifferentiated podocytes while synaptopodin was only expressed in differentiated podocytes. (2) CD2AP and nephrin mRNA and protein expressions were up-regulated during podocyte differentiation (P<0.05). (3) CD2AP and tubulin were distributed in the cytoplasm and perinulcear region in undifferentiated podocytes, and F-actin was predominantly localized to a cortical belt and paralleled to the cell axis. Under differentiation condition, CD2AP distribution profile was presented as peripheral accumulation, tubulin took on fascicular style and F-actin extended into foot processes in podocytes. CD2AP colocalized with nephrin and F-actin in undifferentiated podocytes. (4) After transfection with CD2AP siRNA, the expression of CD2AP was partially inhibited and cell growth was arrested; Synaptopodin, the differentiation podocyte marker, was apparently down-regulated; The differentiation of podocytes was delayed. The results demonstrate that podocyte differentiation is accompanied by cytoskeleton rearrangement and cell morphology change. CD2AP might play an essential role in podocyte differentiation.

Mitochondrial-Associated Cell Death Mechanisms Are Reset to an Embryonic-Like State in Aged Donor-Derived iPS Cells Harboring Chromosomal Aberrations

PubMed Central

Prigione, Alessandro; Hossini, Amir M.; Lichtner, Björn; Serin, Akdes; Fauler, Beatrix; Megges, Matthias; Lurz, Rudi; Lehrach, Hans; Zouboulis, Christos C.

2011-01-01

Somatic cells reprogrammed into induced pluripotent stem cells (iPSCs) acquire features of human embryonic stem cells (hESCs) and thus represent a promising source for cellular therapy of debilitating diseases, such as age-related disorders. However, reprogrammed cell lines have been found to harbor various genomic alterations. In addition, we recently discovered that the mitochondrial DNA of human fibroblasts also undergoes random mutational events upon reprogramming. Aged somatic cells might possess high susceptibility to nuclear and mitochondrial genome instability. Hence, concerns over the oncogenic potential of reprogrammed cells due to the lack of genomic integrity may hinder the applicability of iPSC-based therapies for age-associated conditions. Here, we investigated whether aged reprogrammed cells harboring chromosomal abnormalities show resistance to apoptotic cell death or mitochondrial-associated oxidative stress, both hallmarks of cancer transformation. Four iPSC lines were generated from dermal fibroblasts derived from an 84-year-old woman, representing the oldest human donor so far reprogrammed to pluripotency. Despite the presence of karyotype aberrations, all aged-iPSCs were able to differentiate into neurons, re-establish telomerase activity, and reconfigure mitochondrial ultra-structure and functionality to a hESC-like state. Importantly, aged-iPSCs exhibited high sensitivity to drug-induced apoptosis and low levels of oxidative stress and DNA damage, in a similar fashion as iPSCs derived from young donors and hESCs. Thus, the occurrence of chromosomal abnormalities within aged reprogrammed cells might not be sufficient to over-ride the cellular surveillance machinery and induce malignant transformation through the alteration of mitochondrial-associated cell death. Taken together, we unveiled that cellular reprogramming is capable of reversing aging-related features in somatic cells from a very old subject, despite the presence of genomic alterations. Nevertheless, we believe it will be essential to develop reprogramming protocols capable of safeguarding the integrity of the genome of aged somatic cells, before employing iPSC-based therapy for age-associated disorders. PMID:22110631

Fluoxetine Increases the Expression of miR-572 and miR-663a in Human Neuroblastoma Cell Lines.

PubMed

Mundalil Vasu, Mahesh; Anitha, Ayyappan; Takahashi, Taro; Thanseem, Ismail; Iwata, Keiko; Asakawa, Tetsuya; Suzuki, Katsuaki

2016-01-01

Evidence suggests neuroprotective effects of fluoxetine, a selective serotonin reuptake inhibitor (SSRI), on the developed neurons in the adult brain. In contrast, the drug may be deleterious to immature or undifferentiated neural cells, although the mechanism is unclear. Recent investigations have suggested that microRNAs (miRNA) may be critical for effectiveness of psychotropic drugs including SSRI. We investigated whether fluoxetine could modulate expressions of neurologically relevant miRNAs in two neuroblastoma SK-N-SH and SH-SY5Y cell lines. Initial screening results revealed that three (miR-489, miR-572 and miR-663a) and four (miR-320a, miR-489, miR-572 and miR-663a) miRNAs were up-regulated in SK-N-SH cells and SH-SY5Y cells, respectively, after 24 hours treatment of fluoxetine (1-25 μM). Cell viability was reduced according to the dose of fluoxetine. The upregulation of miR-572 and miR-663a was consistent in both the SH-SY5Y and SK-N-SH cells, confirmed by a larger scale culture condition. Our data is the first in vitro evidence that fluoxetine could increase the expression of miRNAs in undifferentiated neural cells, and that putative target genes of those miRNAs have been shown to be involved in fundamental neurodevelopmental processes.

Conserved and non-conserved characteristics of porcine glial cell line-derived neurotrophic factor expressed in the testis.

PubMed

Kakiuchi, Kazue; Taniguchi, Kazumi; Kubota, Hiroshi

2018-05-16

Glial cell line-derived neurotrophic factor (GDNF) is essential for the self-renewal and proliferation of spermatogonial stem cells (SSCs) in mice, rats, and rabbits. Although the key extrinsic factors essential for spermatogonial proliferation in other mammals have not been determined, GDNF is one of the potential candidates. In this study, we isolated porcine GDNF (pGDNF) cDNAs from neonatal testis and generated recombinant pGDNF to investigate its biological activity on gonocytes/undifferentiated spermatogonia, including SSCs. In porcine testis, long and short forms of GDNF transcripts, the counterparts of pre-(α)pro and pre-(β)pro GDNF identified in humans and rodents, were expressed. The two transcripts encode identical mature proteins. Recombinant pGDNF supported proliferation of murine SSCs in culture, and their stem cell activity was confirmed by a transplantation assay. Subsequently, porcine gonocytes/undifferentiated spermatogonia were cultured with pGDNF; however, pGDNF did not affect their proliferation. Furthermore, GDNF expression was localised to the vascular smooth muscle cells, and its cognate receptor GFRA1 expression was negligible during spermatogonial proliferation in the testes. These results indicate that although pGDNF retains structural similarity with those of other mammals and conserves the biological activity on the self-renewal of murine SSCs, porcine SSCs likely require extrinsic factors other than GDNF for their proliferation.

Diamine Oxidase from White Pea (Lathyrus sativus) Combined with Catalase Protects the Human Intestinal Caco-2 Cell Line from Histamine Damage.

PubMed

Jumarie, Catherine; Séïde, Marilyne; Marcocci, Lucia; Pietrangeli, Paola; Mateescu, Mircea Alexandru

2017-07-01

Diamine oxidase (DAO) administration has been proposed to treat certain gastrointestinal dysfunctions induced by histamine, an immunomodulator, signaling, and pro-inflammatory factor. However, H 2 O 2 resulting from the oxidative deamination of histamine by DAO may be toxic. The purpose of this study was to investigate to which extent DAO from white pea (Lathyrus sativus), alone or in combination with catalase, may modulate histamine toxicity in the human intestinal Caco-2 cell line. The results show that histamine at concentrations higher than 1 mM is toxic to the Caco-2 cells, independently of the cell differentiation status, with a LC 50 of ≅ 10 mM following a 24-h exposure. Depending on its concentration, DAO increased histamine toxicity to a greater extent in differentiated cells compared to undifferentiated cultures. In the presence of catalase, the DAO-induced increase in histamine toxicity was completely abolished in the undifferentiated cells and only partially decreased in differentiated cells, showing differences in the sensitivity of Caco-2 cells to the products resulting from histamine degradation by DAO (H 2 O 2 , NH 3 , or imidazole aldehyde). It appears that treatment of food histaminosis using a combination of vegetal DAO and catalase would protect against histamine toxicity and prevent H 2 O 2 -induced damage that may occur during histamine oxidative deamination.

Proteomic analysis of plasma membranes isolated from undifferentiated and differentiated HepaRG cells

PubMed Central

2012-01-01

Liver infection with hepatitis B virus (HBV), a DNA virus of the Hepadnaviridae family, leads to severe disease, such as fibrosis, cirrhosis and hepatocellular carcinoma. The early steps of the viral life cycle are largely obscure and the host cell plasma membrane receptors are not known. HepaRG is the only proliferating cell line supporting HBV infection in vitro, following specific differentiation, allowing for investigation of new host host-cell factors involved in viral entry, within a more robust and reproducible environment. Viral infection generally begins with receptor recognition at the host cell surface, following highly specific cell-virus interactions. Most of these interactions are expected to take place at the plasma membrane of the HepaRG cells. In the present study, we used this cell line to explore changes between the plasma membrane of undifferentiated (−) and differentiated (+) cells and to identify differentially-regulated proteins or signaling networks that might potentially be involved in HBV entry. Our initial study identified a series of proteins that are differentially expressed in the plasma membrane of (−) and (+) cells and are good candidates for potential cell-virus interactions. To our knowledge, this is the first study using functional proteomics to study plasma membrane proteins from HepaRG cells, providing a platform for future experiments that will allow us to understand the cell-virus interaction and mechanism of HBV viral infection. PMID:22857383

Morphological Analysis of Live Undifferentiated Cells Derived from Induced Pluripotent Stem Cells.

PubMed

Osawa, Yukihiko; Miyamoto, Tomoyuki; Ohno, Setsuyo; Ohno, Eiji

2018-01-01

Induced pluripotent stem (iPS) cells possess pluripotency and self-renewal ability. Therefore, iPS cells are expected to be useful in regenerative medicine. However, iPS cells form malignant immature teratomas after transplantation into animals, even after differentiation induction. It has been suggested that undifferentiated cells expressing Nanog that remain after differentiation induction are responsible for teratoma formation. Various methods of removing these undifferentiated cells have therefore been investigated, but few methods involve morphological approaches, which may induce less cell damage. In addition, for cells derived from iPS cells to be applied in regenerative medicine, they must be alive. However, detailed morphological analysis of live undifferentiated cells has not been performed. For the above reasons, we assessed the morphological features of live undifferentiated cells remaining after differentiation induction as a basic investigation into the clinical application of iPS cells. As a result, live undifferentiated cells remaining after differentiation induction exhibited a round or oval cytoplasm about 12 μm in diameter and a nucleus. They exhibited nucleo-cytoplasmic (N/C) ratio of about 60% and eccentric nuclei, and they possessed partially granule-like structures in the cytoplasm and prominent nucleoli. Although they were similar to iPS cells, they were smaller than live iPS cells. Furthermore, very small cells were present among undifferentiated cells after differentiation induction. These results suggest that the removal of undifferentiated cells may be possible using the morphological features of live iPS cells and undifferentiated cells after differentiation induction. In addition, this study supports safe regenerative medicine using iPS cells.

Enhanced expression of extracellular calcium sensing receptor in monocyte-differentiated versus undifferentiated HL-60 cells: potential role in regulation of a nonselective cation channel

NASA Technical Reports Server (NTRS)

Yamaguchi, T.; Ye, C.; Chattopadhyay, N.; Sanders, J. L.; Vassilev, P. M.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

2000-01-01

Human promyelocytic leukemia cells (HL-60) have been used widely as a model for studying the differentiation of hematopoietic progenitor cells in vitro. After treatment with phorbol-12-myristate-13-acetate (PMA) or 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], HL-60 cells differentiate into cells with the phenotype of monocytes/macrophages. We previously showed that peripheral blood monocytes and the murine J774 monocytic cell line express the CaR, and myeloid progenitors in the bone marrow and myeloid cells in peripheral blood other than monocytes express lower levels of the CaR. Therefore, we investigated whether undifferentiated HL-60 cells express a functional G protein-coupled, extracellular calcium (Ca(2+)(o))-sensing receptor (CaR) and if the expression of the CaR increases as these cells differentiate along the monocytic lineage. The use of reverse transcription-polymerase chain reaction (RT-PCR) with CaR-specific primers, followed by sequencing of the amplified products, identified an authentic CaR transcript in undifferentiated HL-60 cells. Both immunocytochemistry and Western blot analysis using a CaR-specific antiserum detected low levels of CaR protein expression in undifferentiated HL-60 cells. The levels of CaR protein increased considerably following treatment of the cells with PMA (50 nM) or 1,25(OH)(2)D(3) (100 nM) for 5 days. Northern analysis using a CaR-specific riboprobe identified CaR transcripts in undifferentiated HL-60 cells, but CaR mRNA levels did not change appreciably after treatment with either agent, suggesting that upregulation of CaR protein occurs at a translational level. PMA-treated HL-60 cells expressed a nonselective cation channel (NCC), and the calcimimetic CaR activator, NPS R-467, but not its less active stereoisomer, NPS S-467, as well as the polycationic CaR agonist, neomycin, activated this NCC, demonstrating that the CaR expressed in these cells is functionally active. Therefore, HL-60 cells exhibit an increase in CaR protein expression, occurring at a translational level during their differentiation into cells with a monocyte/macrophage phenotype in response to treatment with PMA or 1, 25(OH)(2)D(3), which is functionally linked to activation of a nonselective cation channel.

Ceftaroline modulates the innate immune and host defense responses of immunocompetent cells exposed to cigarette smoke.

PubMed

Bruno, A; Cipollina, C; Di Vincenzo, S; Siena, L; Dino, P; Di Gaudio, F; Gjomarkaj, M; Pace, E

2017-09-05

Cigarette smoke, the principal risk factor for chronic obstructive pulmonary disease (COPD), negatively influences the effectiveness of the immune system's response to a pathogen. The antibiotic ceftaroline exerts immune-modulatory effects in bronchial epithelial cells exposed to cigarette smoke. The present study aims to assess the effects of ceftaroline on TLR2 and TLR4 expression, LPS binding and TNF-α and human beta defensin (HBD2) release in an undifferentiated and PMA-differentiated human monocyte cell line (THP-1) exposed or not to cigarette smoke extracts (CSE). TLR2, TLR4, and LPS binding were assessed by flow cytometry, TNF-α and HBD2 release were evaluated by ELISA. The constitutive expression of TLR2 and TLR4 and LPS binding were higher in differentiated compared to undifferentiated THP-1 cells. In undifferentiated THP-1 cells, CSE increased TLR2 and TLR4 protein levels, LPS binding and TNF-α release and reduced HBD2 release and ceftaroline counteracted all these effects. In differentiated THP-1, CSE did not significantly affect TLR2 and TLR4 expression and LPS binding but reduced HBD2 release and increased TNF-α release. Ceftaroline counteracted the effects of CSE on HBD2 release in differentiated THP-1. Ceftaroline counteracts the effect of CSE in immune cells by increasing the effectiveness of the innate immune system. This effect may also assist in reducing pathogen activity and recurrent exacerbations in COPD patients. Copyright © 2017 Elsevier B.V. All rights reserved.

Noninvasive Detection and Imaging of Molecular Markers in Live Cardiomyocytes Derived from Human Embryonic Stem Cells

PubMed Central

Pascut, Flavius C.; Goh, Huey T.; Welch, Nathan; Buttery, Lee D.; Denning, Chris; Notingher, Ioan

2011-01-01

Raman microspectroscopy (RMS) was used to detect and image molecular markers specific to cardiomyocytes (CMs) derived from human embryonic stem cells (hESCs). This technique is noninvasive and thus can be used to discriminate individual live CMs within highly heterogeneous cell populations. Principal component analysis (PCA) of the Raman spectra was used to build a classification model for identification of individual CMs. Retrospective immunostaining imaging was used as the gold standard for phenotypic identification of each cell. We were able to discriminate CMs from other phenotypes with >97% specificity and >96% sensitivity, as calculated with the use of cross-validation algorithms (target 100% specificity). A comparison between Raman spectral images corresponding to selected Raman bands identified by the PCA model and immunostaining of the same cells allowed assignment of the Raman spectral markers. We conclude that glycogen is responsible for the discrimination of CMs, whereas myofibril proteins have a lesser contribution. This study demonstrates the potential of RMS for allowing the noninvasive phenotypic identification of hESC progeny. With further development, such label-free optical techniques may enable the separation of high-purity cell populations with mature phenotypes, and provide repeated measurements to monitor time-dependent molecular changes in live hESCs during differentiation in vitro. PMID:21190678

Nucleosome eviction along with H3K9ac deposition enhances Sox2 binding during human neuroectodermal commitment

PubMed Central

Du, Yanhua; Liu, Zhenping; Cao, Xinkai; Chen, Xiaolong; Chen, Zhenyu; Zhang, Xiaobai; Zhang, Xiaoqing; Jiang, Cizhong

2017-01-01

Neuroectoderm is an important neural precursor. However, chromatin remodeling and its epigenetic regulatory roles during the differentiation of human neuroectodermal cells (hNECs) from human embryonic stem cells (hESCs) remain largely unexplored. Here, we obtained hNECs through directed differentiation from hESCs, and determined chromatin states in the two cell types. Upon differentiation, H2A.Z-mediated nucleosome depletion leads to an open chromatin structure in promoters and upregulates expression of neuroectodermal genes. Increase in H3K9ac signals and decrease in H3K27me3 signals in promoters result in an active chromatin state and activate neuroectodermal genes. Conversely, decrease in H3K9ac signals and increase in H3K27me3 signals in promoters repress pluripotency genes. Moreover, H3K9ac signals facilitate the pluripotency factor Sox2 binding to target sites unique to hNECs. Knockdown of the acetyltransferase Kat2b erases H3K9ac signals, disrupts Sox2 binding, and fails the differentiation. Our results demonstrate a hierarchy of epigenetic regulation of gene expression during the differentiation of hNECs from hESCs through chromatin remodeling. PMID:28475175

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids.

PubMed

Meraviglia, Viviana; Zanon, Alessandra; Lavdas, Alexandros A; Schwienbacher, Christine; Silipigni, Rosamaria; Di Segni, Marina; Chen, Huei-Sheng Vincent; Pramstaller, Peter P; Hicks, Andrew A; Rossini, Alessandra

2015-06-05

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation. In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

PRMT8 Controls the Pluripotency and Mesodermal Fate of Human Embryonic Stem Cells By Enhancing the PI3K/AKT/SOX2 Axis.

PubMed

Jeong, Ho-Chang; Park, Soon-Jung; Choi, Jong-Jin; Go, Young-Hyun; Hong, Soon-Ki; Kwon, Ok-Seon; Shin, Joong-Gon; Kim, Rae-Kwon; Lee, Mi-Ok; Lee, Su-Jae; Shin, Hyoung Doo; Moon, Sung-Hwan; Cha, Hyuk-Jin

2017-09-01

Basic fibroblast growth factor (bFGF) supplementation is critical to maintain the pluripotency of human pluripotent stem cells (hPSCs) through activation of PI3K/AKT, rather than MEK/ERK pathway. Thus, elaborate molecular mechanisms that preserve PI3K/AKT signaling upon bFGF stimulation may exist in hPSCs. Protein arginine methyltransferase 8 (PRMT8) was expressed and then its level gradually decreased during spontaneous differentiation of human embryonic stem cells (hESCs). PRMT8 loss- or gain-of-function studies demonstrated that PRMT8 contributed to longer maintenance of hESC pluripotency, even under bFGF-deprived conditions. Direct interaction of membrane-localized PRMT8 with p85, a regulatory subunit of PI3K, was associated with accumulation of phosphoinositol 3-phosphate and consequently high AKT activity. Furthermore, the SOX2 induction, which was controlled by the PRMT8/PI3K/AKT axis, was linked to mesodermal lineage differentiation. Thus, we propose that PRMT8 in hESCs plays an important role not only in maintaining pluripotency but also in controlling mesodermal differentiation through bFGF signaling toward the PI3K/AKT/SOX2 axis. Stem Cells 2017;35:2037-2049. © 2017 AlphaMed Press.

The expression of the class 1 glucose transporter isoforms in human embryonic stem cells, and the potential use of GLUT2 as a marker for pancreatic progenitor enrichment.

PubMed

Segev, Hana; Fishman, Betina; Schulman, Rita; Itskovitz-Eldor, Joseph

2012-07-01

Even before the first appearance of the developing pancreas, glucose is the major substrate in the growing embryo. The transport of glucose across cell membranes is facilitated by a family of membranal glucose transporters (GLUT). We analyzed changes in expression of class 1 glucose transporters (GLUT1-4) during human embryonic stem cell (hESC) and human induced pluripotent stem cell (hiPSC) differentiation, from undifferentiated cells to 28-day-old embryoid bodies (EBs). We also examined the potential use of GLUT2 as a marker for differentiating pancreatic progenitor cells. Using quantitative real time polymerase chain reaction (qPCR), western blot, and immunofluorescence, we observed enhanced expression of GLUT1 and GLUT2 during differentiation, but only minor change in GLUT3 expression. GLUT4 expression was found to be very low both at the RNA and in the protein levels. Expression of the early pancreatic transcription factor, pancreatic duodenal homeobox gene 1 (PDX1), correlated with GLUT2 expression, suggesting the potential use of GLUT2 as a surface marker for tracking pancreatic precursor cells. After sorting EBs according to their membranal GLUT2 expression, GLUT2 and PDX1 expression were found elevated, as was expression of other endodermal markers such as PAX4, NGN3, CXCR4, and SOX17. This simple method may be used to differentiate embryonic stem cells and to isolate from them, using GLUT2 as a surface marker, an enriched pancreatic progenitor cell population in order to achieve insulin-producing cells. The sorted GLUT2 cells may potentially be used in the future as insulin-producing cells for beta cell therapies.

Undifferentiated carcinoma of parotid gland.

PubMed Central

López, J I; Alfaro, J; Ballestin, C

1991-01-01

Two cases of undifferentiated carcinomas of the major salivary glands were studied using immunohistochemical techniques. Results showed that this entity was a high grade malignant neoplasm arising from the excretory duct. Despite the undifferentiated appearance multiple immunophenotypes were evident in both cases. PMID:2045506

Disruption of cardiogenesis in human embryonic stem cells exposed to trichloroethylene.

PubMed

Jiang, Yan; Wang, Dan; Zhang, Guoxing; Wang, Guoqing; Tong, Jian; Chen, Tao

2016-11-01

Trichloroethylene (TCE) is ubiquitous in our living environment, and prenatal exposure to TCE is reported to cause congenital heart disease in humans. Although multiple studies have been performed using animal models, they have limited value in predicting effects on humans due to the unknown species-specific toxicological effects. To test whether exposure to low doses of TCE induces developmental toxicity in humans, we investigated the effect of TCE on human embryonic stem cells (hESCs) and cardiomyocytes (derived from the hESCs). In the current study, hESCs cardiac differentiation was achieved by using differentiation medium consisting of StemPro-34. We examined the effects of TCE on cell viability by cell growth assay and cardiac inhibition by analysis of spontaneously beating cluster. The expression levels of genes associated with cardiac differentiation and Ca 2+ channel pathways were measured by immunofluorescence and qPCR. The overall data indicated the following: (1) significant cardiac inhibition, which was characterized by decreased beating clusters and beating rates, following treatment with low doses of TCE; (2) significant up-regulation of the Nkx2.5/Hand1 gene in cardiac progenitors and down regulation of the Mhc-7/cTnT gene in cardiac cells; and (3) significant interference with Ca 2+ channel pathways in cardiomyocytes, which contributes to the adverse effect of TCE on cardiac differentiation during early embryo development. Our results confirmed the involvement of Ca 2+ turnover network in TCE cardiotoxicity as reported in animal models, while the inhibition effect of TCE on the transition of cardiac progenitors to cardiomyocytes is unique to hESCs, indicating a species-specific effect of TCE on heart development. This study provides new insight into TCE biology in humans, which may help explain the development of congenital heart defects after TCE exposure. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1372-1380, 2016. © 2015 Wiley Periodicals, Inc.

Development of a pluripotent stem cell derived neuronal model to identify chemically induced pathway perturbations in relation to neurotoxicity: Effects of CREB pathway inhibition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pistollato, Francesca; Louisse, Jochem; Scelfo, Bibiana

2014-10-15

According to the advocated paradigm shift in toxicology, acquisition of knowledge on the mechanisms underlying the toxicity of chemicals, such as perturbations of biological pathways, is of primary interest. Pluripotent stem cells (PSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), offer a unique opportunity to derive physiologically relevant human cell types to measure molecular and cellular effects of such pathway modulations. Here we compared the neuronal differentiation propensity of hESCs and hiPSCs with the aim to develop novel hiPSC-based tools for measuring pathway perturbation in relation to molecular and cellular effects in vitro.more » Among other fundamental pathways, also, the cAMP responsive element binding protein (CREB) pathway was activated in our neuronal models and gave us the opportunity to study time-dependent effects elicited by chemical perturbations of the CREB pathway in relation to cellular effects. We show that the inhibition of the CREB pathway, using 2-naphthol-AS-E-phosphate (KG-501), induced an inhibition of neurite outgrowth and synaptogenesis, as well as a decrease of MAP2{sup +} neuronal cells. These data indicate that a CREB pathway inhibition can be related to molecular and cellular effects that may be relevant for neurotoxicity testing, and, thus, qualify the use of our hiPSC-derived neuronal model for studying chemical-induced neurotoxicity resulting from pathway perturbations. - Highlights: • HESCs derived neuronal cells serve as benchmark for iPSC based neuronal toxicity test development. • Comparisons between hESCs and hiPSCs demonstrated variability of the epigenetic state • CREB pathway modulation have been explored in relation to the neurotoxicant exposure KG-501 • hiPSC might be promising tools to translate theoretical AoPs into toxicological in vitro tests.« less

Video bioinformatics analysis of human embryonic stem cell colony growth.

PubMed

Lin, Sabrina; Fonteno, Shawn; Satish, Shruthi; Bhanu, Bir; Talbot, Prue

2010-05-20

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software. Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT. To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images. To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time. The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.

ZEB1 overexpression associated with E-cadherin and microRNA-200 downregulation is characteristic of undifferentiated endometrial carcinoma.

PubMed

Romero-Pérez, Laura; López-García, M Ángeles; Díaz-Martín, Juan; Biscuola, Michele; Castilla, M Ángeles; Tafe, Laura J; Garg, Karuna; Oliva, Esther; Matias-Guiu, Xavier; Soslow, Robert A; Palacios, José

2013-11-01

Undifferentiated endometrial carcinomas are very aggressive high-grade endometrial carcinomas that are frequently under-recognized. This study aimed to analyze the molecular alterations underlying the development of these endometrial carcinomas, focusing on those related to dedifferentiation. We assessed a series of 120 tumors: 57 grade 1 and 2 endometrioid endometrial carcinomas, 15 grade 3 endometrioid endometrial carcinomas, 27 endometrial serous carcinomas, and 21 undifferentiated endometrial carcinomas. We found a high frequency of DNA mismatch repair deficiency (38%) and moderate rate of p53 overexpression (∼33%) in undifferentiated carcinomas. In contrast to the characteristic endometrioid phenotype, there was a dramatic downregulation of E-cadherin expression in the undifferentiated subtype. Quantitative methylation studies dismissed CDH1 promoter hypermethylation as the mechanism responsible for this change in gene expression, while immunohistochemistry revealed that the E-cadherin repressor ZEB1 was frequently overexpressed (62%) in undifferentiated endometrial carcinomas. This finding was accompanied by a sharp downregulation in the expression of the miR-200 family of microRNAs, well-known targets of ZEB1. Furthermore, there was enhanced expression of epithelial-to-mesenchymal transition markers in undifferentiated endometrial carcinomas, such as N-cadherin, cytoplasmic p120, and osteonectin. In addition, HMGA2, a regulator of epithelial-to-mesenchymal transition that is expressed in aggressive endometrial tumors, such as endometrial serous carcinomas and carcinosarcomas, was expressed in >20% of undifferentiated carcinomas. These results suggest that ZEB1 overexpression, associated with E-cadherin and miR-200s downregulation, and the expression of mesenchymal markers might enhance the metastatic potential of undifferentiated endometrial carcinomas, leading to a poor prognosis. In addition, our observations suggest that the immnohistochemical analysis of E-cadherin and ZEB1 can aid in the differential diagnosis of the more agressive undifferentiated endometrial carcinomas from grade 3 endometrioid carcinomas.

Mechanism-based facilitated maturation of human pluripotent stem cell-derived cardiomyocytes

PubMed Central

Lieu, Deborah K.; Fu, Ji-Dong; Chiamvimonvat, Nipavan; Chan Tung, Kelvin W.; McNerney, Gregory P.; Huser, Thomas; Keller, Gordon; Kong, Chi-Wing

2013-01-01

Background Human embryonic stem cells (hESCs) can be efficiently and reproducibly directed into cardiomyocytes (CMs) using stage-specific induction protocols. However, their functional properties and suitability for clinical and other applications have not been evaluated. Methods and Results Here we showed that CMs derived from multiple pluripotent human stem cell lines (hESC: H1, HES2) and types (induced pluripotent stem cell or iPSC) using different in vitro differentiation protocols (embryoid body formation, endodermal induction, directed differentiation) commonly displayed immature, pro-arrhythmic action potential (AP) properties such as high-degree of automaticity, depolarized resting membrane potential (RMP), Phase 4- depolarization and delayed after-depolarization (DAD). Among the panoply of sarcolemmal ionic currents investigated (INa+/ICaL2+/IKr+/INCX+/If+/Ito+/IK1-/IKs-), we pinpointed the lack of the Kir2.1-encoded inwardly rectifying K+ current (IK1) as the single mechanistic contributor to the observed immature electrophysiological properties in hESC-CMs. Forced expression of Kir2.1 in hESC-CMs led to robust expression of Ba2+-sensitive IK1 and more importantly, completely ablated all the pro-arrhythmic AP traits, rendering the electrophysiological phenotype indistinguishable from the adult counterparts. These results provided the first link of a complex developmentally arrested phenotype to a major effector gene, and importantly, further led us to develop a biomimetic culturing strategy for enhancing maturation. Conclusions By providing the environmental cues that are missing in conventional culturing method, this approach did not require any genetic or pharmacological interventions. Our findings can facilitate clinical applications, drug discovery and cardiotoxicity screening by improving the yield, safety and efficacy of derived CMs. PMID:23392582

Identification of stable reference genes in differentiating human pluripotent stem cells.

PubMed

Holmgren, Gustav; Ghosheh, Nidal; Zeng, Xianmin; Bogestål, Yalda; Sartipy, Peter; Synnergren, Jane

2015-06-01

Reference genes, often referred to as housekeeping genes (HKGs), are frequently used to normalize gene expression data based on the assumption that they are expressed at a constant level in the cells. However, several studies have shown that there may be a large variability in the gene expression levels of HKGs in various cell types. In a previous study, employing human embryonic stem cells (hESCs) subjected to spontaneous differentiation, we observed that the expression of commonly used HKG varied to a degree that rendered them inappropriate to use as reference genes under those experimental settings. Here we present a substantially extended study of the HKG signature in human pluripotent stem cells (hPSC), including nine global gene expression datasets from both hESC and human induced pluripotent stem cells, obtained during directed differentiation toward endoderm-, mesoderm-, and ectoderm derivatives. Sets of stably expressed genes were compiled, and a handful of genes (e.g., EID2, ZNF324B, CAPN10, and RABEP2) were identified as generally applicable reference genes in hPSCs across all cell lines and experimental conditions. The stability in gene expression profiles was confirmed by reverse transcription quantitative PCR analysis. Taken together, the current results suggest that differentiating hPSCs have a distinct HKG signature, which in some aspects is different from somatic cell types, and underscore the necessity to validate the stability of reference genes under the actual experimental setup used. In addition, the novel putative HKGs identified in this study can preferentially be used for normalization of gene expression data obtained from differentiating hPSCs. Copyright © 2015 the American Physiological Society.

Cationic Surface Charge Combined with Either Vitronectin or Laminin Dictates the Evolution of Human Embryonic Stem Cells/Microcarrier Aggregates and Cell Growth in Agitated Cultures

PubMed Central

Lam, Alan Tin-Lun; Li, Jian; Chen, Allen Kuan-Liang; Reuveny, Shaul

2014-01-01

The expansion of human pluripotent stem cells (hPSC) for biomedical applications generally compels a defined, reliable, and scalable platform. Bioreactors offer a three-dimensional culture environment that relies on the implementation of microcarriers (MC), as supports for cell anchorage and their subsequent growth. Polystyrene microspheres/MC coated with adhesion-promoting extracellular matrix (ECM) protein, vitronectin (VN), or laminin (LN) have been shown to support hPSC expansion in a static environment. However, they are insufficient to promote human embryonic stem cells (hESC) seeding and their expansion in an agitated environment. The present study describes an innovative technology, consisting of a cationic charge that underlies the ECM coatings. By combining poly-L-lysine (PLL) with a coating of ECM protein, cell attachment efficiency and cell spreading are improved, thus enabling seeding under agitation in a serum-free medium. This coating combination also critically enables the subsequent formation and evolution of hPSC/MC aggregates, which ensure cell viability and generate high yields. Aggregate dimensions of at least 300 μm during early cell growth give rise to ≈15-fold expansion at 7 days' culture. Increasing aggregate numbers at a quasi-constant size of ≈300 μm indicates hESC growth within a self-regulating microenvironment. PLL+LN enables cell seeding and aggregate evolution under constant agitation, whereas PLL+VN requires an intermediate 2-day static pause to attain comparable aggregate sizes and correspondingly high expansion yields. The cells' highly reproducible bioresponse to these defined and characterized MC surface properties is universal across multiple cell lines, thus confirming the robustness of this scalable expansion process in a defined environment. PMID:24641164

Undifferentiated Endometrial Carcinomas Show Frequent Loss of Core Switch/Sucrose Nonfermentable Complex Proteins.

PubMed

Köbel, Martin; Hoang, Lien N; Tessier-Cloutier, Basile; Meng, Bo; Soslow, Robert A; Stewart, Colin J R; Lee, Cheng-Han

2018-01-01

Undifferentiated endometrial carcinoma is an aggressive type of endometrial carcinoma that typically presents with advanced stage disease and rapid clinical progression. In contrast to dedifferentiated endometrial carcinoma, undifferentiated carcinoma lacks a concurrent differentiated (typically low-grade endometrioid) carcinoma component, though the undifferentiated component of dedifferentiated carcinoma is similar histologically and immunophenotypically to pure undifferentiated carcinoma. We recently identified 3 mutually exclusive mechanisms of switch/sucrose nonfermentable (SWI/SNF) complex inactivation (BRG1 inactivation, INI1 inactivation or ARID1A/ARID1B co-inactivation) that are associated with histologic dedifferentiation in the majority of dedifferentiated endometrial carcinoma. In the current study, we aimed to determine by immunohistochemistry whether these patterns of SWI/SNF inactivation also occur in undifferentiated endometrial carcinomas. Of the 34 undifferentiated carcinomas examined, 17 (50%) exhibited SWI/SNF complex inactivation, with 11 tumors showing complete loss of both ARID1A and ARID1B, 5 showing complete loss of BRG1 and 1 showing complete loss of INI1. Ten of the remaining 17 undifferentiated carcinomas showed the following alterations: 5 tumors (15%) showed loss of ARID1A only with intact ARID1B, BRG1, and INI1 expression, 4 tumors (12%) showed mutated patterns of p53 staining with intact SWI/SNF protein expression, and 1 tumor (3%) harbored a POLE exonuclease domain mutation (P286R). SWI/SNF complex-inactivated tumors presented more frequently with extrauterine disease spread than those with intact expression (88% vs. 41%, respectively). In addition, patients with SWI/SNF complex-inactivated tumors had a significantly worse disease-specific survival (P=0.02). The findings here demonstrate frequent SWI/SNF complex inactivation in undifferentiated endometrial carcinomas, which has future implications regarding therapies that target chromatin remodelling and epigenetic control.

Undifferentiated and Dedifferentiated Endometrial Carcinomas With POLE Exonuclease Domain Mutations Have a Favorable Prognosis.

PubMed

Espinosa, Iñigo; Lee, Cheng-Han; D'Angelo, Emanuela; Palacios, José; Prat, Jaime

2017-08-01

POLE exonuclease domain mutations have recently been described in undifferentiated endometrial carcinoma but, because of the rarity of this aggressive type of endometrial cancer, their prognostic significance is unknown. We have analyzed the immunophenotype (ARID1A, MLH1, PMS2, MSH2, MSH6, p53, β-catenin, and SMARCB1) and mutational status (POLE, PIK3CA, and PTEN) of 21 undifferentiated carcinomas (8 undifferentiated and 13 dedifferentiated carcinomas). Loss of ARID1A expression was observed in 9 of 19 cases (47%), loss of expression of at least 1 DNA mismatch repair protein in 7 (7/21; 33%), and p53 immunoreaction was aberrant (mutated/inactivated) in 11 cases (11/21; 52%). All tumors were negative for β-catenin. Normal nuclear SMARCB1 (INI1) staining was found in all but 1 dedifferentiated case. Two undifferentiated and 7 dedifferentiated carcinomas showed POLE exonuclease domain mutations (9/21; 42%). PIK3CA mutations occurred in six tumors (6/21; 28%) (2 undifferentiated and 4 dedifferentiated carcinomas). PTEN mutations were found in 7 of 15 cases (47%) (4 undifferentiated and 3 dedifferentiated carcinomas). POLE-mutated undifferentiated and dedifferentiated endometrial carcinomas were more frequently stage I tumors than similar carcinomas lacking exonuclease domain mutations (7/9; 78% vs. 3/12; 25%; P=0.023) and patients had significantly better outcome (disease-specific survival) than those without POLE exonuclease domain mutations (P=0.02). Determination of the POLE mutation status is important for the management of these patients.

Stepping Into and Out of the Void: Funding Dynamics of Human Embryonic Stem Cell Research in California, Sweden, and South Korea.

PubMed

Weinryb, Noomi; Bubela, Tania

2016-02-01

Nonprofit organizations and philanthropists stepped into a funding void caused by controversies over public funding of human embryonic stem cell (hESC) research. Based on interviews of 83 representatives of 53 funders, we examine the motivations and accountability structures of public agencies, corporations, fundraising dependent nonprofit organizations and philanthropic organizations that funded hESC research in three jurisdictions: California, Sweden, and South Korea. While non-traditional forms of funding are essential in the early stages of research advancement, they are unreliable for the long timeframes necessary to advance cell therapies. Such funding sources may enter the field based on high expectations, but may exit just as rapidly based on disappointing rates of progress.

Characterization of three human cell line models for high-throughput neuronal cytotoxicity screening.

PubMed

Tong, Zhi-Bin; Hogberg, Helena; Kuo, David; Sakamuru, Srilatha; Xia, Menghang; Smirnova, Lena; Hartung, Thomas; Gerhold, David

2017-02-01

More than 75 000 man-made chemicals contaminate the environment; many of these have not been tested for toxicities. These chemicals demand quantitative high-throughput screening assays to assess them for causative roles in neurotoxicities, including Parkinson's disease and other neurodegenerative disorders. To facilitate high throughput screening for cytotoxicity to neurons, three human neuronal cellular models were compared: SH-SY5Y neuroblastoma cells, LUHMES conditionally-immortalized dopaminergic neurons, and Neural Stem Cells (NSC) derived from human fetal brain. These three cell lines were evaluated for rapidity and degree of differentiation, and sensitivity to 32 known or candidate neurotoxicants. First, expression of neural differentiation genes was assayed during a 7-day differentiation period. Of the three cell lines, LUHMES showed the highest gene expression of neuronal markers after differentiation. Both in the undifferentiated state and after 7 days of neuronal differentiation, LUHMES cells exhibited greater cytotoxic sensitivity to most of 32 suspected or known neurotoxicants than SH-SY5Y or NSCs. LUHMES cells were also unique in being more susceptible to several compounds in the differentiating state than in the undifferentiated state; including known neurotoxicants colchicine, methyl-mercury (II), and vincristine. Gene expression results suggest that differentiating LUHMES cells may be susceptible to apoptosis because they express low levels of anti-apoptotic genes BCL2 and BIRC5/survivin, whereas SH-SY5Y cells may be resistant to apoptosis because they express high levels of BCL2, BIRC5/survivin, and BIRC3 genes. Thus, LUHMES cells exhibited favorable characteristics for neuro-cytotoxicity screening: rapid differentiation into neurons that exhibit high level expression neuronal marker genes, and marked sensitivity of LUHMES cells to known neurotoxicants. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Lingual Kinematics and Coordination in Speech-Disordered Children Exhibiting Differentiated versus Undifferentiated Lingual Gestures

ERIC Educational Resources Information Center

Goozee, Justine; Murdoch, Bruce; Ozanne, Anne; Cheng, Yan; Hill, Anne; Gibbon, Fiona

2007-01-01

Background: Electropalatographic investigations have revealed that a proportion of children with articulation/phonological disorders exhibit undifferentiated lingual gestures, whereby the whole of the tongue contacts the palate simultaneously during lingual consonant production. These undifferentiated lingual gestures have been interpreted to…

ARG1 Functions in the Physiological Adaptation of Undifferentiated Plant Cells to Spaceflight.

PubMed

Zupanska, Agata K; Schultz, Eric R; Yao, JiQiang; Sng, Natasha J; Zhou, Mingqi; Callaham, Jordan B; Ferl, Robert J; Paul, Anna-Lisa

2017-11-01

Scientific access to spaceflight and especially the International Space Station has revealed that physiological adaptation to spaceflight is accompanied or enabled by changes in gene expression that significantly alter the transcriptome of cells in spaceflight. A wide range of experiments have shown that plant physiological adaptation to spaceflight involves gene expression changes that alter cell wall and other metabolisms. However, while transcriptome profiling aptly illuminates changes in gene expression that accompany spaceflight adaptation, mutation analysis is required to illuminate key elements required for that adaptation. Here we report how transcriptome profiling was used to gain insight into the spaceflight adaptation role of Altered response to gravity 1 (Arg1), a gene known to affect gravity responses in plants on Earth. The study compared expression profiles of cultured lines of Arabidopsis thaliana derived from wild-type (WT) cultivar Col-0 to profiles from a knock-out line deficient in the gene encoding ARG1 (ARG1 KO), both on the ground and in space. The cell lines were launched on SpaceX CRS-2 as part of the Cellular Expression Logic (CEL) experiment of the BRIC-17 spaceflight mission. The cultured cell lines were grown within 60 mm Petri plates in Petri Dish Fixation Units (PDFUs) that were housed within the Biological Research In Canisters (BRIC) hardware. Spaceflight samples were fixed on orbit. Differentially expressed genes were identified between the two environments (spaceflight and comparable ground controls) and the two genotypes (WT and ARG1 KO). Each genotype engaged unique genes during physiological adaptation to the spaceflight environment, with little overlap. Most of the genes altered in expression in spaceflight in WT cells were found to be Arg1-dependent, suggesting a major role for that gene in the physiological adaptation of undifferentiated cells to spaceflight. Key Words: ARG1-Spaceflight-Gene expression-Physiological adaptation-BRIC. Astrobiology 17, 1077-1111.

Use of Stirred Suspension Bioreactors for Male Germ Cell Enrichment.

PubMed

Sakib, Sadman; Dores, Camila; Rancourt, Derrick; Dobrinski, Ina

2016-01-01

Spermatogenesis is a stem cell based system. Both therapeutic and biomedical research applications of spermatogonial stem cells require a large number of cells. However, there are only few germ line stem cells in the testis, contained in the fraction of undifferentiated spermatogonia. The lack of specific markers makes it difficult to isolate these cells. The long term maintenance and proliferation of nonrodent germ cells in culture has so far been met with limited success, partially due to the lack of highly enriched starting populations. Differential plating, which depends on the differential adhesion properties of testicular somatic and germ cells to tissue culture dishes, has been the method of choice for germ cell enrichment, especially for nonrodent germ cells. However, for large animals, this process becomes labor intensive and increases variability due to the need for extensive handling. Here, we describe the use of stirred suspension bioreactors, as a novel system for enriching undifferentiated germ cells from 1-week-old pigs. This method capitalizes on the adherent properties of somatic cells within a controlled environment, thus promoting the enrichment of progenitor cells with minimal handling and variability.

Molecular genetic heterogeneity in undifferentiated endometrial carcinomas.

PubMed

Rosa-Rosa, Juan M; Leskelä, Susanna; Cristóbal-Lana, Eva; Santón, Almudena; López-García, Ma Ángeles; Muñoz, Gloria; Pérez-Mies, Belen; Biscuola, Michele; Prat, Jaime; Esther, Oliva E; Soslow, Robert A; Matias-Guiu, Xavier; Palacios, Jose

2016-11-01

Undifferentiated and dedifferentiated endometrial carcinomas are rare and highly aggressive subtypes of uterine cancer, not well characterized at a molecular level. To investigate whether dedifferentiated carcinomas carry molecular genetic alterations similar to those of pure undifferentiated carcinomas, and to gain insight into the pathogenesis of these tumors, we selected a cohort of 18 undifferentiated endometrial carcinomas, 8 of them with a well-differentiated endometrioid carcinoma component (dedifferentiated endometrioid carcinomas), and studied them by immunohistochemistry and massive parallel and Sanger sequencing. Whole-exome sequencing of the endometrioid and undifferentiated components, as well as normal myometrium, was also carried out in one case. According to The Cancer Genome Atlas classification, we distributed 95% of the undifferentiated carcinomas in this series as follows: (a) hypermutated tumors with loss of any mismatch repair protein expression and microsatellite instability (eight cases, 45%); (b) ultramutated carcinomas carrying mutations in the exonuclease domain of POLE (two cases, 11%); (c) high copy number alterations (copy-number high) tumors group exhibiting only TP53 mutations and high number of alterations detected by FISH (two cases, 11%); and (d) low copy number alterations (copy-number low) tumors with molecular alterations typical of endometrioid endometrial carcinomas (five cases, 28%). Two of the latter cases, however, also had TP53 mutations and higher number of alterations detected by FISH and could have progressed to a copy-number high phenotype. Most dedifferentiated carcinomas belonged to the hypermutated group, whereas pure undifferentiated carcinomas shared molecular genetic alterations with copy-number low or copy-number high tumors. These results indicate that undifferentiated and dedifferentiated endometrial carcinomas are molecularly heterogeneous tumors, which may have prognostic value.

Molecular genetic heterogeneity in undifferentiated endometrial carcinomas

PubMed Central

Rosa-Rosa, J.M.; Leskelä, S.; Cristóbal-Lana, E.; Santón, A.; López-García, M.A.; Muñoz, G.; Pérez-Mies, B.; Biscuola, M; Prat, J.; Oliva, E.; Soslow, R.A.; Matias-Guiu, X.; Palacios, J.

2017-01-01

Undifferentiated and dedifferentiated endometrial carcinomas are rare and highly aggressive subtypes of uterine cancer, not well characterized at a molecular level. To investigate whether dedifferentiated carcinomas carry molecular genetic alterations similar to those of pure undifferentiated carcinomas, and to gain insight into the pathogenesis of these tumours, we selected a cohort of 18 undifferentiated endometrial carcinomas, 8 of them with a well differentiated endometrioid carcinoma component (dedifferentiated endometrioid carcinomas), and studied them by immunohistochemistry and massive parallel and Sanger sequencing. Whole exome sequencing of the endometrioid and undifferentiated components as well as normal myometrium, was also carried out in one case. According to The Cancer Genome Atlas classification, we distributed 95% of the undifferentiated carcinomas in this series as follows: a) hypermutated tumours with loss of any mismatch repair protein expression and microsatellite instability (eight cases, 45%); b) ultramutated carcinomas carrying mutations in the exonuclease domain of POLE (two cases, 11%); c) high copy number alterations (copy-number high) tumours group exhibiting only TP53 mutations and high number of alterations detected by FISH (two cases, 11%) ; and d) low copy number alterations (copy-number low) tumours with molecular alterations typical of endometrioid endometrial carcinomas (five cases, 28%). Two of the latter cases, however, also had TP53 mutations and higher number of alterations detected by FISH and could have progressed to a copy-number high phenotype. Most dedifferentiated carcinomas belonged to the hypermutated group whereas pure undifferentiated carcinomas shared molecular genetic alterations with copy-number low or copy-number high tumours. These results indicate that undifferentiated and dedifferentiated endometrial carcinomas are molecularly heterogeneous tumours, which may have prognostic value. PMID:27491810

Mouse decellularised liver scaffold improves human embryonic and induced pluripotent stem cells differentiation into hepatocyte-like cells

PubMed Central

Scottoni, Federico; Crowley, Claire; Fiadeiro, Rebeca; Maghsoudlou, Panagiotis; Pellegata, Alessandro Filippo; Mazzacuva, Francesca; Gjinovci, Asllan; Lyne, Anne-Marie; Zulini, Justine; Little, Daniel; Mosaku, Olukunbi; Kelly, Deirdre; De Coppi, Paolo; Gissen, Paul

2017-01-01

Liver transplantation is the definitive treatment of liver failure but donor organ shortage limits its availability. Stem cells are highly expandable and have the potential to differentiate into any specialist cell. Use of patient-derived induced Pluripotent Stem Cells (hiPSCs) has the additional advantage for organ regeneration therapies by removing the need for immunosuppression. We compared hepatocyte differentiation of human embryonic stem cells (hESCs) and hiPSCs in a mouse decellularised liver scaffold (3D) with standard in vitro protocol (2D). Mouse livers were decellularised preserving micro-architecture, blood vessel network and extracellular matrix. hESCs and hiPSCs were primed towards the definitive endoderm. Cells were then seeded either in 3D or 2D cultures and the hepatocyte differentiation was continued. Both hESCs and hiPSCs differentiated more efficiently in 3D than in 2D, with higher and earlier expression of mature hepatocyte marker albumin, lipid and glycogen synthesis associated with a decrease in expression of fetal hepatocyte marker alpha-fetoprotein. Thus we conclude that stem cell hepatocyte differentiation in 3D culture promotes faster cell maturation. This finding suggests that optimised 3D protocols could allow generation of mature liver cells not achieved so far in standard 2D conditions and lead to improvement in cell models of liver disease and regenerative medicine applications. PMID:29261712

Immune modulatory mesenchymal stem cells derived from human embryonic stem cells through a trophoblast-like stage.

PubMed

Wang, Xiaofang; Lazorchak, Adam S; Song, Li; Li, Enqin; Zhang, Zhenwu; Jiang, Bin; Xu, Ren-He

2016-02-01

Mesenchymal stem/stromal cells (MSCs) have great clinical potential in modulating inflammation and promoting tissue repair. Human embryonic stem cells (hESCs) have recently emerged as a potentially superior cell source for MSCs. However, the generation methods reported so far vary greatly in quality and efficiency. Here, we describe a novel method to rapidly and efficiently produce MSCs from hESCs via a trophoblast-like intermediate stage in approximately 11-16 days. We term these cells "T-MSCs" and show that T-MSCs express a phenotype and differentiation potential minimally required to define MSCs. T-MSCs exhibit potent immunomodulatory activity in vitro as they can remarkably inhibit proliferation of cocultured T and B lymphocytes. Unlike bone marrow MSCs, T-MSCs do not have increased expression of inflammatory mediators in response to IFNγ. Moreover, T-MSCs constitutively express a high level of the immune inhibitory ligand PD-L1 and elicit strong and durable efficacy in two distinct animal models of autoimmune disease, dextran sulfate sodium induced colitis, and experimental autoimmune encephalomyelitis, at doses near those approved for clinical trials. Together, we present a simple and fast derivation method to generate MSCs from hESCs, which possess potent immunomodulatory properties in vitro and in vivo and may serve as a novel and ideal candidate for MSC-based therapies. © 2015 AlphaMed Press.

Three-Step Method for Proliferation and Differentiation of Human Embryonic Stem Cell (hESC)-Derived Male Germ Cells

PubMed Central

Lim, Jung Jin; Shim, Myung Sun; Lee, Jeoung Eun; Lee, Dong Ryul

2014-01-01

The low efficiency of differentiation into male germ cell (GC)-like cells and haploid germ cells from human embryonic stem cells (hESCs) reflects the culture method employed in the two-dimensional (2D)-microenvironment. In this study, we applied a three-step media and calcium alginate-based 3D-culture system for enhancing the differentiation of hESCs into male germ stem cell (GSC)-like cells and haploid germ cells. In the first step, embryoid bodies (EBs) were derived from hESCs cultured in EB medium for 3 days and re-cultured for 4 additional days in EB medium with BMP4 and RA to specify GSC-like cells. In the second step, the resultant cells were cultured in GC-proliferation medium for 7 days. The GSC-like cells were then propagated after selection using GFR-α1 and were further cultured in GC-proliferation medium for 3 weeks. In the final step, a 3D-co-culture system using calcium alginate encapsulation and testicular somatic cells was applied to induce differentiation into haploid germ cells, and a culture containing approximately 3% male haploid germ cells was obtained after 2 weeks of culture. These results demonstrated that this culture system could be used to efficiently induce GSC-like cells in an EB population and to promote the differentiation of ESCs into haploid male germ cells. PMID:24690677

Bem Sex Role Inventory Undifferentiated Score: A Comparison of Sexual Dysfunction Patients with Sexual Offenders.

ERIC Educational Resources Information Center

Dwyer, Margretta; And Others

1988-01-01

Examined Bem Sex Role undifferentiated scores on 93 male sex offenders as compared with 50 male sexually dysfunctional patients. Chi-square analyses revealed significant difference: offenders obtained undifferentiated scores more often than did sexual dysfunctional population. Concluded that Bem Sex Role Inventory is useful in identifying sexual…

Superficial EWSR1-negative undifferentiated small round cell sarcoma with CIC/DUX4 gene fusion: a new variant of Ewing-like tumors with locoregional lymph node metastasis.

PubMed

Machado, Isidro; Cruz, Julia; Lavernia, Javier; Rubio, Luis; Campos, Jorge; Barrios, María; Grison, Camille; Chene, Virginie; Pierron, Gaelle; Delattre, Olivier; Llombart-Bosch, Antonio

2013-12-01

The present study describes a new case of EWSR1-negative undifferentiated sarcoma with CIC/DUX4 gene fusion. This case is similar to tumors described as primitive undifferentiated round cell sarcomas that occur mainly in the trunk and display an aggressive behavior. To our knowledge, this is the first report of such a tumor presenting locoregional lymph node metastasis. In view of previous studies that prove the existence of a particular variant of undifferentiated sarcoma with Ewing-like morphology and CIC/DUX-4 gene fusion, a search for this gene fusion in all undifferentiated round cell sarcomas should be considered if a conclusive diagnosis cannot be reached following other conventional studies. Although additional cases with more extensive follow-up studies are needed, we believe that EWSR1-negative undifferentiated small round cell sarcoma with CIC/DUX4 gene fusion should be added to the list of new sarcoma variants with the possibility of lymph node metastasis.

ETV4 is a useful marker for the diagnosis of CIC-rearranged undifferentiated round-cell sarcomas: a study of 127 cases including mimicking lesions.

PubMed

Le Guellec, Sophie; Velasco, Valérie; Pérot, Gaëlle; Watson, Sarah; Tirode, Franck; Coindre, Jean-Michel

2016-12-01

Subsets of primitive round-cell sarcomas remain difficult to diagnose and classify. Among these is a rare round-cell sarcoma that harbors a CIC gene rearrangement known as CIC-rearranged undifferentiated round-cell sarcoma, which is most commonly fused to the DUX4 gene. Owing to its aggressive clinical behavior and potential therapeutic implications, accurate identification of this novel soft tissue sarcoma is necessary. Definitive diagnosis requires molecular confirmation, but only a few centers are as yet able to perform this test. Several studies have shown that PEA3 subfamily genes, notably ETV4 (belonging to the family of ETS transcription factors), are upregulated in CIC-rearranged undifferentiated round-cell sarcomas. We performed a detailed immunohistochemical analysis to investigate ETV4 expression in CIC-rearranged undifferentiated round-cell sarcomas and their potential mimics (especially Ewing sarcomas). The study cohort included 17 cases of CIC-rearranged undifferentiated round-cell sarcomas, and 110 tumors that morphologically mimic CIC-rearranged undifferentiated round-cell sarcomas: 43 Ewing sarcomas, 25 alveolar rhabdomyosarcomas, 20 poorly differentiated round-cell synovial sarcomas, 10 desmoplastic round-cell tumors, 5 BCOR-CCNB3 sarcomas, 5 lymphoblastic lymphomas, and 2 rhabdoid tumors. All CIC-rearranged undifferentiated round-cell sarcomas (on core needle biopsies and open biopsies) were ETV4-positive with a strong diffuse nuclear pattern. Among the other 110 tumors, only six cases (four Ewing sarcomas, one alveolar rhabdomyosarcoma, and one desmoplastic round-cell tumor) showed focal (<5% of tumor cells) and very weak nuclear expression of ETV4; all other tumors were completely negative for ETV4. We conclude that systematic immunohistochemical analysis of ETV4 makes it possible to diagnose undifferentiated round-cell sarcomas (with no molecular markers for sarcoma-associated translocation) such as CIC-rearranged undifferentiated round-cell sarcoma.

Expression profile of undifferentiated cell transcription factor 1 in normal and cancerous human epithelia.

PubMed

Mouallif, Mustapha; Albert, Adelin; Zeddou, Mustapha; Ennaji, My Mustapha; Delvenne, Philippe; Guenin, Samuel

2014-08-01

Undifferentiated cell Transcription Factor 1 (UTF1) is a chromatin-bound protein involved in stem cell differentiation. It was initially reported to be restricted to stem cells or germinal tissues. However, recent work suggests that UTF1 is also expressed in somatic cells and that its expression may increase during carcinogenesis. To further clarify the expression profile of UTF1, we evaluated UTF1 expression levels immunohistochemically in eight normal human epithelia (from breast, prostate, endometrium, bladder, colon, oesophagus, lung and kidney) and their corresponding tumours as well as in several epithelial cell lines. We showed UTF1 staining in normal and tumour epithelial tissues, but with varying intensities according to the tissue location. In vitro analyses also revealed that UTF1 is expressed in somatic epithelial cell lines even in the absence of Oct4A and Sox2, its two main known regulators. The comparison of UTF1 levels in normal and tumoral tissues revealed significant overexpression in endometrial and prostatic adenocarcinomas, whereas lower intensity of the staining was observed in renal and colic tumours, suggesting a potential tissue-specific function of UTF1. Altogether, these results highlight a potential dual role for UTF1, acting either as an oncogene or as a tumour suppressor depending on the tissue. These findings also question its role as a specific marker for stem cells. © 2014 The Authors. International Journal of Experimental Pathology © 2014 International Journal of Experimental Pathology.

Putative porcine embryonic stem cell lines derived from aggregated four-celled cloned embryos produced by oocyte bisection cloning.

PubMed

Siriboon, Chawalit; Lin, Yu-Hsuan; Kere, Michel; Chen, Chun-Da; Chen, Lih-Ren; Chen, Chien-Hong; Tu, Ching-Fu; Lo, Neng-Wen; Ju, Jyh-Cherng

2015-01-01

We attempted to isolate ES cell lines using inner cell masses from high-quality cloned porcine blastocysts. After being seeded onto feeders, embryos had better (P < 0.05) attachment, outgrowth formation and primary colonization in both 2× and 3× aggregated cloned embryos (62.8, 42.6 and 12.8% vs. 76.2, 55.2 and 26.2%, respectively) compared to the non-aggregated group (41.6, 23.4 and 3.9%). Effects of feeder types (STO vs. MEF) and serum sources (FBS vs. KSR) on extraction of cloned embryo-derived porcine ES cells were examined. More (17.1%) ntES cell lines over Passage 3 were generated in the MEF/KSR group. However, ntES cells cultured in KSR-supplemented medium had a low proliferation rate with defective morphology, and eventually underwent differentiation or apoptosis subsequently. Approximately 26.1, 22.7 and 35.7% of primary colonies were formed after plating embryos in DMEM, DMEM/F12 and α-MEM media, respectively. Survival rates of ntES cells cultured in α-MEM, DMEM and DMEM/F12 were 16.7, 4.3 and 6.8%, respectively (P > 0.05). We further examined the beneficial effect of TSA treatment of 3× aggregated cloned embryos on establishment of ntES cell lines. Primary colony numbers and survival rates of ntES cells beyond passage 3 were higher (P < 0.05) in those derived from TSA-treated 3× blastocysts (36.7 and 26.7%) than from the non-treated aggregated group (23.1 and 11.5%). These cells, remaining undifferentiated over 25 passages, had alkaline phosphatase activity and expressed ES specific markers Oct4, Nanog, Sox2, and Rex01. Moreover, these ntES cells successfully differentiated into embryoid bodies (EBs) that expressed specific genes of all three germ layers after being cultured in LIF-free medium. In conclusion, we have successfully derived putative porcine ntES cells with high efficiency from quality cloned embryos produced by embryo aggregation, and optimized the ES cell culture system suitable for establishing and maintaining ntES cell lines in undifferentiated state.

Putative Porcine Embryonic Stem Cell Lines Derived from Aggregated Four-Celled Cloned Embryos Produced by Oocyte Bisection Cloning

PubMed Central

Siriboon, Chawalit; Lin, Yu-Hsuan; Kere, Michel; Chen, Chun-Da; Chen, Lih-Ren; Chen, Chien-Hong; Tu, Ching-Fu; Lo, Neng-Wen; Ju, Jyh-Cherng

2015-01-01

We attempted to isolate ES cell lines using inner cell masses from high-quality cloned porcine blastocysts. After being seeded onto feeders, embryos had better (P < 0.05) attachment, outgrowth formation and primary colonization in both 2× and 3× aggregated cloned embryos (62.8, 42.6 and12.8% vs. 76.2, 55.2 and 26.2%, respectively) compared to the non-aggregated group (41.6, 23.4 and 3.9%). Effects of feeder types (STO vs. MEF) and serum sources (FBS vs. KSR) on extraction of cloned embryo-derived porcine ES cells were examined. More (17.1%) ntES cell lines over Passage 3 were generated in the MEF/KSR group. However, ntES cells cultured in KSR-supplemented medium had a low proliferation rate with defective morphology, and eventually underwent differentiation or apoptosis subsequently. Approximately 26.1, 22.7 and 35.7% of primary colonies were formed after plating embryos in DMEM, DMEM/F12 and α-MEM media, respectively. Survival rates of ntES cells cultured in α-MEM, DMEM and DMEM/F12 were 16.7, 4.3 and 6.8%, respectively (P > 0.05). We further examined the beneficial effect of TSA treatment of 3× aggregated cloned embryos on establishment of ntES cell lines. Primary colony numbers and survival rates of ntES cells beyond passage 3 were higher (P < 0.05) in those derived from TSA-treated 3× blastocysts (36.7 and 26.7%) than from the non-treated aggregated group (23.1 and 11.5%). These cells, remaining undifferentiated over 25 passages, had alkaline phosphatase activity and expressed ES specific markers Oct4, Nanog, Sox2, and Rex01. Moreover, these ntES cells successfully differentiated into embryoid bodies (EBs) that expressed specific genes of all three germ layers after being cultured in LIF-free medium. In conclusion, we have successfully derived putative porcine ntES cells with high efficiency from quality cloned embryos produced by embryo aggregation, and optimized the ES cell culture system suitable for establishing and maintaining ntES cell lines in undifferentiated state. PMID:25680105

mRNA-binding protein TIA-1 reduces cytokine expression in human endometrial stromal cells and is down-regulated in ectopic endometrium.

PubMed

Karalok, Hakan Mete; Aydin, Ebru; Saglam, Ozlen; Torun, Aysenur; Guzeloglu-Kayisli, Ozlem; Lalioti, Maria D; Kristiansson, Helena; Duke, Cindy M P; Choe, Gina; Flannery, Clare; Kallen, Caleb B; Seli, Emre

2014-12-01

Cytokines and growth factors play important roles in endometrial function and the pathogenesis of endometriosis. mRNAs encoding cytokines and growth factors undergo rapid turnover; primarily mediated by adenosine- and uridine-rich elements (AREs) located in their 3'-untranslated regions. T-cell intracellular antigen (TIA-1), an mRNA-binding protein, binds to AREs in target transcripts, leading to decreased gene expression. The purpose of this article was to determine whether TIA-1 plays a role in the regulation of endometrial cytokine and growth factor expression during the normal menstrual cycle and whether TIA-1 expression is altered in women with endometriosis. Eutopic endometrial tissue obtained from women without endometriosis (n = 30) and eutopic and ectopic endometrial tissues from women with endometriosis (n = 17) were immunostained for TIA-1. Staining intensities were evaluated by histological scores (HSCOREs). The regulation of endometrial TIA-1 expression by immune factors and steroid hormones was studied by treating primary cultured human endometrial stromal cells (HESCs) with vehicle, lipopolysaccharide, TNF-α, IL-6, estradiol, or progesterone, followed by protein blot analyses. HESCs were engineered to over- or underexpress TIA-1 to test whether TIA-1 regulates IL-6 or TNF-α expression in these cells. We found that TIA-1 is expressed in endometrial stromal and glandular cells throughout the menstrual cycle and that this expression is significantly higher in the perimenstrual phase. In women with endometriosis, TIA-1 expression in eutopic and ectopic endometrium was reduced compared with TIA-1 expression in eutopic endometrium of unaffected control women. Lipopolysaccharide and TNF-α increased TIA-1 expression in HESCs in vitro, whereas IL-6 or steroid hormones had no effect. In HESCs, down-regulation of TIA-1 resulted in elevated IL-6 and TNF-α expression, whereas TIA-1 overexpression resulted in decreased IL-6 and TNF-α expression. Endometrial TIA-1 is regulated throughout the menstrual cycle, TIA-1 modulates the expression of immune factors in endometrial cells, and downregulation of TIA-1 may contribute to the pathogenesis of endometriosis.

mRNA-Binding Protein TIA-1 Reduces Cytokine Expression in Human Endometrial Stromal Cells and Is Down-Regulated in Ectopic Endometrium

PubMed Central

Karalok, Hakan Mete; Aydin, Ebru; Saglam, Ozlen; Torun, Aysenur; Guzeloglu-Kayisli, Ozlem; Lalioti, Maria D.; Kristiansson, Helena; Duke, Cindy M. P.; Choe, Gina; Flannery, Clare; Kallen, Caleb B.

2014-01-01

Background: Cytokines and growth factors play important roles in endometrial function and the pathogenesis of endometriosis. mRNAs encoding cytokines and growth factors undergo rapid turnover; primarily mediated by adenosine- and uridine-rich elements (AREs) located in their 3′-untranslated regions. T-cell intracellular antigen (TIA-1), an mRNA-binding protein, binds to AREs in target transcripts, leading to decreased gene expression. Objective: The purpose of this article was to determine whether TIA-1 plays a role in the regulation of endometrial cytokine and growth factor expression during the normal menstrual cycle and whether TIA-1 expression is altered in women with endometriosis. Methods: Eutopic endometrial tissue obtained from women without endometriosis (n = 30) and eutopic and ectopic endometrial tissues from women with endometriosis (n = 17) were immunostained for TIA-1. Staining intensities were evaluated by histological scores (HSCOREs). The regulation of endometrial TIA-1 expression by immune factors and steroid hormones was studied by treating primary cultured human endometrial stromal cells (HESCs) with vehicle, lipopolysaccharide, TNF-α, IL-6, estradiol, or progesterone, followed by protein blot analyses. HESCs were engineered to over- or underexpress TIA-1 to test whether TIA-1 regulates IL-6 or TNF-α expression in these cells. Results: We found that TIA-1 is expressed in endometrial stromal and glandular cells throughout the menstrual cycle and that this expression is significantly higher in the perimenstrual phase. In women with endometriosis, TIA-1 expression in eutopic and ectopic endometrium was reduced compared with TIA-1 expression in eutopic endometrium of unaffected control women. Lipopolysaccharide and TNF-α increased TIA-1 expression in HESCs in vitro, whereas IL-6 or steroid hormones had no effect. In HESCs, down-regulation of TIA-1 resulted in elevated IL-6 and TNF-α expression, whereas TIA-1 overexpression resulted in decreased IL-6 and TNF-α expression. Conclusions: Endometrial TIA-1 is regulated throughout the menstrual cycle, TIA-1 modulates the expression of immune factors in endometrial cells, and downregulation of TIA-1 may contribute to the pathogenesis of endometriosis. PMID:25140393

Mechanism and characteristics of stimuli-dependent ROS generation in undifferentiated HL-60 cells.

PubMed

Muranaka, Shikibu; Fujita, Hirofumi; Fujiwara, Takuzo; Ogino, Tetsuya; Sato, Eisuke F; Akiyama, Jitsuo; Imada, Isuke; Inoue, Masayasu; Utsumi, Kozo

2005-01-01

It has been widely believed that undifferentiated human promyelocytic leukemia cells (HL-60) have no ability to generate reactive oxygen species (ROS) responding to stimuli. We report here that undifferentiated HL-60 cells possess NADPH oxidase and that generation of superoxide can be measured using a highly sensitive chemiluminescence dye, L-012. Five subunits of NADPH oxidase, namely, gp91(phox), p22(phox), p67(phox), p47(phox), and Rac 2, were detected in undifferentiated HL-60 cells by immunoblotting analysis. The contents of these NADPH oxidase components in the cells were increased with the differentiation induced by phorbol myristate acetate (PMA), except for p22(phox). Messenger RNAs of these subunits were also detected by the RT-PCR method, and their expressions increased except that of p22(phox) with the differentiation induced by PMA. Kinetic analysis using L-012 revealed that HL-60 cells generated substantial amounts of ROS by various stimulants, including formylmethionyl-leucyl-phenylalanine, PMA, myristic acid, and a Ca2+ ionophore, A23187. Both diphenyleneiodonium (an inhibitor of FAD-dependent oxidase) and apocynin (a specific inhibitor of NADPH oxidase) suppressed this stimuli-dependent ROS generation. Genistein, staurosporine, uric acid, and sodium azide inhibited the ROS generation in undifferentiated HL-60 cells in a similar way to that in undifferentiated neutrophils. These results suggested that the mechanism of ROS generation in undifferentiated HL-60 cells is the same as that in primed neutrophils.

A lentiviral vector with a short troponin-I promoter for tracking cardiomyocyte differentiation of human embryonic stem cells.

PubMed

Gallo, P; Grimaldi, S; Latronico, M V G; Bonci, D; Pagliuca, A; Gallo, P; Ausoni, S; Peschle, C; Condorelli, G

2008-02-01

Human embryonic stem cells (hESCs) may become important for cardiac repair due to their potentially unlimited ability to generate cardiomyocytes (CMCs). Moreover, genetic manipulation of hESC-derived CMCs would be a very promising technique for curing myocardial disorders. At the present time, however, inducing the differentiation of hESCs into CMCs is extremely difficult and, therefore, an easy and standardizable technique is needed to evaluate differentiation strategies. Vectors driving cardiac-specific expression may represent an important tool not only for monitoring new cardiac-differentiation strategies, but also for the manipulation of cardiac differentiation of ESCs. To this aim, we generated cardiac-specific lentiviral vectors (LVVs) in which expression is driven by a short fragment of the cardiac troponin-I proximal promoter (TNNI3) with a human cardiac alpha-actin enhancer, and tested its suitability in inducing tissue-specific gene expression and ability to track the CMC lineage during differentiation of ESCs. We determined that (1) TNNI3-LVVs efficiently drive cardiac-specific gene expression and mark the cardiomyogenic lineage in human and mouse ESC differentiation systems (2) the cardiac alpha-actin enhancer confers a further increase in gene-expression specificity of TNNI3-LVVs in hESCs. Although this technique may not be useful in tracking small numbers of cells, data suggested that TNNI3-based LVVs are a powerful tool for manipulating human ESCs and modifying hESC-derived CMCs.

The RUNX1 +24 enhancer and P1 promoter identify a unique subpopulation of hematopoietic progenitor cells derived from human pluripotent stem cells

PubMed Central

Ferrell, Patrick I; Xi, Jiafei; Ma, Chao; Adlakha, Mitali; Kaufman, Dan S.

2016-01-01

Derivation of hematopoietic stem cells from human pluripotent stem cells remains a key goal for the fields of developmental biology and regenerative medicine. Here, we use a novel genetic reporter system to prospectively identify and isolate early hematopoietic cells derived from human embryonic stem cells (hESCs) and human induced pluripotent cells (iPSCs). Cloning the human RUNX1c P1 promoter and +24 enhancer to drive expression of tdTomato (tdTom) in hESCs and iPSCs, we demonstrate that tdTom expression faithfully enriches for RUNX1c-expressing hematopoietic progenitor cells. Time-lapse microscopy demonstrated the tdTom+ hematopoietic cells to emerge from adherent cells. Furthermore, inhibition of primitive hematopoiesis by blocking Activin/Nodal signaling promoted the expansion and/or survival of tdTom+ population. Notably, RUNX1c/tdTom+ cells represent only a limited subpopuation of CD34+CD45+ and CD34+CD43+ cells with a unique genetic signature. Using gene array analysis, we find significantly lower expression of Let-7 and mir181a microRNAs in the RUNX1c/tdTom+ cell population. These phenotypic and genetic analyses comparing the RUNX1c/tdTom+ population to CD34+CD45+ umbilical cord blood and fetal liver demonstrate several key differences that likely impact the development of HSCs capable of long-term multilineage engraftment from hESCs and iPSCs. PMID:25546363

Pazopanib Hydrochloride in Treating Patients With Advanced Thyroid Cancer

ClinicalTrials.gov

2018-05-08

Recurrent Thyroid Gland Carcinoma; Stage III Differentiated Thyroid Gland Carcinoma AJCC v7; Stage III Thyroid Gland Medullary Carcinoma AJCC v7; Stage IVA Differentiated Thyroid Gland Carcinoma AJCC v7; Stage IVA Thyroid Gland Medullary Carcinoma AJCC v7; Stage IVA Thyroid Gland Undifferentiated (Anaplastic) Carcinoma AJCC v7; Stage IVB Differentiated Thyroid Gland Carcinoma AJCC v7; Stage IVB Thyroid Gland Medullary Carcinoma AJCC v7; Stage IVB Thyroid Gland Undifferentiated (Anaplastic) Carcinoma AJCC v7; Stage IVC Differentiated Thyroid Gland Carcinoma AJCC v7; Stage IVC Thyroid Gland Medullary Carcinoma AJCC v7; Stage IVC Thyroid Gland Undifferentiated (Anaplastic) Carcinoma AJCC v7; Thyroglobulin Antibody Negative; Thyroid Gland Undifferentiated (Anaplastic) Carcinoma

Disease-specific induced pluripotent stem cells: a platform for human disease modeling and drug discovery.

PubMed

Jang, Jiho; Yoo, Jeong-Eun; Lee, Jeong-Ah; Lee, Dongjin R; Kim, Ji Young; Huh, Yong Jun; Kim, Dae-Sung; Park, Chul-Yong; Hwang, Dong-Youn; Kim, Han-Soo; Kang, Hoon-Chul; Kim, Dong-Wook

2012-03-31

The generation of disease-specific induced pluripotent stem cell (iPSC) lines from patients with incurable diseases is a promising approach for studying disease mechanisms and drug screening. Such innovation enables to obtain autologous cell sources in regenerative medicine. Herein, we report the generation and characterization of iPSCs from fibroblasts of patients with sporadic or familial diseases, including Parkinson's disease (PD), Alzheimer's disease (AD), juvenile-onset, type I diabetes mellitus (JDM), and Duchenne type muscular dystrophy (DMD), as well as from normal human fibroblasts (WT). As an example to modeling disease using disease-specific iPSCs, we also discuss the previously established childhood cerebral adrenoleukodystrophy (CCALD)- and adrenomyeloneuropathy (AMN)-iPSCs by our group. Through DNA fingerprinting analysis, the origins of generated disease-specific iPSC lines were identified. Each iPSC line exhibited an intense alkaline phosphatase activity, expression of pluripotent markers, and the potential to differentiate into all three embryonic germ layers: the ectoderm, endoderm, and mesoderm. Expression of endogenous pluripotent markers and downregulation of retrovirus-delivered transgenes [OCT4 (POU5F1), SOX2, KLF4, and c-MYC] were observed in the generated iPSCs. Collectively, our results demonstrated that disease-specific iPSC lines characteristically resembled hESC lines. Furthermore, we were able to differentiate PD-iPSCs, one of the disease-specific-iPSC lines we generated, into dopaminergic (DA) neurons, the cell type mostly affected by PD. These PD-specific DA neurons along with other examples of cell models derived from disease-specific iPSCs would provide a powerful platform for examining the pathophysiology of relevant diseases at the cellular and molecular levels and for developing new drugs and therapeutic regimens.

Human neuroblastoma SH-SY5Y cells show increased resistance to hyperthermic stress after differentiation, associated with elevated levels of Hsp72.

PubMed

Cheng, Lesley; Smith, Danielle J; Anderson, Robin L; Nagley, Phillip

2011-01-01

Terminally differentiated neurones in the central nervous system need to be protected from stress. We ask here whether differentiation of progenitor cells to neurones is accompanied by up-regulation of Hsp72, with acquisition of enhanced thermotolerance. Human neuroblastoma SH-SY5Y cells were propagated in an undifferentiated form and subsequently differentiated into neurone-like cells. Thermotolerance tests were carried out by exposure of cells to various temperatures, monitoring nuclear morphology as index of cell death. Abundance of Hsp72 was measured in cell lysates by western immunoblotting. The differentiation of SH-SY5Y cells was accompanied by increased expression of Hsp72. Further, in both cell states, exposure to mild hyperthermic stress (43°C for 30 min) increased Hsp72 expression. After differentiation, SH-SY5Y cells were more resistant to hyperthermic stress compared to their undifferentiated state, correlating with levels of Hsp72. Stable exogenous expression of Hsp72 in SH-SY5Y cells (transfected line 5YHSP72.1, containing mildly elevated levels of Hsp72), led to enhanced resistance to hyperthermic stress. Hsp72 was found to be inducible in undifferentiated 5YHSP72.1 cells; such heat-treated cells displayed enhanced thermotolerance. Treatment of cells with KNK437, a suppressor of Hsp72 induction, resulted in acute thermosensitisation of all cell types tested here. Hsp72 has a major role in the enhanced hyperthermic resistance acquired during neuronal differentiation of SH-SY5Y cells. These findings model the requirement in intact organisms for highly differentiated neurones to be specially protected against thermal stress.

A prospective study of ketamine versus haloperidol for severe prehospital agitation.

PubMed

Cole, Jon B; Moore, Johanna C; Nystrom, Paul C; Orozco, Benjamin S; Stellpflug, Samuel J; Kornas, Rebecca L; Fryza, Brandon J; Steinberg, Lila W; O'Brien-Lambert, Alex; Bache-Wiig, Peter; Engebretsen, Kristin M; Ho, Jeffrey D

2016-08-01

Ketamine is an emerging drug for the treatment of acute undifferentiated agitation in the prehospital environment, however no prospective comparative studies have evaluated its effectiveness or safety in this clinical setting. We hypothesized 5 mg/kg of intramuscular ketamine would be superior to 10 mg of intramuscular haloperidol for severe prehospital agitation, with time to adequate sedation as the primary outcome measure. This was a prospective open label study of all patients in an urban EMS system requiring chemical sedation for severe acute undifferentiated agitation that were subsequently transported to the EMS system's primary Emergency Department. All paramedics were trained in the Altered Mental Status Scale and prospectively recorded agitation scores on all patients. Two 6-month periods where either ketamine or haloperidol was the first-line therapy for severe agitation were prospectively compared primarily for time to adequate sedation. Secondary outcomes included laboratory data and adverse medication events. 146 subjects were enrolled; 64 received ketamine, 82 received haloperidol. Median time to adequate sedation for the ketamine group was 5 minutes (range 0.4-23) vs. 17 minutes (range 2-84) in the haloperidol group (difference 12 minutes, 95% CI 9-15). Complications occurred in 49% (27/55) of patients receiving ketamine vs. 5% (4/82) in the haloperidol group. Complications specific to the ketamine group included hypersalivation (21/56, 38%), emergence reaction (5/52, 10%), vomiting (5/57, 9%), and laryngospasm (3/55, 5%). Intubation was also significantly higher in the ketamine group; 39% of patients receiving ketamine were intubated vs. 4% of patients receiving haloperidol. Ketamine is superior to haloperidol in terms of time to adequate sedation for severe prehospital acute undifferentiated agitation, but is associated with more complications and a higher intubation rate.

Disorders as undifferentiated selfobject formations: treatment of a multidisordered patient.

PubMed

Rowe, Crayton E

2014-06-01

This paper offers a new understanding of disorders as undifferentiated selfobject formations. A treatment example of a multipledisordered patient is presented to illustrate how disorders diminished as a result of this understanding. This paper highlights the developmental importance of the undifferentiated selfobject and suggests that early interruptions of this discovery experience that take place during the infant's positive moments of freedom and enthusiasm are traumatic. If they go beyond the tolerance of the infant, they can be imprinted as unconscious core traumatic experiences. They remain as implicit memories that can act as warnings of repetitions of the trauma that occurred at the time of freedom and enthusiasm in the act of discovering. It can be suggested that the threat of repetitions of the traumatic loss is associated with these positive moments of discovery. This threat directs the needed self-sustaining undifferentiated selfobject discovery experience away from the positive, thereby leaving the posttraumatic effects of the loss as the focus of discovery. This focus leads to destructive preoccupations and obsessions that are considered disorders such as depression, suicidal thinking, self-mutilation, and eating disorders. Once patients understand the importance of the undifferentiated selfobject discovery need, the delinking of the undifferentiated selfobject from the negative preoccupations takes place. As a result, disorders diminish, and patients begin to consider positive possibilities for their lives. This paper suggests that early interferences in the development of the undifferentiated selfobject lead to the formation of disorders. A treatment of a multidisordered patient is presented to illustrate how this understanding was central to the diminishing of the disorders.

Gastrin-releasing peptide-induced down-regulation of tumor suppressor protein PTEN (phosphatase and tensin homolog deleted on chromosome ten) in neuroblastomas.

PubMed

Qiao, Jingbo; Kang, Junghee; Cree, Jeremy; Evers, B Mark; Chung, Dai H

2005-05-01

To evaluate whether aggressive, undifferentiated neuroblastomas express tumor suppressor protein PTEN (phosphatase and tensin homolog deleted on chromosome ten) and to examine the effects of gastrin-releasing peptide (GRP) on PTEN gene and protein expression. We have previously shown that neuroblastomas secrete GRP, which binds to its cell surface receptor (GRP-R) to stimulate cell growth in an autocrine fashion. However, the effects of GRP on expression of the tumor suppressor gene PTEN have not been elucidated in neuroblastomas. Paraffin-embedded sections from human neuroblastomas were analyzed for PTEN and phospho-Akt protein expression by immunohistochemistry. Human neuroblastoma cell lines (SK-N-SH and SH-SY5Y) were stably transfected with the plasmid pEGFP-GRP-R to establish GRP-R overexpression cell lines, and the effects of GRP on PTEN gene and protein expression were determined. A decrease in the ratio of PTEN to phospho-Akt protein expression was identified in poorly differentiated neuroblastomas. An increase in GRP binding capacity was confirmed in GRP-R overexpressing cells, which demonstrated an accelerated constitutive cell growth rate. PTEN gene and protein expression was significantly decreased in GRP-R overexpressing cells when compared with controls. Our findings demonstrate decreased expression of the tumor suppressor protein PTEN in more aggressive undifferentiated neuroblastomas. An increase in GRP binding capacity, as a result of GRP-R overexpression, down-regulates PTEN expression. These findings suggest that an inhibition of the tumor suppressor gene PTEN may be an important regulatory mechanism involved in GRP-induced cell proliferation in neuroblastomas.

Cellular network entropy as the energy potential in Waddington's differentiation landscape

PubMed Central

Banerji, Christopher R. S.; Miranda-Saavedra, Diego; Severini, Simone; Widschwendter, Martin; Enver, Tariq; Zhou, Joseph X.; Teschendorff, Andrew E.

2013-01-01

Differentiation is a key cellular process in normal tissue development that is significantly altered in cancer. Although molecular signatures characterising pluripotency and multipotency exist, there is, as yet, no single quantitative mark of a cellular sample's position in the global differentiation hierarchy. Here we adopt a systems view and consider the sample's network entropy, a measure of signaling pathway promiscuity, computable from a sample's genome-wide expression profile. We demonstrate that network entropy provides a quantitative, in-silico, readout of the average undifferentiated state of the profiled cells, recapitulating the known hierarchy of pluripotent, multipotent and differentiated cell types. Network entropy further exhibits dynamic changes in time course differentiation data, and in line with a sample's differentiation stage. In disease, network entropy predicts a higher level of cellular plasticity in cancer stem cell populations compared to ordinary cancer cells. Importantly, network entropy also allows identification of key differentiation pathways. Our results are consistent with the view that pluripotency is a statistical property defined at the cellular population level, correlating with intra-sample heterogeneity, and driven by the degree of signaling promiscuity in cells. In summary, network entropy provides a quantitative measure of a cell's undifferentiated state, defining its elevation in Waddington's landscape. PMID:24154593

Essential role of citron kinase in cytokinesis of spermatogenic precursors.

PubMed

Cunto, Ferdinando Di; Imarisio, Sara; Camera, Paola; Boitani, Carla; Altruda, Fiorella; Silengo, Lorenzo

2002-12-15

During spermatogenesis, the first morphological indication of spermatogonia differentiation is incomplete cytokinesis, followed by the assembly of stable intercellular cytoplasmic communications. This distinctive feature of differentiating male germ cells has been highly conserved during evolution, suggesting that regulation of the cytokinesis endgame is a crucial aspect of spermatogenesis. However, the molecular mechanisms underlying testis-specific regulation of cytokinesis are still largely unknown. Citron kinase is a myotonin-related protein acting downstream of the GTPase Rho in cytokinesis control. We previously reported that Citron kinase knockout mice are affected by a complex neurological syndrome caused by cytokinesis block and apoptosis of specific neuronal precursors. In this report we show that, in addition, these mice display a dramatic testicular impairment, with embryonic and postnatal loss of undifferentiated germ cells and complete absence of mature spermatocytes. By contrast, the ovaries of mutant females appear essentially normal. Developmental analysis revealed that the cellular depletion observed in mutant testes is caused by increased apoptosis of undifferentiated and differentiating precursors. The same cells display a severe cytokinesis defect, resulting in the production of multinucleated cells and apoptosis. Our data indicate that Citron kinase is specifically required for cytokinesis of the male germ line.

Surface presentation of biochemical cues for stem cell expansion - Spatial distribution of growth factors and self-assembly of extracellular matrix

NASA Astrophysics Data System (ADS)

Liu, Xingyu

Despite its great potential applications to stem cell technology and tissue engineering, matrix presentation of biochemical cues such as growth factors and extracellular matrix (ECM) components remains undefined. This is largely due to the difficulty in preserving the bioactivities of signaling molecules and in controlling the spatial distribution, cellular accessibility, molecular orientation and intermolecular assembly of the biochemical cues. This dissertation comprises of two parts that focuses on understanding surface presentation of a growth factor and ECM components, respectively. This dissertation addresses two fundamental questions in stem cell biology using two biomaterials platforms. How does nanoscale distribution of growth factor impact signaling activation and cellular behaviors of adult neural stem cells? How does ECM self-assembly impact human embryonic stem cell survival and proliferation? The first question was addressed by the design of a novel quantitative platform that allows the control of FGF-2 molecular presentation locally as either monomers or clusters when tethered to a polymeric substrate. This substrate-tethered FGF-2 enables a switch-like signaling activation in response to dose titration of FGF-2. This is in contrast to a continuous MAPK activation pattern elicited by soluble FGF-2. Consequently, cell proliferation, and spreading were also consistent with this FGF-2 does-response pattern. We demonstrated that the combination of FGF-2 concentration and its cluster size, rather than concentration alone, serves as the determinants to govern its biological effect on neural stem cells. The second part of this dissertation was inspired by the challenge that hESCs have extremely low clonal efficiency and hESC survival is critically dependent on cell substrate adhesion. We postulated that ECM integrity is a critical factor in preventing hESC anchorage-dependent apoptosis, and that the matrix for feeder-free culture need to be properly assembled in order to mimic the stem cell niche in vivo. First, we established assays that allow high-throughput quantification of hESC proliferation and ECM deposition. Human ESC survival was found to be highly sensitive to ECM assembly, and was improved by at least 20 times on substrates with well-assembled ECM. ECM polymerization alone improves clonal efficiency by at least 20 fold, from less than 0.1% to be 3-5%. This ratio is further improved to greater than 35% when combined with ROCK inhibitor, suggesting ECM polymerization underlines another critical factor in dictating hESC survival and growth. Given that many important signaling molecules including growth factors and extracellular matrix are highly enriched and restricted at the stem cell niche, we anticipate that our investigation into these questions provides better insight into the physiological roles of the stem cell niche components, and helps us to rationally direct stem cell fates in future stem cell-based therapeutic interventions.

NKX2-5 regulates human cardiomyogenesis via a HEY2 dependent transcriptional network.

PubMed

Anderson, David J; Kaplan, David I; Bell, Katrina M; Koutsis, Katerina; Haynes, John M; Mills, Richard J; Phelan, Dean G; Qian, Elizabeth L; Leitoguinho, Ana Rita; Arasaratnam, Deevina; Labonne, Tanya; Ng, Elizabeth S; Davis, Richard P; Casini, Simona; Passier, Robert; Hudson, James E; Porrello, Enzo R; Costa, Mauro W; Rafii, Arash; Curl, Clare L; Delbridge, Lea M; Harvey, Richard P; Oshlack, Alicia; Cheung, Michael M; Mummery, Christine L; Petrou, Stephen; Elefanty, Andrew G; Stanley, Edouard G; Elliott, David A

2018-04-10

Congenital heart defects can be caused by mutations in genes that guide cardiac lineage formation. Here, we show deletion of NKX2-5, a critical component of the cardiac gene regulatory network, in human embryonic stem cells (hESCs), results in impaired cardiomyogenesis, failure to activate VCAM1 and to downregulate the progenitor marker PDGFRα. Furthermore, NKX2-5 null cardiomyocytes have abnormal physiology, with asynchronous contractions and altered action potentials. Molecular profiling and genetic rescue experiments demonstrate that the bHLH protein HEY2 is a key mediator of NKX2-5 function during human cardiomyogenesis. These findings identify HEY2 as a novel component of the NKX2-5 cardiac transcriptional network, providing tangible evidence that hESC models can decipher the complex pathways that regulate early stage human heart development. These data provide a human context for the evaluation of pathogenic mutations in congenital heart disease.

Quantitative Analysis of Human Pluripotency and Neural Specification by In-Depth (Phospho)Proteomic Profiling.

PubMed

Singec, Ilyas; Crain, Andrew M; Hou, Junjie; Tobe, Brian T D; Talantova, Maria; Winquist, Alicia A; Doctor, Kutbuddin S; Choy, Jennifer; Huang, Xiayu; La Monaca, Esther; Horn, David M; Wolf, Dieter A; Lipton, Stuart A; Gutierrez, Gustavo J; Brill, Laurence M; Snyder, Evan Y

2016-09-13

Controlled differentiation of human embryonic stem cells (hESCs) can be utilized for precise analysis of cell type identities during early development. We established a highly efficient neural induction strategy and an improved analytical platform, and determined proteomic and phosphoproteomic profiles of hESCs and their specified multipotent neural stem cell derivatives (hNSCs). This quantitative dataset (nearly 13,000 proteins and 60,000 phosphorylation sites) provides unique molecular insights into pluripotency and neural lineage entry. Systems-level comparative analysis of proteins (e.g., transcription factors, epigenetic regulators, kinase families), phosphorylation sites, and numerous biological pathways allowed the identification of distinct signatures in pluripotent and multipotent cells. Furthermore, as predicted by the dataset, we functionally validated an autocrine/paracrine mechanism by demonstrating that the secreted protein midkine is a regulator of neural specification. This resource is freely available to the scientific community, including a searchable website, PluriProt. Published by Elsevier Inc.

A sequential EMT-MET mechanism drives the differentiation of human embryonic stem cells towards hepatocytes.

PubMed

Li, Qiuhong; Hutchins, Andrew P; Chen, Yong; Li, Shengbiao; Shan, Yongli; Liao, Baojian; Zheng, Dejin; Shi, Xi; Li, Yinxiong; Chan, Wai-Yee; Pan, Guangjin; Wei, Shicheng; Shu, Xiaodong; Pei, Duanqing

2017-05-03

Reprogramming has been shown to involve EMT-MET; however, its role in cell differentiation is unclear. We report here that in vitro differentiation of hESCs to hepatic lineage undergoes a sequential EMT-MET with an obligatory intermediate mesenchymal phase. Gene expression analysis reveals that Activin A-induced formation of definitive endoderm (DE) accompanies a synchronous EMT mediated by autocrine TGFβ signalling followed by a MET process. Pharmacological inhibition of TGFβ signalling blocks the EMT as well as DE formation. We then identify SNAI1 as the key EMT transcriptional factor required for the specification of DE. Genetic ablation of SNAI1 in hESCs does not affect the maintenance of pluripotency or neural differentiation, but completely disrupts the formation of DE. These results reveal a critical mesenchymal phase during the acquisition of DE, highlighting a role for sequential EMT-METs in both differentiation and reprogramming.

Comparative analysis of cardiomyocyte differentiation from human embryonic stem cells under 3-D and 2-D culture conditions.

PubMed

Pal, Rajarshi; Mamidi, Murali Krishna; Das, Anjan Kumar; Bhonde, Ramesh

2013-02-01

Post-myocardial infarction cardiomyocytes are the most important target cell types for cardiac repair. Many of the applications envisaged for human embryonic stem cells (hESC)-derived cardiomyocytes demand that the differentiation procedure be robust, cost effective and high yielding. Various lines of evidence including our earlier study suggest that hESCs have distinct preferences to become heart cells. However, a direct comparison between different protocols has not yet been reported to date. Here, we performed a logical and systematic comparison of cardiomyocytes obtained from hESCs via embryoid bodies (EBs) in suspension versus adherent static cultures of feeder-free hES colonies representing three-dimensional (3-D) and two-dimensional (2-D) culture systems, respectively. An in-depth characterization of the beating cells revealed appropriate cardiac marker expression both at gene and protein levels. Despite using similar media, 3-D and 2-D cultures showed significant variation in growth and ability to form beating areas. While the expression of pre-cardiac mesoderm markers like GATA-4, HAND1, Myf5, Msx1, and BMP-IIR remained unaltered; levels of functional heart-specific markers such as MLC-2A/2V, cTnT, ANP, Phospholamban, α-MHC and KV4.3 were substantially up-regulated in 3-D compared to 2-D cultures. Concurrently we observed a sharp decline in the expression of ESC, ectoderm and endoderm markers including Oct-4, Sox-2, NFH, Sox-1, Sox-17 and AFP. Further immunocytochemistry and flow cytometry demonstrated a higher percentage of cells positive for Brachyury, desmin and cardiac troponin in 3-D cultures. Our results underscore the higher efficiency of cardiomyocytes derived via 3-D cultures. This finding enriches our basic understanding of the differentiation pattern in hESC-derived cardiomyocytes. Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Carboplatin, Paclitaxel and Gemcitabine Hydrochloride With or Without Bevacizumab After Surgery in Treating Patients With Recurrent Ovarian, Epithelial, Primary Peritoneal, or Fallopian Tube Cancer

ClinicalTrials.gov

2018-06-06

Clear Cell Adenocarcinoma; Fallopian Tube Clear Cell Adenocarcinoma; Fallopian Tube Endometrioid Adenocarcinoma; Fallopian Tube Mucinous Adenocarcinoma; Fallopian Tube Serous Adenocarcinoma; Mucinous Adenocarcinoma; Ovarian Brenner Tumor; Ovarian Clear Cell Adenocarcinofibroma; Ovarian Endometrioid Adenocarcinoma; Ovarian Seromucinous Carcinoma; Ovarian Serous Adenocarcinoma; Primary Peritoneal Serous Adenocarcinoma; Recurrent Fallopian Tube Carcinoma; Recurrent Ovarian Carcinoma; Recurrent Primary Peritoneal Carcinoma; Undifferentiated Carcinoma; Undifferentiated Fallopian Tube Carcinoma; Undifferentiated Ovarian Carcinoma

Phosphoproteomics profiling suggests a role for nuclear βΙPKC in transcription processes of undifferentiated murine embryonic stem cells.

PubMed

Costa-Junior, Helio Miranda; Garavello, Nicole Milaré; Duarte, Mariana Lemos; Berti, Denise Aparecida; Glaser, Talita; de Andrade, Alexander; Labate, Carlos A; Ferreira, André Teixeira da Silva; Perales, Jonas Enrique Aguilar; Xavier-Neto, José; Krieger, José Eduardo; Schechtman, Deborah

2010-12-03

Protein kinase C (PKC) plays a key role in embryonic stem cell (ESC) proliferation, self-renewal, and differentiation. However, the function of specific PKC isoenzymes have yet to be determined. Of the PKCs expressed in undifferentiated ESCs, βIPKC was the only isoenzyme abundantly expressed in the nuclei. To investigate the role of βΙPKC in these cells, we employed a phosphoproteomics strategy and used two classical (cPKC) peptide modulators and one βIPKC-specific inhibitor peptide. We identified 13 nuclear proteins that are direct or indirect βΙPKC substrates in undifferentiated ESCs. These proteins are known to be involved in regulating transcription, splicing, and chromatin remodeling during proliferation and differentiation. Inhibiting βΙPKC had no effect on DNA synthesis in undifferentiated ESCs. However, upon differentiation, many cells seized to express βΙPKC and βΙPKC was frequently found in the cytoplasm. Taken together, our results suggest that βIPKC takes part in the processes that maintain ESCs in their undifferentiated state.

Stem Cells and Scaffolds for Vascularizing Engineered Tissue Constructs

NASA Astrophysics Data System (ADS)

Luong, E.; Gerecht, S.

The clinical impact of tissue engineering depends upon our ability to direct cells to form tissues with characteristic structural and mechanical properties from the molecular level up to organized tissue. Induction and creation of functional vascular networks has been one of the main goals of tissue engineering either in vitro, for the transplantation of prevascularized constructs, or in vivo, for cellular organization within the implantation site. In most cases, tissue engineering attempts to recapitulate certain aspects of normal development in order to stimulate cell differentiation and functional tissue assembly. The induction of tissue growth generally involves the use of biodegradable and bioactive materials designed, ideally, to provide a mechanical, physical, and biochemical template for tissue regeneration. Human embryonic stem cells (hESCs), derived from the inner cell mass of a developing blastocyst, are capable of differentiating into all cell types of the body. Specifically, hESCs have the capability to differentiate and form blood vessels de novo in a process called vasculogenesis. Human ESC-derived endothelial progenitor cells (EPCs) and endothelial cells have substantial potential for microvessel formation, in vitro and in vivo. Human adult EPCs are being isolated to understand the fundamental biology of how these cells are regulated as a population and to explore whether these cells can be differentiated and reimplanted as a cellular therapy in order to arrest or even reverse damaged vasculature. This chapter focuses on advances made toward the generation and engineering of functional vascular tissue, focusing on both the scaffolds - the synthetic and biopolymer materials - and the cell sources - hESCs and hEPCs.

Teratoma formation of human embryonic stem cells in three-dimensional perfusion culture bioreactors.

PubMed

Stachelscheid, H; Wulf-Goldenberg, A; Eckert, K; Jensen, J; Edsbagge, J; Björquist, P; Rivero, M; Strehl, R; Jozefczuk, J; Prigione, A; Adjaye, J; Urbaniak, T; Bussmann, P; Zeilinger, K; Gerlach, J C

2013-09-01

Teratoma formation in mice is today the most stringent test for pluripotency that is available for human pluripotent cells, as chimera formation and tetraploid complementation cannot be performed with human cells. The teratoma assay could also be applied for assessing the safety of human pluripotent cell-derived cell populations intended for therapeutic applications. In our study we examined the spontaneous differentiation behaviour of human embryonic stem cells (hESCs) in a perfused 3D multi-compartment bioreactor system and compared it with differentiation of hESCs and human induced pluripotent cells (hiPSCs) cultured in vitro as embryoid bodies and in vivo in an experimental mouse model of teratoma formation. Results from biochemical, histological/immunohistological and ultrastuctural analyses revealed that hESCs cultured in bioreactors formed tissue-like structures containing derivatives of all three germ layers. Comparison with embryoid bodies and the teratomas revealed a high degree of similarity of the tissues formed in the bioreactor to these in the teratomas at the histological as well as transcriptional level, as detected by comparative whole-genome RNA expression profiling. The 3D culture system represents a novel in vitro model that permits stable long-term cultivation, spontaneous multi-lineage differentiation and tissue formation of pluripotent cells that is comparable to in vivo differentiation. Such a model is of interest, e.g. for the development of novel cell differentiation strategies. In addition, the 3D in vitro model could be used for teratoma studies and pluripotency assays in a fully defined, controlled environment, alternatively to in vivo mouse models. Copyright © 2012 John Wiley & Sons, Ltd.

Fresh embryo donation for human embryonic stem cell (hESC) research: the experiences and values of IVF couples asked to be embryo donors

PubMed Central

Haimes, E.; Taylor, K.

2009-01-01

BACKGROUND This article reports on an investigation of the views of IVF couples asked to donate fresh embryos for research and contributes to the debates on: the acceptability of human embryonic stem cell (hESC) research, the moral status of the human embryo and embryo donation for research. METHODS A hypothesis-generating design was followed. All IVF couples in one UK clinic who were asked to donate embryos in 1 year were contacted 6 weeks after their pregnancy result. Forty four in-depth interviews were conducted. RESULTS Interviewees were preoccupied with IVF treatment and the request to donate was a secondary consideration. They used a complex and dynamic system of embryo classification. Initially, all embryos were important but then their focus shifted to those that had most potential to produce a baby. At that point, ‘other’ embryos were less important though they later realise that they did not know what happened to them. Guessing that these embryos went to research, interviewees preferred not to contemplate what that might entail. The embryos that caused interviewees most concern were good quality embryos that might have produced a baby but went to research instead. ‘The’ embryo, the morally laden, but abstract, entity, did not play a central role in their decision-making. CONCLUSIONS This study, despite missing those who refuse to donate embryos, suggests that debates on embryo donation for hESC research should include the views of embryo donors and should consider the social, as well as the moral, status of the human embryo. PMID:19502616

B cell markers in Ph1-positive acute lymphoblastic leukemia.

PubMed

Alimena, G; De Rossi, G; Gastaldi, R; Guglielmi, C; Mandelli, F

1980-01-01

A case of acute lymphoblastic leukemia (ALL) where the blast cells had B cell markers and displayed the presence of a typical Ph1 chromosome, originated by a standard t (9;22) translocation, is reported. Cytological and clinical aspects during the entire course of the disease were consistent with the diagnosis of ALL. Evidence of differentiation along a well-defined lymphoid cell line in a Ph1-positive cell confirms the presence of the Ph1 chromosome in conditions other than chronic granulocytic leukemia and shows that it possibly does not occur in an exclusively undifferentiated totipotent stem cell.

Contribution of transposable elements and distal enhancers to evolution of human-specific features of interphase chromatin architecture in embryonic stem cells.

PubMed

Glinsky, Gennadi V

2018-03-01

Transposable elements have made major evolutionary impacts on creation of primate-specific and human-specific genomic regulatory loci and species-specific genomic regulatory networks (GRNs). Molecular and genetic definitions of human-specific changes to GRNs contributing to development of unique to human phenotypes remain a highly significant challenge. Genome-wide proximity placement analysis of diverse families of human-specific genomic regulatory loci (HSGRL) identified topologically associating domains (TADs) that are significantly enriched for HSGRL and designated rapidly evolving in human TADs. Here, the analysis of HSGRL, hESC-enriched enhancers, super-enhancers (SEs), and specific sub-TAD structures termed super-enhancer domains (SEDs) has been performed. In the hESC genome, 331 of 504 (66%) of SED-harboring TADs contain HSGRL and 68% of SEDs co-localize with HSGRL, suggesting that emergence of HSGRL may have rewired SED-associated GRNs within specific TADs by inserting novel and/or erasing existing non-coding regulatory sequences. Consequently, markedly distinct features of the principal regulatory structures of interphase chromatin evolved in the hESC genome compared to mouse: the SED quantity is 3-fold higher and the median SED size is significantly larger. Concomitantly, the overall TAD quantity is increased by 42% while the median TAD size is significantly decreased (p = 9.11E-37) in the hESC genome. Present analyses illustrate a putative global role for transposable elements and HSGRL in shaping the human-specific features of the interphase chromatin organization and functions, which are facilitated by accelerated creation of novel transcription factor binding sites and new enhancers driven by targeted placement of HSGRL at defined genomic coordinates. A trend toward the convergence of TAD and SED architectures of interphase chromatin in the hESC genome may reflect changes of 3D-folding patterns of linear chromatin fibers designed to enhance both regulatory complexity and functional precision of GRNs by creating predominantly a single gene (or a set of functionally linked genes) per regulatory domain structures. Collectively, present analyses reveal critical evolutionary contributions of transposable elements and distal enhancers to creation of thousands primate- and human-specific elements of a chromatin folding code, which defines the 3D context of interphase chromatin both restricting and facilitating biological functions of GRNs.

Transposable Elements and DNA Methylation Create in Embryonic Stem Cells Human-Specific Regulatory Sequences Associated with Distal Enhancers and Noncoding RNAs

PubMed Central

Glinsky, Gennadi V.

2015-01-01

Despite significant progress in the structural and functional characterization of the human genome, understanding of the mechanisms underlying the genetic basis of human phenotypic uniqueness remains limited. Here, I report that transposable element-derived sequences, most notably LTR7/HERV-H, LTR5_Hs, and L1HS, harbor 99.8% of the candidate human-specific regulatory loci (HSRL) with putative transcription factor-binding sites in the genome of human embryonic stem cells (hESC). A total of 4,094 candidate HSRL display selective and site-specific binding of critical regulators (NANOG [Nanog homeobox], POU5F1 [POU class 5 homeobox 1], CCCTC-binding factor [CTCF], Lamin B1), and are preferentially located within the matrix of transcriptionally active DNA segments that are hypermethylated in hESC. hESC-specific NANOG-binding sites are enriched near the protein-coding genes regulating brain size, pluripotency long noncoding RNAs, hESC enhancers, and 5-hydroxymethylcytosine-harboring regions immediately adjacent to binding sites. Sequences of only 4.3% of hESC-specific NANOG-binding sites are present in Neanderthals’ genome, suggesting that a majority of these regulatory elements emerged in Modern Humans. Comparisons of estimated creation rates of novel TF-binding sites revealed that there was 49.7-fold acceleration of creation rates of NANOG-binding sites in genomes of Chimpanzees compared with the mouse genomes and further 5.7-fold acceleration in genomes of Modern Humans compared with the Chimpanzees genomes. Preliminary estimates suggest that emergence of one novel NANOG-binding site detectable in hESC required 466 years of evolution. Pathway analysis of coding genes that have hESC-specific NANOG-binding sites within gene bodies or near gene boundaries revealed their association with physiological development and functions of nervous and cardiovascular systems, embryonic development, behavior, as well as development of a diverse spectrum of pathological conditions such as cancer, diseases of cardiovascular and reproductive systems, metabolic diseases, multiple neurological and psychological disorders. A proximity placement model is proposed explaining how a 33–47% excess of NANOG, CTCF, and POU5F1 proteins immobilized on a DNA scaffold may play a functional role at distal regulatory elements. PMID:25956794

[Value of immunologic phenotyping of acute leukemias in children].

PubMed

Vannier, J P; Bene, M C

1989-10-01

Immunologic typing has demonstrated considerable heterogeneity among the acute leukemias. The most significant recent advance has been development of monoclonal antibody techniques. Some markers identified using these techniques seem to be specific for a given stage of maturation of one lymphoid or myeloid cell line. Most acute lymphoblastic leukemias (ALLs) are malignant proliferations whose differentiation appears to have become 'stuck' at one stage of maturation. Results of immunologic typing correlate well with the other clinical and biological data. For prognostic purposes, several patterns can be identified. Among B line ALLs, four varieties have been differentiated, i.e., CD10 negative ALLs, common ALLs, pre-B ALLs, and B ALLs. T ALLs include a broad spectrum of heterogeneous proliferations whose immunologic classification is made difficult by the large number of phenotypes encountered. Among acute myeloblastic leukemias (AMLs), some highly undifferentiated forms have been recognized, by means of immunologic typing, as originating in one of the myeloid cell lines. However, the nosologic and prognostic significance of these studies is less obvious than in ALLs.

Increased reprogramming of human fetal hepatocytes compared with adult hepatocytes in feeder-free conditions.

PubMed

Hansel, Marc C; Gramignoli, Roberto; Blake, William; Davila, Julio; Skvorak, Kristen; Dorko, Kenneth; Tahan, Veysel; Lee, Brian R; Tafaleng, Edgar; Guzman-Lepe, Jorge; Soto-Gutierrez, Alejandro; Fox, Ira J; Strom, Stephen C

2014-01-01

Hepatocyte transplantation has been used to treat liver disease. The availability of cells for these procedures is quite limited. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) may be a useful source of hepatocytes for basic research and transplantation if efficient and effective differentiation protocols were developed and problems with tumorigenicity could be overcome. Recent evidence suggests that the cell of origin may affect hiPSC differentiation. Thus, hiPSCs generated from hepatocytes may differentiate back to hepatocytes more efficiently than hiPSCs from other cell types. We examined the efficiency of reprogramming adult and fetal human hepatocytes. The present studies report the generation of 40 hiPSC lines from primary human hepatocytes under feeder-free conditions. Of these, 37 hiPSC lines were generated from fetal hepatocytes, 2 hiPSC lines from normal hepatocytes, and 1 hiPSC line from hepatocytes of a patient with Crigler-Najjar syndrome, type 1. All lines were confirmed reprogrammed and expressed markers of pluripotency by gene expression, flow cytometry, immunocytochemistry, and teratoma formation. Fetal hepatocytes were reprogrammed at a frequency over 50-fold higher than adult hepatocytes. Adult hepatocytes were only reprogrammed with six factors, while fetal hepatocytes could be reprogrammed with three (OCT4, SOX2, NANOG) or four factors (OCT4, SOX2, NANOG, LIN28 or OCT4, SOX2, KLF4, C-MYC). The increased reprogramming efficiency of fetal cells was not due to increased transduction efficiency or vector toxicity. These studies confirm that hiPSCs can be generated from adult and fetal hepatocytes including those with genetic diseases. Fetal hepatocytes reprogram much more efficiently than adult hepatocytes, although both could serve as useful sources of hiPSC-derived hepatocytes for basic research or transplantation.

Characterization of Differentiated SH-SY5Y as Neuronal Screening Model Reveals Increased Oxidative Vulnerability

PubMed Central

Forster, J. I.; Köglsberger, S.; Trefois, C.; Boyd, O.; Baumuratov, A. S.; Buck, L.; Balling, R.; Antony, P. M. A.

2016-01-01

The immortalized and proliferative cell line SH-SY5Y is one of the most commonly used cell lines in neuroscience and neuroblastoma research. However, undifferentiated SH-SY5Y cells share few properties with mature neurons. In this study, we present an optimized neuronal differentiation protocol for SH-SY5Y that requires only two work steps and 6 days. After differentiation, the cells present increased levels of ATP and plasma membrane activity but reduced expression of energetic stress response genes. Differentiation results in reduced mitochondrial membrane potential and decreased robustness toward perturbations with 6-hydroxydopamine. We are convinced that the presented differentiation method will leverage genetic and chemical high-throughput screening projects targeting pathways that are involved in the selective vulnerability of neurons with high energetic stress levels. PMID:26738520

Seven diverse human embryonic stem cell-derived chondrogenic clonal embryonic progenitor cell lines display site-specific cell fates.

PubMed

Sternberg, Hal; Kidd, Jennifer; Murai, James T; Jiang, Jianjie; Rinon, Ariel; Erickson, Isaac E; Funk, Walter D; Wang, Qian; Chapman, Karen B; Vangsness, C Thomas; West, Michael D

2013-03-01

The transcriptomes of seven diverse clonal human embryonic progenitor cell lines with chondrogenic potential were compared with that of bone marrow-derived mesenchymal stem cells (MSCs). The cell lines 4D20.8, 7PEND24, 7SMOO32, E15, MEL2, SK11 and SM30 were compared with MSCs using immunohistochemical methods, gene expression microarrays and quantitative real-time PCR. In the undifferentiated progenitor state, each line displayed unique combinations of site-specific markers, including AJAP1, ALDH1A2, BMP5, BARX1, HAND2, HOXB2, LHX1, LHX8, PITX1, TBX15 and ZIC2, but none of the lines expressed the MSC marker CD74. The lines showed diverse responses when differentiated in the presence of combinations of TGF-β3, BMP2, 4, 6 and 7 and GDF5, with the lines 4D20.8, SK11, SM30 and MEL2 showing osteogenic markers in some differentiation conditions. The line 7PEND24 showed evidence of regenerating articular cartilage and, in some conditions, markers of tendon differentiation. The scalability of site-specific clonal human embryonic stem cell-derived embryonic progenitor cell lines may provide novel models for the study of differentiation and methods for preparing purified and identified cells types for use in therapy.

A monoclonal antibody recognizes undifferentiation-specific carbohydrate moieties expressed on cell surface of the human dental pulp cells.

PubMed

Kang, Kyung-Jung; Ko, Seon-Yle; Ryu, Chun-Jeih; Jang, Young-Joo

2017-05-01

Human dental pulp cells are obtained from dental pulp tissue, and have the ability to form dentin and a pulp-like complex. Although adult stem cells have been identified from the primary culture by using specific cell surface markers, the identity of surface markers for the purification of stem cells within the dental pulp population are still unclear. Previously, we had constructed monoclonal antibodies against the undifferentiated cell-specific surface markers of human dental pulp cells (hDPCs) by performing decoy immunization. Among them, a monoclonal antibody against the cell surface antigen of the undifferentiated hDPCs (named UPSA-1) was purified and its heavy and light chain consensus regions were analyzed. The cell surface binding affinity of UPSA-1 mAb on the undifferentiated hDPCs was stronger than that on the differentiated cells. When tunicamycin was applied to hDPSCs during culture, the cell surface binding affinity of the antibody was dramatically decreased, and dentinogenic differentiation was reduced. The purified UPSA-1 antigen band resulting from immunoprecipitation disappeared or shifted down on the SDS-PAGE by deglycosylation. These data suggested that glycosylation on the cell surface might be a marker of an undifferentiated state, and that UPSA-1 mAb might be useful for identifying the carbohydrate moiety on the cell surface of undifferentiated pulp cells. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Primary undifferentiated sarcoma of the meninges: A case report and comprehensive review of the literature.

PubMed

Wapshott, Taylor; Schammel, Christine M G; Schammel, David P; Rezeanu, Luminita; Lynn, Michael

2018-05-21

Sarcomas make up 1% of all cases of adult cancer, with 5-10% of those classified as undifferentiated pleomorphic sarcomas (UPS/PUS) and 0.1-4.3% primary intracranial sarcomas. Intracranial undifferentiated sarcoma is characterized by an earlier age of onset and generally poorer prognosis compared to extracranial undifferentiated sarcomas. Current therapies involve surgical excision with wide margins and radiotherapy, with minimal data available regarding the efficacy of chemotherapy. A 79-year-old man with a history of remote superficial bladder cancer presented with a large frontal scalp lesion. A biopsy was initially attempted by a dermatologist in the outpatient setting, but a follow-up CT scan revealed a skull-eroding, enhancing soft tissue lesion. Neurosurgical treatment revealed an undifferentiated sarcoma. The patient underwent adjuvant radiation therapy of 59.4 Gy fractionated over 45 days following surgery. Follow-up brain MRIs at 1-, 6-, 9-, 12-, 15-, 21-, and 27 months after surgery have not shown any indications of local recurrence or tumor metastasis. Despite the high propensity that undifferentiated sarcomas have for recurrence and metastasis and the patient's advanced age, this patient remains uniquely disease-free. We provide a description of an unusual case and comprehensive literature review of UPS to clarify the hallmarks of the disease, identify the difficulties in diagnosis, and provide a summary of therapies employed in the literature with their corresponding patient outcomes. Copyright © 2018 Elsevier Ltd. All rights reserved.

Activation of the mouse Oct4 promoter in medaka embryonic stem cells and its use for ablation of spontaneous differentiation.

PubMed

Hong, Yunhan; Winkler, Christoph; Liu, Tongming; Chai, Guixuan; Schartl, Manfred

2004-07-01

The determination and maintenance of the cell fate is ultimately due to differential gene activity. In the mouse, expression of the transcription factor Oct4 is high in totipotent inner cell mass, germ cells and undifferentiated embryonic stem (ES) cells, but dramatically reduced or extinct upon differentiation. Here, we show that medaka blastula embryos and cells of the ES cell line MES1 are able to activate the Oct4 promoter. Ectopic expression of a fusion gene for beta-galactosidase and neomycin resistance from the Oct4 promoter conferred resistance to G418. G418 selection led to a homogeneous population of undifferentiated ES cells which were able to undergo induced or directed differentiation into various cell types including neuron-like cells and melanocytes. Furthermore, GFP-labeled GOF18geo-MES1 cells after differentiation ablation were able to contribute to a wide variety of organ systems derived from all the three germ layers. Most importantly, we show that drug ablation of differentiation on the basis of Oct4 promoter is a useful tool to improve ES cell cultivation and chimera formation: MES1 cells after differentiation ablation appeared to be better donors than the parental MES1 line, as the permissive number of input donor cells increases from 100 to 200, resulting in an enhanced degree of chimerism. Taken together, some transcription factors and cis-acting regulatory sequences controlling totipotency-specific gene expression appear to be conserved between mammals and fish, and medaka ES cells offer an in vitro system for characterizing the expression of totipotency-specific genes such as putative Oct4 homologs from fish.

Two stages of enteropathogenic Escherichia coli intestinal pathogenicity are up and down-regulated by the epithelial cell differentiation.

PubMed

Gabastou, J M; Kernéis, S; Bernet-Camard, M F; Barbat, A; Coconnier, M H; Kaper, J B; Servin, A L

1995-09-01

Pathogens and eucaryotic cells are active partners during the process of pathogenicity. To gain access to enterocytes and to cross the epithelial membrane, many enterovirulent microorganisms interact with the brush border membrane-associated components as receptors. Recent reports provide evidence that intestinal cell differentiation plays a role in microbial pathogenesis. Human enteropathogenic Escherichia coli (EPEC) develop their pathogenicity upon infecting enterocytes. To determine if intestinal epithelial cell differentiation influences EPEC pathogenicity, we examined the infection of human intestinal epithelial cells by JPN 15 (pMAR7) [EAF+ eae+] EPEC strain as a function of the cell differentiation. The human embryonic intestinal INT407 cells, the human colonic T84 cells, the human undifferentiated HT-29 cells (HT-29 Std) and two enterocytic cell lines, HT-29 glc-/+ and Caco-2 cells, were used as cellular models. Cells were infected apically with the EPEC strain and the cell-association and cell-entry were examined by quantitative determination using metabolically radiolabeled bacteria, as well as by light, scanning and transmission electron microscopy. [EAF+ eae+] EPEC bacteria efficiently colonized the cultured human intestinal cells. Diffuse bacterial adhesion occurred to undifferentiated HT-29 Std and INT407 cells, whereas characteristic EPEC cell clusters were observed on fully differentiated enterocytic HT-29 glc-/+ cells and on colonic crypt T84 cells. As shown using the Caco-2 cell line, which spontaneously differentiates in culture, the formation of EPEC clusters increased as a function of the epithelial cell differentiation. In contrast, efficient cell-entry of [EAF+ eae+] EPEC bacteria occurred in recently differentiated Caco-2 cells and decreased when the cells were fully differentiated.(ABSTRACT TRUNCATED AT 250 WORDS)

Frequent loss of claudin-4 expression in dedifferentiated and undifferentiated endometrial carcinomas.

PubMed

Tessier-Cloutier, Basile; Soslow, Robert A; Stewart, Colin J R; Köbel, Martin; Lee, Cheng-Han

2018-04-19

Dedifferentiated endometrial carcinomas (DDECs)/undifferentiated endometrial carcinomas (UECs) are aggressive endometrial cancers with frequent genomic inactivation of core components of switch/sucrose non-fermentable (SWI/SNF) complex proteins. Claudin-4, an epithelial intercellular tight junction protein, was recently found to be expressed in SWI/SNF-deficient undifferentiated carcinomas but not in SWI/SNF-deficient sarcomas. The aim of this study was to examine claudin-4 expression in UECs/DDECs and other high-grade uterine carcinomas. We examined claudin-4 expression by immunohistochemistry (clone 3E2C1) on tissue microarrays that contained 44 UECs/DDECs (24 SWI/SNF-deficient), 50 carcinosarcomas, 164 grade 3 endometrioid carcinomas, 57 serous carcinomas, and 20 clear cell carcinomas. Tumours with <5% claudin-4 expression were considered to be negative. Nearly all SWI/SNF-deficient, and most SWI/SNF-proficient, UECs/DDECs showed a complete absence of claudin-4 expression in the undifferentiated component, whereas the differentiated component in DDECs showed consistent and diffuse claudin-4 expression. Only one SWI/SNF-deficient DDEC showed focal expression of claudin-4 in the undifferentiated component, as compared with diffuse expression in the corresponding differentiated component. Claudin-4 expression was consistently absent in the sarcomatous component of carcinosarcoma, and it was absent in 24% of grade 3 endometrioid carcinomas and serous carcinomas. Claudin-4 expression can be absent or very focal in a subset of high-grade endometrial carcinomas, and is almost always absent in the undifferentiated components of SWI/SNF-deficient UECs/DDECs, despite the apparent epithelial origin in the case of DDECs. Therefore, claudin-4 expression cannot be used to infer mesenchymal or epithelial tumour origin in the endometrium. The consistent loss or down-regulation of claudin-4, a tight junction protein, in SWI/SNF-deficient UECs/DDECs further supports the undifferentiated nature of these tumours. © 2018 John Wiley & Sons Ltd.

A Novel In Vitro Method for Detecting Undifferentiated Human Pluripotent Stem Cells as Impurities in Cell Therapy Products Using a Highly Efficient Culture System

PubMed Central

Tano, Keiko; Yasuda, Satoshi; Kuroda, Takuya; Saito, Hirohisa; Umezawa, Akihiro; Sato, Yoji

2014-01-01

Innovative applications of cell therapy products (CTPs) derived from human pluripotent stem cells (hPSCs) in regenerative medicine are currently being developed. The presence of residual undifferentiated hPSCs in CTPs is a quality concern associated with tumorigencity. However, no simple in vitro method for direct detection of undifferentiated hPSCs that contaminate CTPs has been developed. Here, we show a novel approach for direct and sensitive detection of a trace amount of undifferentiated human induced pluripotent stem cells (hiPSCs) using a highly efficient amplification method in combination with laminin-521 and Essential 8 medium. Essential 8 medium better facilitated the growth of hiPSCs dissociated into single cells on laminin-521 than in mTeSR1 medium. hiPSCs cultured on laminin-521 in Essential 8 medium were maintained in an undifferentiated state and they maintained the ability to differentiate into various cell types. Essential 8 medium allowed robust hiPSC proliferation plated on laminin-521 at low cell density, whereas mTeSR1 did not enhance the cell growth. The highly efficient culture system using laminin-521 and Essential 8 medium detected hiPSCs spiked into primary human mesenchymal stem cells (hMSCs) or human neurons at the ratio of 0.001%–0.01% as formed colonies. Moreover, this assay method was demonstrated to detect residual undifferentiated hiPSCs in cell preparations during the process of hMSC differentiation from hiPSCs. These results indicate that our highly efficient amplification system using a combination of laminin-521 and Essential 8 medium is able to detect a trace amount of undifferentiated hPSCs contained as impurities in CTPs and would contribute to quality assessment of hPSC-derived CTPs during the manufacturing process. PMID:25347300

Contacting co-culture of human retinal microvascular endothelial cells alters barrier function of human embryonic stem cell derived retinal pigment epithelial cells.

PubMed

Skottman, H; Muranen, J; Lähdekorpi, H; Pajula, E; Mäkelä, K; Koivusalo, L; Koistinen, A; Uusitalo, H; Kaarniranta, K; Juuti-Uusitalo, K

2017-10-01

Here we evaluated the effects of human retinal microvascular endothelial cells (hREC) on mature human embryonic stem cell (hESC) derived retinal pigment epithelial (RPE) cells. The hESC-RPE cells (Regea08/017, Regea08/023 or Regea11/013) and hREC (ACBRI 181) were co-cultured on opposite sides of transparent membranes for up to six weeks. Thereafter barrier function, small molecule permeability, localization of RPE and endothelial cell marker proteins, cellular fine structure, and growth factor secretion of were evaluated. After co-culture, the RPE specific CRALBP and endothelial cell specific von Willebrand factor were appropriately localized. In addition, the general morphology, pigmentation, and fine structure of hESC-RPE cells were unaffected. Co-culture increased the barrier function of hESC-RPE cells, detected both with TEER measurements and cumulative permeability of FD4 - although the differences varied among the cell lines. Co-culturing significantly altered VEGF and PEDF secretion, but again the differences were cell line specific. The results of this study showed that co-culture with hREC affects hESC-RPE functionality. In addition, co-culture revealed drastic cell line specific differences, most notably in growth factor secretion. This model has the potential to be used as an in vitro outer blood-retinal barrier model for drug permeability testing. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

nm23 regulates decidualization through the PI3K-Akt-mTOR signaling pathways in mice and humans.

PubMed

Zhang, Xue; Fu, Li-Juan; Liu, Xue-Qing; Hu, Zhuo-Ying; Jiang, Yu; Gao, Ru-Fei; Feng, Qian; Lan, Xi; Geng, Yan-Qing; Chen, Xue-Mei; He, Jun-Lin; Wang, Ying-Xiong; Ding, Yu-Bin

2016-10-01

Does nm23 have functional significance in decidualization in mice and humans? nm23 affects decidualization via the phosphoinositide 3 kinase/mammalian target of rapamycin (PI3K-Akt-mTOR) signaling pathways in mouse endometrial stromal cells (ESCs; mESCs) and human ESCs. The function of nm23 in suppressing metastasis has been demonstrated in a variety of cancer types. nm23 also participates in the control of DNA replication and cell proliferation and differentiation. We first analyzed the expression profile of nm23 in mice during early pregnancy (n = 6/group), pseudopregnancy (n = 6/group) and artificial decidualization (n = 6/group) and in humans during the menstrual cycle phases and the first trimester. We then used primary cultured mESCs and a human ESC line, T-HESC, to explore the hormonal regulation of nm23 and the roles of nm23 in in vitro decidualization, and as a possible mediator of downstream PI3K-Akt-mTOR signaling pathways. We evaluated the dynamic expression of nm23 in mice and humans using immunohistochemistry, western blot and real-time quantitative RT-PCR (RT-qPCR). Regulation of nm23 by steroid hormones was investigated in isolated primary mESCs and T-HESCs by western blot. The effect of nm23 knockdown (using siRNA) on ESC proliferation was analyzed by 5-ethynyl-2'-deoxyuridine staining (EdU) and proliferating cell nuclear antigen protein (PCNA) expression. The influence of nm23 expression on the differentiation of ESCs was determined by RT-qPCR using the mouse differentiation markers decidual/trophoblast PRL-related protein (dtprp, also named prl8a2) and prolactin family 3 subfamily c member 1 (prl3c1) and the human differentiation markers insulin-like growth factor binding protein 1 (IGFBP1) and prolactin (PRL). The effects of nm23 siRNA (si-nm23) and the PI3K inhibitor LY294002 on the downstream effects of nm23 on the PI3K-Akt-mTOR signaling pathway were estimated by western blot. NM23-M1 was specifically expressed in the decidual zone during early pregnancy and in artificially induced deciduoma, and NM23-H1 was strongly expressed in human first trimester decidua. The expression of nm23 was upregulated by oestradiol and progesterone (P < 0.05 versus control) in vitro in mESCs and T-HESC, and this was inhibited by their respective receptor antagonists, ICI 182,780 and RU486. Mouse and human nm23 knockdown decreased ESC proliferation and differentiation (P < 0.05 versus control). The PI3K-Akt-mTOR signaling pathways were downstream mediators of nm23 in mESCs and T-HESCs decidualization. Whether the nm23 regulates decidualization via the activation of AMPK, RAS, PKA, STAT3 or other signaling molecules remains to be determined. The role of nm23 in decidualization was tested in vitro only. Results demonstrate that nm23 plays a vital role in decidualization in mice and humans and that nm23 gene expression is hormonally regulated. The downregulation of nm23 in decidua during the first trimester may be associated with infertility in women. This study was supported by the National Natural Science Foundation of China (grant nos. 81370731, 31571551 and 31571190), the Science and Technology Project of Chongqing Education Committee (KJ130309), open funding by the Chongqing Institute for Family Planning (1201) and the Excellent Young Scholars of Chongqing Medical University (CQYQ201302). The authors have no conflicts of interest to declare. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Viral Vector-Based Innovative Approaches to Directly Abolishing Tumorigenic Pluripotent Stem Cells for Safer Regenerative Medicine.

PubMed

Mitsui, Kaoru; Ide, Kanako; Takahashi, Tomoyuki; Kosai, Ken-Ichiro

2017-06-16

Human pluripotent stem cells (hPSCs) are a promising source of regenerative material for clinical applications. However, hPSC transplant therapies pose the risk of teratoma formation and malignant transformation of undifferentiated remnants. These problems underscore the importance of developing technologies that completely prevent tumorigenesis to ensure safe clinical application. Research to date has contributed to establishing safe hPSC lines, improving the efficiency of differentiation induction, and indirectly ensuring the safety of products. Despite such efforts, guaranteeing the clinical safety of regenerative medicine products remains a key challenge. Given the intrinsic genome instability of hPSCs, selective growth advantage of cancer cells, and lessons learned through failures in previous attempts at hematopoietic stem cell gene therapy, conventional strategies are unlikely to completely overcome issues related to hPSC tumorigenesis. Researchers have recently embarked on studies aimed at locating and directly treating hPSC-derived tumorigenic cells. In particular, novel approaches to directly killing tumorigenic cells by transduction of suicide genes and oncolytic viruses are expected to improve the safety of hPSC-based therapy. This article discusses the current status and future perspectives of methods aimed at directly eradicating undifferentiated tumorigenic hPSCs, with a focus on viral vector transduction.

Primary Culture of Undifferentiated Pleomorphic Sarcoma: Molecular Characterization and Response to Anticancer Agents

PubMed Central

Recine, Federica; Mercatali, Laura; Miserocchi, Giacomo; Spadazzi, Chiara; Liverani, Chiara; Bongiovanni, Alberto; Pieri, Federica; Casadei, Roberto; Riva, Nada; Fausti, Valentina; Amadori, Dino; Ibrahim, Toni

2017-01-01

Undifferentiated pleomorphic sarcoma (UPS) is an aggressive mesenchymal neoplasm with no specific line of differentiation. Eribulin, a novel synthetic microtubule inhibitor, has shown anticancer activity in several tumors, including soft tissue sarcomas (STS). We investigated the molecular biology of UPS, and the mechanisms of action of this innovative microtubule-depolymerizing drug. A primary culture from a patient with UPS was established and characterized in terms of gene expression. The activity of eribulin was also compared with that of other drugs currently used for STS treatment, including trabectedin. Finally, Western blot analysis was performed to better elucidate the activity of eribulin. Our results showed an upregulation of epithelial mesenchymal transition-related genes, and a downregulation of epithelial markers. Furthermore, genes involved in chemoresistance were upregulated. Pharmacological analysis confirmed limited sensitivity to chemotherapy. Interestingly, eribulin exhibited a similar activity to that of standard treatments. Molecular analysis revealed the expression of cell cycle arrest-related and pro-apoptotic-related proteins. These findings are suggestive of aggressive behavior in UPS. Furthermore, the identification of chemoresistance-related genes could facilitate the development of innovative drugs to improve patient outcome. Overall, the results from the present study furnish a rationale for elucidating the role of eribulin for the treatment of UPS. PMID:29292724

TRANSCRIPTIONAL PROFILES FOR GLUTAMATE TRANSPORTERS REVEAL DIFFERENCES BETWEEN ORGANOPHOSPHATES BUT SIMILARITIES WITH UNRELATED NEUROTOXICANTS

PubMed Central

Slotkin, Theodore A.; Lobner, Doug; Seidler, Frederic J.

2010-01-01

The developmental neurotoxicity of organophosphates involves mechanisms other than their shared property as cholinesterase inhibitors, among which are excitotoxicity and oxidative stress. We used PC12 cells as a neurodevelopmental model to compare the effects of chlorpyrifos and diazinon on the expression of genes encoding glutamate transporters. Chlorpyrifos had a greater effect in cells undergoing nerve growth factor-induced neurodifferentiation as compared to undifferentiated PC12 cells, with peak sensitivity at the initiation of differentiation, reflecting a global upregulation of all the glutamate transporter genes expressed in this cell line. In differentiating cells, chlorpyrifos had a significantly greater effect than did diazinon and concordance analysis indicated no resemblance in their expression patterns. At the same time, the smaller effects of diazinon were highly concordant with those of an organochlorine pesticide (dieldrin) and a metal (divalent nickel). We also performed similar evaluations for the cystine/glutamate exchanger, which provides protection against oxidative stress by moving cystine into the cell; again, chlorpyrifos had the greatest effect, in this case reducing expression in undifferentiated and differentiating cells. Our results point to excitotoxicity and oxidative stress as major contributors to the noncholinesterase mechanisms that distinguish the neurodevelopmental outcomes betweem different organophosphates while providing a means whereby apparently unrelated neurotoxicants may produce similar outcomes. PMID:20600679

Synthesis of glycosaminoglycans by undifferentiated and differentiated HT29 human colonic cancer cells.

PubMed

Simon-Assmann, P; Bouziges, F; Daviaud, D; Haffen, K; Kedinger, M

1987-08-15

Among the extracellular matrix components which have been suggested to be involved in developmental and neoplastic changes are glycosaminoglycans (GAGs). To try to correlate their amount and nature with the process of enterocytic differentiation, we studied glycosaminoglycan synthesis of human colonic adenocarcinoma cells (HT29 cell line) by [3H]glucosamine and [35S]sulfate incorporation. Enterocytic differentiation of the cells obtained in a sugar-free medium (for review, see A. Zweibaum et al. In: Handbook of Physiology. Intestinal Transport of the Gastrointestinal System, in press, 1987) resulted in a marked increase in total incorporation of labeled precursors (20-fold for [3H]glucosamine, 4.5-fold for [35S]sulfate) as well as in uronic acid content (5-fold); most of the synthesized GAGs were found associated with the cell pellet. Chromatographic and electrophoretic analysis of the labeled GAGs revealed that undifferentiated cells synthesized and secreted hyaluronic acid, heparan sulfate, and one class of chondroitin sulfate. Differentiation of HT29 cells because associated with the synthesis of an additional class of chondroitin sulfate (CS4) concomitant to a decrease in heparan sulfate which is no longer found secreted in the medium. Furthermore, the charge density of this latter GAG component varied as assessed by a shift of its affinity on ion-exchange chromatography.

CARM1 modulators affect epigenome of stem cells and change morphology of nucleoli.

PubMed

Franek, M; Legartová, S; Suchánková, J; Milite, C; Castellano, S; Sbardella, G; Kozubek, S; Bártová, E

2015-01-01

CARM1 interacts with numerous transcription factors to mediate cellular processes, especially gene expression. This is important for the maintenance of ESC pluripotency or intervention to tumorigenesis. Here, we studied epigenomic effects of two potential CARM1 modulators: an activator (EML159) and an inhibitor (ellagic acid dihydrate, EA). We examined nuclear morphology in human and mouse embryonic stem cells (hESCs, mESCs), as well as in iPS cells. The CARM1 modulators did not function similarly in all cell types. EA decreased the levels of the pluripotency markers, OCT4 and NANOG, particularly in iPSCs, whereas the levels of these proteins increased after EML159 treatment. EML159 treatment of mouse ESCs led to decreased levels of OCT4 and NANOG, which was accompanied by an increased level of Endo-A. The same trend was observed for NANOG and Endo-A in hESCs affected by EML159. Interestingly, EA mainly changed epigenetic features of nucleoli because a high level of arginine asymmetric di-methylation in the nucleoli of hESCs was reduced after EA treatment. ChIP-PCR of ribosomal genes confirmed significantly reduced levels of H3R17me2a, in both the promoter region of ribosomal genes and rDNA encoding 28S rRNA, after EA addition. Moreover, EA treatment changed the nuclear pattern of AgNORs (silver-stained nucleolus organizer regions) in all cell types studied. In EA-treated ESCs, AgNOR pattern was similar to the pattern of AgNORs after inhibition of RNA pol I by actinomycin D. Together, inhibitory effect of EA on arginine methylation and effect on related morphological parameters was especially observed in compartment of nucleoli.

Microbioreactor Arrays for Full Factorial Screening of Exogenous and Paracrine Factors in Human Embryonic Stem Cell Differentiation

PubMed Central

Titmarsh, Drew M.; Hudson, James E.; Hidalgo, Alejandro; Elefanty, Andrew G.; Stanley, Edouard G.; Wolvetang, Ernst J.; Cooper-White, Justin J.

2012-01-01

Timed exposure of pluripotent stem cell cultures to exogenous molecules is widely used to drive differentiation towards desired cell lineages. However, screening differentiation conditions in conventional static cultures can become impractical in large parameter spaces, and is intrinsically limited by poor spatiotemporal control of the microenvironment that also makes it impossible to determine whether exogenous factors act directly or through paracrine-dependent mechanisms. We detail here the development of a continuous flow microbioreactor array platform that combines full-factorial multiplexing of input factors with progressive accumulation of paracrine factors through serially-connected culture chambers, and further, the use of this system to explore the combinatorial parameter space of both exogenous and paracrine factors involved in human embryonic stem cell (hESC) differentiation to a MIXL1-GFP+ primitive streak-like population. We show that well known inducers of primitive streak (BMP, Activin and Wnt signals) do not simply act directly on hESC to induce MIXL1 expression, but that this requires accumulation of surplus, endogenous factors; and, that conditioned medium or FGF-2 supplementation is able to offset this. Our approach further reveals the presence of a paracrine, negative feedback loop to the MIXL1-GFP+ population, which can be overcome with GSK-3β inhibitors (BIO or CHIR99021), implicating secreted Wnt inhibitory signals such as DKKs and sFRPs as candidate effectors. Importantly, modulating paracrine effects identified in microbioreactor arrays by supplementing FGF-2 and CHIR in conventional static culture vessels resulted in improved differentiation outcomes. We therefore demonstrate that this microbioreactor array platform uniquely enables the identification and decoding of complex soluble factor signalling hierarchies, and that this not only challenges prevailing strategies for extrinsic control of hESC differentiation, but also is translatable to conventional culture systems. PMID:23300662

Loss of spastin function results in disease-specific axonal defects in human pluripotent stem cell-based models of hereditary spastic paraplegia

PubMed Central

Denton, Kyle R.; Lei, Ling; Grenier, Jeremy; Rodionov, Vladimir; Blackstone, Craig; Li, Xue-Jun

2013-01-01

Human neuronal models of hereditary spastic paraplegias (HSP) that recapitulate disease-specific axonal pathology hold the key to understanding why certain axons degenerate in patients and to developing therapies. SPG4, the most common form of HSP, is caused by autosomal dominant mutations in the SPAST gene, which encodes the microtubule-severing ATPase spastin. Here, we have generated a human neuronal model of SPG4 by establishing induced pluripotent stem cells (iPSCs) from an SPG4 patient and differentiating these cells into telencephalic glutamatergic neurons. The SPG4 neurons displayed a significant increase in axonal swellings, which stained strongly for mitochondria and tau, indicating the accumulation of axonal transport cargoes. In addition, mitochondrial transport was decreased in SPG4 neurons, revealing that these patient iPSC-derived neurons recapitulate disease-specific axonal phenotypes. Interestingly, spastin protein levels were significantly decreased in SPG4 neurons, supporting a haploinsufficiency mechanism. Furthermore, cortical neurons derived from spastin-knockdown human embryonic stem cells (hESCs) exhibited similar axonal swellings, confirming that the axonal defects can be caused by loss of spastin function. These spastin-knockdown hESCs serve as an additional model for studying HSP. Finally, levels of stabilized acetylated-tubulin were significantly increased in SPG4 neurons. Vinblastine, a microtubule-destabilizing drug, rescued this axonal swelling phenotype in neurons derived from both SPG4 iPSCs and spastin-knockdown hESCs. Thus, this study demonstrates the successful establishment of human pluripotent stem cell-based neuronal models of SPG4, which will be valuable for dissecting the pathogenic cellular mechanisms and screening compounds to rescue the axonal degeneration in HSP. PMID:24123785

Inconsistent formation and nonfunction of insulin-positive cells from pancreatic endoderm derived from human embryonic stem cells in athymic nude rats.

PubMed

Matveyenko, Aleksey V; Georgia, Senta; Bhushan, Anil; Butler, Peter C

2010-11-01

Embryonic stem cell therapy has been proposed as a therapeutic strategy to restore β-cell mass and function in T1DM. Recently, a group from Novocell (now ViaCyte) reported successful development of glucose-responsive islet-like structures after implantation of pancreatic endoderm (PE) derived from human embryonic stem cells (hESC) into immune-deficient mice. Our objective was to determine whether implantation of hESC-derived pancreatic endoderm from Novocell into athymic nude rats results in development of viable glucose-responsive pancreatic endocrine tissue. Athymic nude rats were implanted with PE derived from hESC either via implantation into the epididymal fat pads or by subcutaneous implantation into TheraCyte encapsulation devices for 20 wk. Blood glucose, weight, and human insulin/C-peptide secretion were monitored by weekly blood draws. Graft β-cell function was assessed by a glucose tolerance test, and graft morphology was assessed by immunohistochemistry and immunofluorescence. At 20 wk postimplantation, epididymal fat-implanted PE progressed to develop islet-like structures in 50% of implants, with a mean β-cell fractional area of 0.8 ± 0.3%. Human C-peptide and insulin were detectable, but at very low levels (C-peptide = 50 ± 26 pmol/l and insulin = 15 ± 7 pmol/l); however, there was no increase in human C-peptide/insulin levels after glucose challenge. There was no development of viable pancreatic tissue or meaningful secretory function when human PE was implanted in the TheraCyte encapsulation devices. These data confirm that islet-like structures develop from hESC differentiated to PE by the protocol developed by NovoCell. However, the extent of endocrine cell formation and secretory function is not yet sufficient to be clinically relevant.

Inconsistent formation and nonfunction of insulin-positive cells from pancreatic endoderm derived from human embryonic stem cells in athymic nude rats

PubMed Central

Matveyenko, Aleksey V.; Georgia, Senta; Bhushan, Anil

2010-01-01

Embryonic stem cell therapy has been proposed as a therapeutic strategy to restore β-cell mass and function in T1DM. Recently, a group from Novocell (now ViaCyte) reported successful development of glucose-responsive islet-like structures after implantation of pancreatic endoderm (PE) derived from human embryonic stem cells (hESC) into immune-deficient mice. Our objective was to determine whether implantation of hESC-derived pancreatic endoderm from Novocell into athymic nude rats results in development of viable glucose-responsive pancreatic endocrine tissue. Athymic nude rats were implanted with PE derived from hESC either via implantation into the epididymal fat pads or by subcutaneous implantation into TheraCyte encapsulation devices for 20 wk. Blood glucose, weight, and human insulin/C-peptide secretion were monitored by weekly blood draws. Graft β-cell function was assessed by a glucose tolerance test, and graft morphology was assessed by immunohistochemistry and immunofluorescence. At 20 wk postimplantation, epididymal fat-implanted PE progressed to develop islet-like structures in 50% of implants, with a mean β-cell fractional area of 0.8 ± 0.3%. Human C-peptide and insulin were detectable, but at very low levels (C-peptide = 50 ± 26 pmol/l and insulin = 15 ± 7 pmol/l); however, there was no increase in human C-peptide/insulin levels after glucose challenge. There was no development of viable pancreatic tissue or meaningful secretory function when human PE was implanted in the TheraCyte encapsulation devices. These data confirm that islet-like structures develop from hESC differentiated to PE by the protocol developed by NovoCell. However, the extent of endocrine cell formation and secretory function is not yet sufficient to be clinically relevant. PMID:20587750

VEGF induces differentiation of functional endothelium from human embryonic stem cells: implications for tissue engineering

PubMed Central

Nourse, Marilyn B.; Halpin, Daniel E.; Scatena, Marta; Mortisen, Derek J.; Tulloch, Nathaniel L.; Hauch, Kip D.; Torok-Storb, Beverly; Ratner, Buddy D.; Pabon, Lil; Murry, Charles E.

2010-01-01

Objective Human embryonic stem cells (hESCs) offer a sustainable source of endothelial cells for therapeutic vascularization and tissue engineering, but current techniques for generating these cells remain inefficient. We endeavored to induce and isolate functional endothelial cells from differentiating hESCs. Methods and Results To enhance endothelial cell differentiation above a baseline of ∼2% in embryoid body (EB) spontaneous differentiation, three alternate culture conditions were compared. Vascular endothelial growth factor (VEGF) treatment of EBs showed the best induction, with markedly increased expression of endothelial cell proteins CD31, VE-Cadherin, and von Willebrand Factor, but not the hematopoietic cell marker CD45. CD31 expression peaked around days 10-14. Continuous VEGF treatment resulted in a four- to five-fold enrichment of CD31+ cells but did not increase endothelial proliferation rates, suggesting a primary effect on differentiation. CD31+ cells purified from differentiating EBs upregulated ICAM-1 and VCAM-1 in response to TNFα, confirming their ability to function as endothelial cells. These cells also expressed multiple endothelial genes and formed lumenized vessels when seeded onto porous poly(2-hydroxyethyl methacrylate) scaffolds and implanted in vivo subcutaneously in athymic rats. Collagen gel constructs containing hESC-derived endothelial cells and implanted into infarcted nude rat hearts formed robust networks of patent vessels filled with host blood cells. Conclusions VEGF induces functional endothelial cells from hESCs independent of endothelial cell proliferation. These enrichment methods increase endothelial cell yield, enabling applications for revascularization as well as basic studies of human endothelial biology. We demonstrate the ability of hESC-derived endothelial cells to facilitate vascularization of tissue-engineered implants. PMID:19875721

Ketamine induces toxicity in human neurons differentiated from embryonic stem cells via mitochondrial apoptosis pathway

PubMed Central

Bosnjak, Zeljko J.; Yan, Yasheng; Canfield, Scott; Muravyeva, Maria Y.; Kikuchi, Chika; Wells, Clive; Corbett, John; Bai, Xiaowen

2013-01-01

Ketamine is widely used for anesthesia in pediatric patients. Growing evidence indicates that ketamine causes neurotoxicity in a variety of developing animal models. Our understanding of anesthesia neurotoxicity in humans is currently limited by difficulties in obtaining neurons and performing developmental toxicity studies in fetal and pediatric populations. It may be possible to overcome these challenges by obtaining neurons from human embryonic stem cells (hESCs) in vitro. hESCs are able to replicate indefinitely and differentiate into every cell type. In this study, we investigated the toxic effect of ketamine on neurons differentiated from hESCs. Two-week-old neurons were treated with different doses and durations of ketamine with or without the reactive oxygen species (ROS) scavenger, Trolox. Cell viability, ultrastructure, mitochondrial membrane potential (ΔΨm), cytochrome c distribution within cells, apoptosis, and ROS production were evaluated. Here we show that ketamine induced ultrastructural abnormalities and dose- and time-dependently caused cell death. In addition, ketamine decreased ΔΨm and increased cytochrome c release from mitochondria. Ketamine also increased ROS production and induced differential expression of oxidative stress-related genes. Specifically, abnormal ultrastructural and ΔΨm changes occurred earlier than cell death in the ketamine-induced toxicity process. Furthermore, Trolox significantly decreased ROS generation and attenuated cell death caused by ketamine in a dose-dependent manner. In conclusion, this study illustrates that ketamine time- and dose-dependently induces human neurotoxicity via ROS-mediated mitochondrial apoptosis pathway and that these side effects can be prevented by the antioxidant agent Trolox. Thus, hESC-derived neurons might provide a promising tool for studying anesthetic-induced developmental neurotoxicity and prevention strategies. PMID:22873495

DNA methylation and copy number variation analyses of human embryonic stem cell-derived neuroprogenitors after low-dose decabromodiphenyl ether and/or bisphenol A exposure.

PubMed

Du, L; Sun, W; Li, X M; Li, X Y; Liu, W; Chen, D

2018-05-01

The polybrominated diphenyl ether flame retardants decabromodiphenyl ether (BDE-209) and bisphenol A (BPA) are environmental contaminants that can cross the placenta and exert toxicity in the developing fetal nervous system. Copy number variants (CNVs) play a role in a number of genetic disorders and may be implicated in BDE-209/BPA teratogenicity. In this study, we found that BDE-209 and/or BPA exposure decreased neural differentiation efficiency of human embryonic stem cells (hESCs), although there was a >90% induction of neuronal progenitor cells (NPCs) from exposed hESCs. However, the mean of CNV numbers in the NPCs with BDE-209 + BPA treatment was significantly higher compared to the other groups, whereas DNA methylation was lower and DNA methyltransferase(DNMT1 and DNMT3A) expression were significantly decreased in all of the BDE-209 and/or BPA treatment groups compared with the control groups. The number of CNVs in chromosomes 3, 4, 11, 22, and X in NPCs with BDE-209 and/or BPA exposure was higher compared to the control group. In addition, CNVs in chromosomes 7, 8, 14, and 16 were stable in hESCs and hESCs-derived NPCs irrespective of BDE-209/BPA exposure, and CNVs in chromosomes 20 q11.21 and 16 p13.11 might be induced by neural differentiation. Thus, BDE-209/BPA exposure emerges as a potential source of CNVs distinct from neural differentiation by itself. BDE-209 and/or BPA exposure may cause genomic instability in cultured stem cells via reduced activity of DNA methyltransferase, suggesting a new mechanism of human embryonic neurodevelopmental toxicity caused by this class of environmental toxins.

Thalidomide induces apoptosis in undifferentiated human induced pluripotent stem cells.

PubMed

Tachikawa, Saoko; Nishimura, Toshinobu; Nakauchi, Hiromitsu; Ohnuma, Kiyoshi

2017-10-01

Thalidomide, which was formerly available commercially to control the symptoms of morning sickness, is a strong teratogen that causes fetal abnormalities. However, the mechanism of thalidomide teratogenicity is not fully understood; thalidomide toxicity is not apparent in rodents, and the use of human embryos is ethically and technically untenable. In this study, we designed an experimental system featuring human-induced pluripotent stem cells (hiPSCs) to investigate the effects of thalidomide. These cells exhibit the same characteristics as those of epiblasts originating from implanted fertilized ova, which give rise to the fetus. Therefore, theoretically, thalidomide exposure during hiPSC differentiation is equivalent to that in the human fetus. We examined the effects of thalidomide on undifferentiated hiPSCs and early-differentiated hiPSCs cultured in media containing bone morphogenetic protein-4, which correspond, respectively, to epiblast (future fetus) and trophoblast (future extra-embryonic tissue). We found that only the number of undifferentiated cells was reduced. In undifferentiated cells, application of thalidomide increased the number of apoptotic and dead cells at day 2 but not day 4. Application of thalidomide did not affect the cell cycle. Furthermore, immunostaining and flow cytometric analysis revealed that thalidomide exposure had no effect on the expression of specific markers of undifferentiated and early trophectodermal differentiated cells. These results suggest that the effect of thalidomide was successfully detected in our experimental system and that thalidomide eliminated a subpopulation of undifferentiated hiPSCs. This study may help to elucidate the mechanisms underlying thalidomide teratogenicity and reveal potential strategies for safely prescribing this drug to pregnant women.

[Nailfold capillaroscopy in the evaluation of Raynaud's phenomenon and undifferentiated connective tissue disease].

PubMed

Cortes, Sara; Clemente-Coelho, Paulo

2008-01-01

Microvascular abnormalities involved in the pathogenic mechanism of several connective tissue disorders can be detected by nailfold capillaroscopy. Evaluation of the interest of nailfold capillaroscopy results in patients with Raynaud s phenomenon or undifferentiated connective tissue disease and their correlation with diagnostic and therapeutical evolution. Selection of capillaroscopic and laboratory results of patients with the diagnosis of Raynaud s phenomenon (without defined connective tissue disease) or undifferentiated connective tissue disease. Evaluation of the present diagnosis and treatment comparing with the ones existed at the time of capillaroscopy performance. 80 patients were enrolled with an age of 51.4+/-14.3 years (mean+/-SD) 78 females (97.5%) with Raynaud s phenomenon and undifferentiated connective tissue disease 27 patients (33.8%); Raynaud s Phenomenon 46 patients (57.5%); undifferentiated connective tissue disease 7 patients (8.7%). The capillaroscopic results were normal 30 patients (37.5%); minor changes tortuosity enlargement 16 patients (20.0%) major changes 34 patients (42.5%) hemorrhages 25 patients (31.3%) megacapillaries 26 patients (32.5%) avascular areas 3 patients (3.8%). The introduction of new treatments after the capillaroscopy occurred in 32 patients (40.0%) and a new diagnosis was done in 39 patients (48.8%). Major changes in capillaroscopy correlated with the change of diagnosis and the introduction of a new treatment (p<0.0001). Nailfold capillaroscopy performed in patients with isolated Raynaud s phenomenon or undifferentiated connective tissue disease has a role in the prognostic evaluation related to the possibility of an evolution of the diagnosis or to the need of the introduction of new treatments.

Apoptosis and cell proliferation in the development of gastric carcinomas: associations with c-myc and p53 protein expression.

PubMed

Ishii, Hideaki H; Gobé, Glenda C; Pan, Wenshen; Yoneyama, Juichi; Ebihara, Yoshiro

2002-09-01

Patients with gastric carcinomas have a poor prognosis and low survival rates. The aim of the present paper was to characterize cellular and molecular properties to provide insight into aspects of tumor progression in early compared with advanced gastric cancers. One hundred and nine graded gastric carcinomas (early or advanced stage, undifferentiated or differentiated type) with paired non-cancer tissue were studied to define the correlation between apoptosis (morphology, terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labeling), cell proliferation (Ki-67 expression, morphology) and expression and localization of two proteins frequently having altered expression in cancers, namely p53 and c-myc. Overall, apoptosis was lower in early stage, differentiated and undifferentiated gastric carcinomas compared with advanced-stage cancers. Cell proliferation was comparatively high in all stages. There was a high level of p53 positivity in all stages. Only the early- and advanced-stage undifferentiated cancers that were p53 positive had a significantly higher level of apoptosis (P < 0.05). Cell proliferation was significantly greater (P < 0.05) only in the early undifferentiated cancers that had either c-myc or p53-positivity. The results indicate that low apoptosis and high cell proliferation combine to drive gastric cancer development. The molecular controls for high cell proliferation of the early stage undifferentiated gastric cancers involve overexpression of both p53 and c-myc. Overexpression of p53 may also control cancer development in that its expression is associated with higher levels of apoptosis in early and late-stage undifferentiated, cancers. Copyright 2002 Blackwell Publishing Asia Pty Ltd

Rhabdoid and Undifferentiated Phenotype in Renal Cell Carcinoma: Analysis of 32 Cases Indicating a Distinctive Common Pathway of Dedifferentiation Frequently Associated With SWI/SNF Complex Deficiency.

PubMed

Agaimy, Abbas; Cheng, Liang; Egevad, Lars; Feyerabend, Bernd; Hes, Ondřej; Keck, Bastian; Pizzolitto, Stefano; Sioletic, Stefano; Wullich, Bernd; Hartmann, Arndt

2017-02-01

Undifferentiated (anaplastic) and rhabdoid cell features are increasingly recognized as adverse prognostic findings in renal cell carcinoma (RCC), but their molecular pathogenesis has not been studied sufficiently. Recent studies identified alterations in the Switch Sucrose nonfermentable (SWI/SNF) chromatin remodeling complex as molecular mechanisms underlying dedifferentiation and rhabdoid features in carcinomas of different organs. We herein have analyzed 32 undifferentiated RCCs having in common an undifferentiated (anaplastic) phenotype, prominent rhabdoid features, or both, irrespective of the presence or absence of conventional RCC component. Cases were stained with 6 SWI/SNF pathway members (SMARCB1, SMARCA2, SMARCA4, ARID1A, SMARCC1, and SMARCC2) in addition to conventional RCC markers. Patients were 20 males and 12 females aged 32 to 85 years (mean, 59). A total of 22/27 patients with known stage presented with ≥pT3. A differentiated component varying from microscopic to major component was detected in 20/32 cases (16 clear cell and 2 cases each chromophobe and papillary RCC). The undifferentiated component varied from rhabdoid dyscohesive cells to large epithelioid to small monotonous anaplastic cells. Variable loss of at least 1 SWI/SNF complex subunit was noted in the undifferentiated/rhabdoid component of 21/32 cases (65%) compared with intact or reduced expression in the differentiated component. A total of 15/17 patients (88%) with follow-up died of metastatic disease (mostly within 1 y). Only 2 patients were disease free at last follow-up (1 and 6 y). No difference in survival, age distribution, or sex was observed between the SWI/SNF-deficient and the SWI/SNF-intact group. This is the first study exploring the role of SWI/SNF deficiency as a potential mechanism underlying undifferentiated and rhabdoid phenotype in RCC. Our results highlight the association between the aggressive rhabdoid phenotype and the SWI/SNF complex deficiency, consistent with studies on similar neoplasms in other organs. Thorough sampling of such tumors that are usually huge and locally advanced is necessary for recognizing the clone of origin and hence for proper subtyping and also for differentiating them from undifferentiated urothelial carcinoma.

Endometrial and ovarian carcinomas with undifferentiated components: clinically aggressive and frequently underrecognized neoplasms.

PubMed

Tafe, Laura J; Garg, Karuna; Chew, Ivy; Tornos, Carmen; Soslow, Robert A

2010-06-01

Carcinomas of the endometrium and ovary with undifferentiated components are uncommon neoplasms that are likely underdiagnosed. They are important to recognize as they have been shown to be clinically aggressive. We identified 32 carcinomas with undifferentiated components as defined by Silva and co-workers, 26 endometrial and 6 of ovarian origin. The patient age ranged from 21 to 76 years (median 55); 40% of patients were

Designer babies on tap? Medical students' attitudes to pre-implantation genetic screening.

PubMed

Meisenberg, Gerhard

2009-03-01

This paper describes two studies about the determinants of attitudes to pre-implantation genetic screening in a multicultural sample of medical students from the United States. Sample sizes were 292 in study 1 and 1464 in study 2. Attitudes were of an undifferentiated nature, but respondents did make a major distinction between use for disease prevention and use for enhancement. No strong distinctions were made between embryo selection and germ line gene manipulations, and between somatic gene therapy and germ line gene manipulations. Religiosity was negatively associated with acceptance of "designer baby" technology for Christians and Muslims but not Hindus. However, the strongest and most consistent influence was an apparently moralistic stance against active and aggressive interference with natural processes in general. Trust in individuals and institutions was unrelated to acceptance of the technology, indicating that fear of abuse by irresponsible individuals and corporations is not an important determinant of opposition.

SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells.

PubMed

Dubois, Nicole C; Craft, April M; Sharma, Parveen; Elliott, David A; Stanley, Edouard G; Elefanty, Andrew G; Gramolini, Anthony; Keller, Gordon

2011-10-23

To identify cell-surface markers specific to human cardiomyocytes, we screened cardiovascular cell populations derived from human embryonic stem cells (hESCs) against a panel of 370 known CD antibodies. This screen identified the signal-regulatory protein alpha (SIRPA) as a marker expressed specifically on cardiomyocytes derived from hESCs and human induced pluripotent stem cells (hiPSCs), and PECAM, THY1, PDGFRB and ITGA1 as markers of the nonmyocyte population. Cell sorting with an antibody against SIRPA allowed for the enrichment of cardiac precursors and cardiomyocytes from hESC/hiPSC differentiation cultures, yielding populations of up to 98% cardiac troponin T-positive cells. When plated in culture, SIRPA-positive cells were contracting and could be maintained over extended periods of time. These findings provide a simple method for isolating populations of cardiomyocytes from human pluripotent stem cell cultures, and thereby establish a readily adaptable technology for generating large numbers of enriched cardiomyocytes for therapeutic applications.

A small molecule-based strategy for endothelial differentiation and three-dimensional morphogenesis from human embryonic stem cells.

PubMed

Geng, Yijie; Feng, Bradley

2016-07-01

The emerging models of human embryonic stem cell (hESC) self-organizing organoids provide a valuable in vitro platform for studying self-organizing processes that presumably mimic in vivo human developmental events. Here we report that through a chemical screen, we identified two novel and structurally similar small molecules BIR1 and BIR2 which robustly induced the self-organization of a balloon-shaped three-dimensional structure when applied to two-dimensional adherent hESC cultures in the absence of growth factors. Gene expression analyses and functional assays demonstrated an endothelial identity of this balloon-like structure, while cell surface marker analyses revealed a VE-cadherin(+)CD31(+)CD34(+)KDR(+)CD43(-) putative endothelial progenitor population. Furthermore, molecular marker labeling and morphological examinations characterized several other distinct DiI-Ac-LDL(+) multi-cellular modules and a VEGFR3(+) sprouting structure in the balloon cultures that likely represented intermediate structures of balloon-formation.

Long-term culture and cryopreservation does not affect the stability and functionality of human embryonic stem cell-derived hepatocyte-like cells.

PubMed

Mandal, Arundhati; Raju, Sheena; Viswanathan, Chandra

2016-02-01

Human embryonic stem cells (hESCs) are predicted to be an unlimited source of hepatocytes which can pave the way for applications such as cell replacement therapies or as a model of human development or even to predict the hepatotoxicity of drug compounds. We have optimized a 23-d differentiation protocol to generate hepatocyte-like cells (HLCs) from hESCs, obtaining a relatively pure population which expresses the major hepatic markers and is functional and mature. The stability of the HLCs in terms of hepato-specific marker expression and functionality was found to be intact even after an extended period of in vitro culture and cryopreservation. The hESC-derived HLCs have shown the capability to display sensitivity and an alteration in the level of CYP enzyme upon drug induction. This illustrates the potential of such assays in predicting the hepatotoxicity of a drug compound leading to advancement of pharmacology.

Time-Lapse Analysis of Human Embryonic Stem Cells Reveals Multiple Bottlenecks Restricting Colony Formation and Their Relief upon Culture Adaptation

PubMed Central

Barbaric, Ivana; Biga, Veronica; Gokhale, Paul J.; Jones, Mark; Stavish, Dylan; Glen, Adam; Coca, Daniel; Andrews, Peter W.

2014-01-01

Summary Using time-lapse imaging, we have identified a series of bottlenecks that restrict growth of early-passage human embryonic stem cells (hESCs) and that are relieved by karyotypically abnormal variants that are selected by prolonged culture. Only a minority of karyotypically normal cells divided after plating, and these were mainly cells in the later stages of cell cycle at the time of plating. Furthermore, the daughter cells showed a continued pattern of cell death after division, so that few formed long-term proliferating colonies. These colony-forming cells showed distinct patterns of cell movement. Increasing cell density enhanced cell movement facilitating cell:cell contact, which resulted in increased proportion of dividing cells and improved survival postplating of normal hESCs. In contrast, most of the karyotypically abnormal cells reentered the cell cycle on plating and gave rise to healthy progeny, without the need for cell:cell contacts and independent of their motility patterns. PMID:25068128

Characterization of acute undifferentiated leukemia by combined analysis of plasma membrane-associated gamma-glutamyltranspeptidase and soluble terminal transferase.

PubMed

Heumann, D; Losa, G; Barras, C; Morell, A; von Fliedner, V

1985-08-01

gamma-Glutamyltranspeptidase (gamma-GT) is a plasma membrane-associated enzyme present in blasts of certain acute leukemias. We analyzed 90 cases of undifferentiated and differentiated acute leukemias for gamma-GT, using a colorimetric assay. Blasts of all patients with common acute lymphoblastic leukemia (ALL) and T-ALL were negative for gamma-GT (less than 5 units). In contrast, gamma-GT was significantly elevated in acute myeloblastic or monoblastic leukemia blasts (P less than .001). In 16 cases of acute undifferentiated leukemia (AUL) studied, the levels of gamma-GT ranged from 0 to 93 units; in eight cases, gamma-GT was positive (greater than 5 units), and six of these had 2% to 5% Sudan black-positive leukemic cells in the blast-enriched suspension. Combined gamma-GT/TdT analysis revealed that both enzyme markers were mutually exclusive in 75% of AUL cases, suggesting that gamma-GT+/TdT-blasts are of nonlymphoid origin, and gamma-GT-/TdT+ blasts are of lymphoid origin. Two cases were devoid of both enzyme activities and could represent truly undifferentiated leukemia. Thus, combined gamma-GT/TdT analysis underlines the heterogeneity of AUL and appears to be useful in defining the lineage commitment of undifferentiated leukemic blasts.

Etiologies of Acute Undifferentiated Fever and Clinical Prediction of Scrub Typhus in a Non-Tropical Endemic Area

PubMed Central

Jung, Ho-Chul; Chon, Sung-Bin; Oh, Won Sup; Lee, Dong-Hyun; Lee, Ho-Jin

2015-01-01

Scrub typhus usually presents as acute undifferentiated fever. This cross-sectional study included adult patients presenting with acute undifferentiated fever defined as any febrile illness for ≤ 14 days without evidence of localized infection. Scrub typhus cases were defined by an antibody titer of a ≥ fourfold increase in paired sera, a ≥ 1:160 in a single serum using indirect immunofluorescence assay, or a positive result of the immunochromatographic test. Multiple regression analysis identified predictors associated with scrub typhus to develop a prediction rule. Of 250 cases with known etiology of acute undifferentiated fever, influenza (28.0%), hepatitis A (25.2%), and scrub typhus (16.4%) were major causes. A prediction rule for identifying suspected cases of scrub typhus consisted of age ≥ 65 years (two points), recent fieldwork/outdoor activities (one point), onset of illness during an outbreak period (two points), myalgia (one point), and eschar (two points). The c statistic was 0.977 (95% confidence interval = 0.960–0.994). At a cutoff value ≥ 4, the sensitivity and specificity were 92.7% (79.0–98.1%) and 90.9% (86.0–94.3%), respectively. Scrub typhus, the third leading cause of acute undifferentiated fever in our region, can be identified early using the prediction rule. PMID:25448236

The Role of an Undifferentiated Component in Submucosal Invasion and Submucosal Invasion Depth After Endoscopic Submucosal Dissection for Early Gastric Cancer.

PubMed

Miyahara, Koji; Hatta, Waku; Nakagawa, Masahiro; Oyama, Tsuneo; Kawata, Noboru; Takahashi, Akiko; Yoshifuku, Yoshikazu; Hoteya, Shu; Hirano, Masaaki; Esaki, Mitsuru; Matsuda, Mitsuru; Ohnita, Ken; Shimoda, Ryo; Yoshida, Motoyuki; Dohi, Osamu; Takada, Jun; Tanaka, Keiko; Yamada, Shinya; Tsuji, Tsuyotoshi; Ito, Hirotaka; Aoyagi, Hiroyuki; Shimosegawa, Tooru

2018-06-05

The role of an undifferentiated component in submucosal invasion and submucosal invasion depth (SID) for lymph node metastasis (LNM) of early gastric cancer (EGC) with deep submucosal invasion (SID ≥500 μm from the muscularis mucosa) after endoscopic submucosal dissection (ESD) has not been fully understood. This study aimed to clarify the risk factors (RFs), including these factors, for LNM in such patients. We enrolled 513 patients who underwent radical surgery after ESD for EGC with deep submucosal invasion. We evaluated RFs for LNM, including an undifferentiated component in submucosal invasion and the SID, which was subdivided into 500-999, 1,000-1,499, 1,500-1,999, and ≥2,000 µm. LNM was detected in 7.6% of patients. Multivariate analysis revealed that an undifferentiated component in submucosal invasion (OR 2.22), in addition to tumor size >30 mm (OR 2.51) and lymphatic invasion (OR 3.07), were the independent RFs for LNM. However, the SID was not significantly associated with LNM. An undifferentiated component in submucosal invasion was one of the RFs for LNM, in contrast to SID, in patients who underwent ESD for EGC with deep submucosal invasion. This insight would be helpful in managing such patients. © 2018 S. Karger AG, Basel.

Acute undifferentiated fever in India: a multicentre study of aetiology and diagnostic accuracy.

PubMed

Mørch, Kristine; Manoharan, Anand; Chandy, Sara; Chacko, Novin; Alvarez-Uria, Gerardo; Patil, Suvarna; Henry, Anil; Nesaraj, Joel; Kuriakose, Cijoy; Singh, Ashita; Kurian, Siby; Gill Haanshuus, Christel; Langeland, Nina; Blomberg, Bjørn; Vasanthan Antony, George; Mathai, Dilip

2017-10-04

The objectives of this study were to determine the proportion of malaria, bacteraemia, scrub typhus, leptospirosis, chikungunya and dengue among hospitalized patients with acute undifferentiated fever in India, and to describe the performance of standard diagnostic methods. During April 2011-November 2012, 1564 patients aged ≥5 years with febrile illness for 2-14 days were consecutively included in an observational study at seven community hospitals in six states in India. Malaria microscopy, blood culture, Dengue rapid NS1 antigen and IgM Combo test, Leptospira IgM ELISA, Scrub typhus IgM ELISA and Chikungunya IgM ELISA were routinely performed at the hospitals. Second line testing, Dengue IgM capture ELISA (MAC-ELISA), Scrub typhus immunofluorescence (IFA), Leptospira Microscopic Agglutination Test (MAT), malaria PCR and malaria immunochromatographic rapid diagnostic test (RDT) Parahit Total™ were performed at the coordinating centre. Convalescence samples were not available. Case definitions were as follows: Leptospirosis: Positive ELISA and positive MAT. Scrub typhus: Positive ELISA and positive IFA. Dengue: Positive RDT and/or positive MAC-ELISA. Chikungunya: Positive ELISA. Bacteraemia: Growth in blood culture excluding those defined as contaminants. Malaria: Positive genus-specific PCR. Malaria was diagnosed in 17% (268/1564) and among these 54% had P. falciparum. Dengue was diagnosed in 16% (244/1564). Bacteraemia was found in 8% (124/1564), and among these Salmonella typhi or S. paratyphi constituted 35%. Scrub typhus was diagnosed in 10%, leptospirosis in 7% and chikungunya in 6%. Fulfilling more than one case definition was common, most frequent in chikungunya where 26% (25/98) also had positive dengue test. Malaria and dengue were the most common causes of fever in this study. A high overlap between case definitions probably reflects high prevalence of prior infections, cross reactivity and subclinical infections, rather than high prevalence of coinfections. Low accuracy of routine diagnostic tests should be taken into consideration when approaching the patient with acute undifferentiated fever in India.

Marker expression, behaviors, and responses vary in different lines of conditionally immortalized cultured podocytes

PubMed Central

Chittiprol, Seetharamaiah; Chen, Phylip; Petrovic-Djergovic, Danica; Eichler, Tad

2011-01-01

The state-of-the-art cultured podocyte is conditionally immortalized by expression of a temperature-sensitive mutant of the SV40 large-T antigen. These cultures proliferate at 33°C and differentiate at 37°C into arborized cells that more closely resemble in vivo podocytes. However, the degree of resemblance remains controversial. In this study, several parameters were measured in podocyte cell lines derived from mouse (JR, KE), human (MS), and rat (HK). In all lines, the quantities of NEPH1 and podocin proteins and NEPH1 and SYNPO mRNAs were comparable to glomeruli, while synaptopodin and nephrin proteins and NPHS1 and NPHS2 mRNAs were <5% of glomerular levels. Expression of Wilms' tumor-1 (WT1) mRNA in mouse lines was comparable to glomeruli, but rat and human lines expressed little WT1. Undifferentiated human and mouse lines had similar proliferation rates that decreased after differentiation, while the rate in rat cells remained constant. The motility of different lines varied as measured by both general motility and wound-healing assays. The toxicity of puromycin aminonucleoside was MS ∼ JR >> KE, and of doxorubicin was JR ∼ KE > MS, while HK cells were almost unaffected. Process formation was largely a result of contractile action after formation of lamellipodia. These findings demonstrate dramatic differences in marker expression, response to toxins, and motility between lines of podocytes from different species and even between similarly-derived mouse lines. PMID:21632959

Marker expression, behaviors, and responses vary in different lines of conditionally immortalized cultured podocytes.

PubMed

Chittiprol, Seetharamaiah; Chen, Phylip; Petrovic-Djergovic, Danica; Eichler, Tad; Ransom, Richard F

2011-09-01

The state-of-the-art cultured podocyte is conditionally immortalized by expression of a temperature-sensitive mutant of the SV40 large-T antigen. These cultures proliferate at 33°C and differentiate at 37°C into arborized cells that more closely resemble in vivo podocytes. However, the degree of resemblance remains controversial. In this study, several parameters were measured in podocyte cell lines derived from mouse (JR, KE), human (MS), and rat (HK). In all lines, the quantities of NEPH1 and podocin proteins and NEPH1 and SYNPO mRNAs were comparable to glomeruli, while synaptopodin and nephrin proteins and NPHS1 and NPHS2 mRNAs were <5% of glomerular levels. Expression of Wilms' tumor-1 (WT1) mRNA in mouse lines was comparable to glomeruli, but rat and human lines expressed little WT1. Undifferentiated human and mouse lines had similar proliferation rates that decreased after differentiation, while the rate in rat cells remained constant. The motility of different lines varied as measured by both general motility and wound-healing assays. The toxicity of puromycin aminonucleoside was MS ∼ JR > KE, and of doxorubicin was JR ∼ KE > MS, while HK cells were almost unaffected. Process formation was largely a result of contractile action after formation of lamellipodia. These findings demonstrate dramatic differences in marker expression, response to toxins, and motility between lines of podocytes from different species and even between similarly-derived mouse lines.

Noun-Verb Ambiguity in Chronic Undifferentiated Schizophrenia

ERIC Educational Resources Information Center

Goldfarb, Robert; Bekker, Natalie

2009-01-01

This study investigated noun-verb retrieval patterns of 30 adults with chronic undifferentiated schizophrenia and 67 typical adults, to determine if schizophrenia affected nouns (associated with temporal lobe function) differently from verbs (associated with frontal lobe function). Stimuli were homophonic homographic homonyms, balanced according…

Sapanisertib or Pazopanib Hydrochloride in Treating Patients With Locally Advanced or Metastatic Sarcoma

ClinicalTrials.gov

2018-06-20

High Grade Sarcoma; Metastatic Leiomyosarcoma; Metastatic Malignant Peripheral Nerve Sheath Tumor; Metastatic Synovial Sarcoma; Metastatic Undifferentiated Pleomorphic Sarcoma; Myxofibrosarcoma; Recurrent Leiomyosarcoma; Recurrent Malignant Peripheral Nerve Sheath Tumor; Recurrent Synovial Sarcoma; Recurrent Undifferentiated Pleomorphic Sarcoma; Uterine Corpus Leiomyosarcoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hirano, Kazumi; Van Kuppevelt, Toin H.; Nishihara, Shoko, E-mail: shoko@soka.ac.jp

Highlights: ► Fas transcript increases during the transition from the naïve to the primed state. ► 3OST-5 transcript, the HS4C3 epitope synthesis gene, increases during the transition. ► Fas signaling regulates the transition from the naïve to the primed state. ► HS4C3-binding epitope regulates the transition from the naïve to the primed state. ► Fas signaling is regulated by the HS4C3 epitope during the transition. -- Abstract: The characteristics of pluripotent embryonic stem cells of human and mouse are different. The properties of human embryonic stem cells (hESCs) are similar to those of mouse epiblast stem cells (mEpiSCs), which aremore » in a later developmental pluripotency state, the so-called “primed state” compared to mouse embryonic stem cells (mESCs) which are in a naïve state. As a result of the properties of the primed state, hESCs proliferate slowly, cannot survive as single cells, and can only be transfected with genes at low efficiency. Generating hESCs in the naïve state is necessary to overcome these problems and allow their application in regenerative medicine. Therefore, clarifying the mechanism of the transition between the naïve and primed states in pluripotent stem cells is important for the establishment of stable methods of generating naïve state hESCs. However, the signaling pathways which contribute to the transition between the naïve and primed states are still unclear. In this study, we carried out induction from mESCs to mEpiSC-like cells (mEpiSCLCs), and observed an increase in the activation of Fas signaling during the induction. The expression of Fgf5, an epiblast marker, was diminished by inhibition of Fas signaling using the caspase-8 and -3 blocking peptides, IETD and DEVD, respectively. Furthermore, during the induction, we observed increased expression of 3-O sulfated heparan sulfate (HS) structures synthesized by HS 3-O-sulfotransferase (3OST), which are recognized by the HS4C3 antibody (HS4C3-binding epitope). Knockdown of 3OST-5 reduced Fas signaling and the potential for the transition to mEpiSCLCs. This indicates that the HS4C3-binding epitope is necessary for the transition to the primed state. We propose that Fas signaling through the HS4C3-binding epitope contributes to the transition from the naïve state to the primed state.« less

Loss of switch/sucrose non-fermenting complex protein expression is associated with dedifferentiation in endometrial carcinomas

PubMed Central

Karnezis, Anthony N.; Hoang, Lien N.; Coatham, Mackenzie; Ravn, Sarah; Almadani, Noorah; Cloutier, Basile; Irving, Julie; Meng, Bo; Li, Xiaodong; Chow, Christine; McAlpine, Jessica; Kuo, Kuan-Ting; Mao, Tsui-Lien; Djordjevic, Bojana; Soslow, Robert A.; Huntsman, David G.; Gilks, C. Blake; Köbel, Martin; Lee, Cheng-Han

2016-01-01

Dedifferentiated endometrial carcinoma is an aggressive type of endometrial cancer that contains a mix of low grade endometrioid and undifferentiated carcinoma components. We performed targeted sequencing of 8 dedifferentiated endometrial carcinomas and identified somatic frameshift/nonsense mutations in SMARCA4, a core member of the switch/sucrose non-fermenting (SWI/SNF) complex, in the undifferentiated components of 4 tumors. Immunohistochemical analysis confirmed the loss of SMARCA4 in the undifferentiated component of these 4 SMARCA4-mutated cases while the corresponding low grade endometrioid component showed retained SMARCA4 expression. An expanded survey of another member of the SWI/SNF complex showed SMARCB1 loss in the undifferentiated component of 2 SMARCA4-intact tumors. Subsequent immunohistochemical analysis of SMARCA4 and SMARCB1 was done in an additional set of 22 centrally reviewed dedifferentiated endometrial carcinomas and 31 grade 3 endometrioid carcinomas. Combining the results from the index and the expansion set, 15 of 30 (50%) of the dedifferentiated endometrial carcinomas examined showed either SMARCA4 loss (37%) or SMARCB1 loss (13%). The loss of SMARCA4 or SMARCB1 was mutually exclusive and occurred only in the undifferentiated component. All 31 grade 3 endometrioid carcinomas showed intact SMARCA4/SMARCB1 expression. The majority (73%) of the SMARCA4-deficient and half of SMARCB1-deficient undifferentiated component developed in a mismatch repair protein (MMR)-deficient molecular context. The observed spatial association between SMARCA4/SMARCB1 loss and histologic dedifferentiation suggests that loss of these SWI/SNF complex proteins may contribute to the development of dedifferentiated endometrial carcinoma. PMID:26743474

Loss of switch/sucrose non-fermenting complex protein expression is associated with dedifferentiation in endometrial carcinomas.

PubMed

Karnezis, Anthony N; Hoang, Lien N; Coatham, Mackenzie; Ravn, Sarah; Almadani, Noorah; Tessier-Cloutier, Basile; Irving, Julie; Meng, Bo; Li, Xiaodong; Chow, Christine; McAlpine, Jessica; Kuo, Kuan-Ting; Mao, Tsui-Lien; Djordjevic, Bojana; Soslow, Robert A; Huntsman, David G; Blake Gilks, C; Köbel, Martin; Lee, Cheng-Han

2016-03-01

Dedifferentiated endometrial carcinoma is an aggressive type of endometrial cancer that contains a mix of low-grade endometrioid and undifferentiated carcinoma components. We performed targeted sequencing of eight dedifferentiated carcinomas and identified somatic frameshift/nonsense mutations in SMARCA4, a core ATPase of the switch/sucrose non-fermenting (SWI/SNF) complex, in the undifferentiated components of four tumors. Immunohistochemical analysis confirmed the loss of SMARCA4 in the undifferentiated component of these four SMARCA4-mutated cases, whereas the corresponding low-grade endometrioid component showed retained SMARCA4 expression. An expanded survey of other members of the SWI/SNF complex showed SMARCB1 loss in the undifferentiated component of two SMARCA4-intact tumors, and all SMARCA4- or SMARCB1-deficient tumors showed concomitant loss of expression of SMARCA2. We subsequently examined the expression of SMARCA2, SMARCA4, and SMARCB1 in an additional set of 22 centrally reviewed dedifferentiated carcinomas and 31 grade 3 endometrioid carcinomas. Combining the results from the index and the expansion set, 15 of 30 (50%) of the dedifferentiated carcinomas examined showed either concurrent SMARCA4 and SMARCA2 loss (37%) or concurrent SMARCB1 and SMARCA2 loss (13%) in the undifferentiated component. The loss of SMARCA4 or SMARCB1 was mutually exclusive. All 31 grade 3 endometrioid carcinomas showed intact expression of these core SWI/SNF proteins. The majority (73%) of the SMARCA4/SMARCA2-deficient and half of SMARCB1/SMARCA2-deficient undifferentiated component developed in a mismatch repair-deficient molecular context. The observed spatial association between SWI/SNF protein loss and histologic dedifferentiation suggests that inactivation of these core SWI/SNF proteins may contribute to the development of dedifferentiated endometrial carcinoma.

Dedifferentiated endometrial carcinomas with neuroendocrine features: a clinicopathologic, immunohistochemical, and molecular genetic study.

PubMed

Espinosa, Iñigo; De Leo, Antonio; D'Angelo, Emanuela; Rosa-Rosa, Juan M; Corominas, Marina; Gonzalez, Alan; Palacios, José; Prat, Jaime

2018-02-01

Undifferentiated endometrial carcinoma is an aggressive type of uterine cancer, which is occasionally associated with a low-grade endometrioid carcinoma component. This combination is referred to as "dedifferentiated endometrioid endometrial carcinoma." Neuroendocrine expression may occur in undifferentiated endometrial carcinoma, but its significance in dedifferentiated endometrial carcinomas is unknown. To gain insight into the pathogenesis of these tumors we have analyzed the immunophenotype (ARID1A, MLH1, PMS2, MSH2, MSH6, p53, β-catenin, SMARCB1, synaptophysin, chromogranin A, and CD56) and mutational status (PTEN, KRAS, PIK3CA, TP53 and POLE) of 4 dedifferentiated endometrial carcinomas with strong and diffuse neuroendocrine expression. All tumors demonstrated neuroendocrine expression in ≥70% of the cells in the undifferentiated carcinoma areas. Loss of expression of at least 1 DNA mismatch repair protein was observed in 2 cases, and p53 immunoreaction was aberrant (mutated/inactivated) in one case. All carcinomas were negative for β-catenin and maintained nuclear SMARCB1 (INI1) and ARID1A expression. Three tumors shared identical endometrioid molecular profile (PTEN and/or PIK3CA mutations) in both components. One tumor had POLE exonuclease domain mutation in the undifferentiated component. In one case, TP53 mutation was found exclusively in the undifferentiated component. Two patients died with peritoneal carcinomatosis and abdominal metastases, respectively; one patient died of a renal failure without evidence of disease, and the last patient is alive and free of disease at 3.3 years. Dedifferentiated endometrial carcinomas with neuroendocrine features are clinically and molecularly heterogeneous tumors. Probably, these carcinomas might acquire undifferentiated phenotype through mutations in TP53 and POLE. Copyright © 2017 Elsevier Inc. All rights reserved.

Phenotypic and Molecular Analysis of Mes-3, a Maternal-Effect Gene Required for Proliferation and Viability of the Germ Line in C. Elegans

PubMed Central

Paulsen, J. E.; Capowski, E. E.; Strome, S.

1995-01-01

mes-3 is one of four maternal-effect sterile genes that encode maternal components required for normal postembryonic development of the germ line in Caenorhabditis elegans. mes-3 mutant mothers produce sterile progeny, which contain few germ cells and no gametes. This terminal phenotype reflects two problems: reduced proliferation of the germ line and germ cell death. Both the appearance of the dying germ cells and the results of genetic tests indicate that germ cells in mes-3 animals undergo a necrotic-like death, not programmed cell death. The few germ cells that appear healthy in mes-3 worms do not differentiate into gametes, even after elimination of the signaling pathway that normally maintains the undifferentiated population of germ cells. Thus, mes-3 encodes a maternally supplied product that is required both for proliferation of the germ line and for maintenance of viable germ cells that are competent to differentiate into gametes. Cloning and molecular characterization of mes-3 revealed that it is the upstream gene in an operon. The genes in the operon display parallel expression patterns; transcripts are present throughout development and are not restricted to germ-line tissue. Both mes-3 and the downstream gene in the operon encode novel proteins. PMID:8601481

PAQ Types and Power Strategies Used in Intimate Relationships.

ERIC Educational Resources Information Center

Falbo, Toni

1982-01-01

Examined kinds of power strategies used by masculine, feminine, androgynous, and undifferentiated people in their intimate relationships. Androgynous people reported using primarily bilateral strategies, such as persuasion. Undifferentiated people reported using primarily unilateral strategies, such as doing what they wanted, regardless of their…

[Undifferentiated blastic cell crisis of chronic myelogenous leukemia with myeloblastic tumor in the skin].

PubMed

Kawakami, K; Kiyosaki, M; Amaya, H; Nakamaki, T; Hino, K; Tomoyasu, S

2000-04-01

A 54-year-old female, who had been treated for 4 years in the chronic phase of chronic myelogenous leukemia (CML) was admitted for management of a CML blastic crisis. Blast cells showed strong positive expression of CD7 and HLA-DR, and weakly expressed CD2, CD5 and CD10, as well. The cells were peroxidase negative in peripheral blood and bone marrow. An undifferentiated blastic crisis was diagnosed and she was treated with Interferon-alpha and VP(vincristine 2 mg/week; prednisolone 30 mg/day). A 5-7 mm in diameter tumor in the skin of the anterior right chest appeared one week after VP therapy. The tumor consisted of blasts which were CD13, CD33 and peroxidase positive, unlike the peripheral undifferentiated blasts. This is a rare case of mixed blast crisis with an increase in undifferentiated blasts in peripheral blood and bone marrow, and myeloblastic tumor formation in the skin.

Ribociclib and Doxorubicin in Treating Patients With Metastatic or Advanced Soft Tissue Sarcomas That Cannot Be Removed by Surgery

ClinicalTrials.gov

2018-05-09

Metastatic Angiosarcoma; Metastatic Epithelioid Sarcoma; Metastatic Fibrosarcoma; Metastatic Leiomyosarcoma; Metastatic Liposarcoma; Metastatic Malignant Peripheral Nerve Sheath Tumor; Metastatic Synovial Sarcoma; Metastatic Undifferentiated Pleomorphic Sarcoma; Myxofibrosarcoma; Pleomorphic Rhabdomyosarcoma; Stage III Soft Tissue Sarcoma; Stage IV Soft Tissue Sarcoma; Undifferentiated (Embryonal) Sarcoma

Vorinostat and Azacitidine in Treating Patients With Locally Recurrent or Metastatic Nasopharyngeal Cancer or Nasal Natural Killer T-Cell Lymphoma

ClinicalTrials.gov

2018-04-20

Adult Nasal Type Extranodal NK/T-Cell Lymphoma; Recurrent Nasopharyngeal Keratinizing Squamous Cell Carcinoma; Recurrent Nasopharyngeal Undifferentiated Carcinoma; Stage IV Nasopharyngeal Keratinizing Squamous Cell Carcinoma AJCC v7; Stage IV Nasopharyngeal Undifferentiated Carcinoma AJCC v7

MicroRNA-195 targets ADP-ribosylation factor-like protein 2 to induce apoptosis in human embryonic stem cell-derived neural progenitor cells

PubMed Central

Zhou, Y; Jiang, H; Gu, J; Tang, Y; Shen, N; Jin, Y

2013-01-01

Neural progenitor cells (NPCs) derived from human embryonic stem cells (hESCs) have great potential in cell therapy, drug screening and toxicity testing of neural degenerative diseases. However, the molecular regulation of their proliferation and apoptosis, which needs to be revealed before clinical application, is largely unknown. MicroRNA miR-195 is known to be expressed in the brain and is involved in a variety of proapoptosis or antiapoptosis processes in cancer cells. Here, we defined the proapoptotic role of miR-195 in NPCs derived from two independent hESC lines (human embryonic stem cell-derived neural progenitor cells, hESC-NPCs). Overexpression of miR-195 in hESC-NPCs induced extensive apoptotic cell death. Consistently, global transcriptional microarray analyses indicated that miR-195 primarily regulated genes associated with apoptosis in hESC-NPCs. Mechanistically, a small GTP-binding protein ADP-ribosylation factor-like protein 2 (ARL2) was identified as a direct target of miR-195. Silencing ARL2 in hESC-NPCs provoked an apoptotic phenotype resembling that of miR-195 overexpression, revealing for the first time an essential role of ARL2 for the survival of human NPCs. Moreover, forced expression of ALR2 could abolish the cell number reduction caused by miR-195 overexpression. Interestingly, we found that paraquat, a neurotoxin, not only induced apoptosis but also increased miR-195 and reduced ARL2 expression in hESC-NPCs, indicating the possible involvement of miR-195 and ARL2 in neurotoxin-induced NPC apoptosis. Notably, inhibition of miR-195 family members could block neurotoxin-induced NPC apoptosis. Collectively, miR-195 regulates cell apoptosis in a context-dependent manner through directly targeting ARL2. The finding of the critical role of ARL2 for the survival of human NPCs and association of miR-195 and ARL2 with neurotoxin-induced apoptosis have important implications for understanding molecular mechanisms that control NPC survival and would facilitate our manipulation of the neurological pathogenesis. PMID:23807224

Protein tyrosine phosphatase 1B (PTP1B) is required for cardiac lineage differentiation of mouse embryonic stem cells.

PubMed

Eshkiki, Zahra Shokati; Ghahremani, Mohammad Hossein; Shabani, Parisa; Firuzjaee, Sattar Gorgani; Sadeghi, Asie; Ghanbarian, Hossein; Meshkani, Reza

2017-01-01

Protein tyrosine phosphatase 1B (PTP1B) has been shown to regulate multiple cellular events such as differentiation, cell growth, and proliferation; however, the role of PTP1B in differentiation of embryonic stem (ES) cells into cardiomyocytes remains unexplored. In the present study, we investigated the effects of PTP1B inhibition on differentiation of ES cells into cardiomyocytes. PTP1B mRNA and protein levels were increased during the differentiation of ES cells into cardiomyocytes. Accordingly, a stable ES cell line expressing PTP1B shRNA was established. In vitro, the number and size of spontaneously beating embryoid bodies were significantly decreased in PTP1B-knockdown cells, compared with the control cells. Decreased expression of cardiac-specific markers Nkx2-5, MHC-α, cTnT, and CX43, as assessed by real-time PCR analysis, was further confirmed by immunocytochemistry of the markers. The results also showed that PTP1B inhibition induced apoptosis in both differentiated and undifferentiated ES cells, as presented by increasing the level of cleaved caspase-3, cytochrome C, and cleaved PARP. Further analyses revealed that PTP1B inhibition did not change proliferation and pluripotency of undifferentiated ES cells. Taken together, the data presented here suggest that PTP1B is essential for proper differentiation of ES cells into cardiomyocytes.

Radiation damage to the microvasculature in the rabbit ear chamber. An electron microscope study. [X radiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, V.V.; Stearner, S.P.; Dimitrievich, G.S.

1977-04-01

Cell aggregates in increased numbers appear along blood vessel walls within a few days after local x irradiation of the tissue within rabbit ear chambers. At 7 days after irradiation with 400 or 700 rad of 250 kVp of x rays, electron microscopic studies of the microvasculature were carried out to determine the morphological characteristics of the cell types involved in the aggregates and the relation of these cells to vascular repair. The cell aggregates usually occur in the interstitial region subjacent to the endothelium. The cells that make up the aggregates show morphological characteristics of relatively undifferentiated mesenchymal cells;more » they have an irregularly rounded shape and contain large amounts of rough endoplasmic reticulum, Golgi vesicles, and mitochondria. In a few instances, cells of similar morphology also occur as part of the lining of the blood vessels. The perivascular cell aggregates may originate from the pericyte population or from undifferentiated mesenchymal cells that occur in the interstitial region surrounding blood vessels; it is improbable that they are dedifferentiated smooth muscle cells. It is suggested that the cells that make up these aggregates contribute to the repair of the microvasculation after radiation injury. The radiosensitivity of vascular endothelium reported by previous investigators seems to preclude endothelial proliferation as the principal repair mechanism at higher radiation doses.« less

Pluripotent Human embryonic stem cell derived neural lineages for in vitro modelling of enterovirus 71 infection and therapy.

PubMed

Yap, May Shin; Tang, Yin Quan; Yeo, Yin; Lim, Wei Ling; Lim, Lee Wei; Tan, Kuan Onn; Richards, Mark; Othman, Iekhsan; Poh, Chit Laa; Heng, Boon Chin

2016-01-06

The incidence of neurological complications and fatalities associated with Hand, Foot & Mouth disease has increased over recent years, due to emergence of newly-evolved strains of Enterovirus 71 (EV71). In the search for new antiviral therapeutics against EV71, accurate and sensitive in vitro cellular models for preliminary studies of EV71 pathogenesis is an essential prerequisite, before progressing to expensive and time-consuming live animal studies and clinical trials. This study thus investigated whether neural lineages derived from pluripotent human embryonic stem cells (hESC) can fulfil this purpose. EV71 infection of hESC-derived neural stem cells (NSC) and mature neurons (MN) was carried out in vitro, in comparison with RD and SH-SY5Y cell lines. Upon assessment of post-infection survivability and EV71 production by the various types, it was observed that NSC were significantly more susceptible to EV71 infection compared to MN, RD (rhabdomyosarcoma) and SH-SY5Y cells, which was consistent with previous studies on mice. The SP81 peptide had significantly greater inhibitory effect on EV71 production by NSC and MN compared to the cancer-derived RD and SH-SY5Y cell lines. Hence, this study demonstrates that hESC-derived neural lineages can be utilized as in vitro models for studying EV71 pathogenesis and for screening of antiviral therapeutics.

Airborne gamma-ray spectrometer and magnetometer survey, Seattle quadrangle (Washington). Final report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

1981-01-01

One uranium anomaly meets the minimum statistical requirements as defined. This anomaly is over the potassium (%K) contact area between undifferentiated Tertiary rocks and Pleistocene glacial deposits. Equivalent uranium (ppM eU), equivalent thorium (ppM eT), eU/eT, eU/eK, eT,K, and magnetic pseudo-contour maps are presented in Appendix E. Stacked profiles showing geologic strip maps along each flight-line, together with sensor data, and ancillary data are presented in Appendix F. All maps and profiles were prepared on a scale of 1:250,000, but have been reduced to 1:500,000 for presentation in this report.

A genetic platform to model sarcomagenesis from primary adult mesenchymal stem cells

PubMed Central

Guarnerio, Jlenia; Riccardi, Luisa; Taulli, Riccardo; Maeda, Takahiro; Wang, Guocan; Hobbs, Robin M.; Song, Min Sup; Sportoletti, Paolo; Bernardi, Rosa; Bronson, Roderick T.; Castillo-Martin, Mireia; Cordon-Cardo, Carlos; Lunardi, Andrea; Pandolfi, Pier Paolo

2015-01-01

The regulatory factors governing adult mesenchymal stem cells (MSCs) physiology and their tumorigenic potential are still largely unknown, which substantially delays the identification of effective therapeutic approaches for the treatment of aggressive and lethal form of MSC-derived mesenchymal tumors, such as undifferentiated sarcomas. Here we have developed a novel platform to screen and quickly identify genes and pathways responsible for adult MSCs transformation, modeled undifferentiated sarcoma in vivo, and, ultimately, tested the efficacy of targeting the identified oncopathways. Importantly, by taking advantage of this new platform, we demonstrate the key role of an aberrant LRF-DLK1-SOX9 pathway in the pathogenesis of undifferentiated sarcoma with important therapeutic implications. PMID:25614485

Acrylamide affects proliferation and differentiation of the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y.

PubMed

Attoff, K; Kertika, D; Lundqvist, J; Oredsson, S; Forsby, A

2016-09-01

Acrylamide is a well-known neurotoxic compound and people get exposed to the compound by food consumption and environmental pollutants. Since acrylamide crosses the placenta barrier, the fetus is also being exposed resulting in a risk for developmental neurotoxicity. In this study, the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y were used to study proliferation and differentiation as alerting indicators for developmental neurotoxicity. For both cell lines, acrylamide reduced the number of viable cells by reducing proliferation and inducing cell death in undifferentiated cells. Acrylamide concentrations starting at 10fM attenuated the differentiation process in SH-SY5Y cells by sustaining cell proliferation and neurite outgrowth was reduced at concentrations from 10pM. Acrylamide significantly reduced the number of neurons starting at 1μM and altered the ratio between the different phenotypes in differentiating C17.2 cell cultures. Ten micromolar of acrylamide also reduced the expression of the neuronal and astrocyte biomarkers. Although the neurotoxic concentrations in the femtomolar range seem to be specific for the SH-SY5Y cell line, the fact that micromolar concentrations of acrylamide seem to attenuate the differentiation process in both cell lines raises the interest to further investigations on the possible developmental neurotoxicity of acrylamide. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Functional characterization of cell hybrids generated by induced fusion of primary porcine mesenchymal stem cells with an immortal murine cell line.

PubMed

Islam, M Q; Ringe, J; Reichmann, E; Migotti, R; Sittinger, M; da S Meirelles, L; Nardi, N B; Magnusson, P; Islam, K

2006-10-01

Bone marrow mesenchymal stem cells (MSC) integrate into various organs and contribute to the regeneration of diverse tissues. However, the mechanistic basis of the plasticity of MSC is not fully understood. The change of cell fate has been suggested to occur through cell fusion. We have generated hybrid cell lines by polyethylene-glycol-mediated cell fusion of primary porcine MSC with the immortal murine fibroblast cell line F7, a derivative of the GM05267 cell line. The hybrid cell lines display fibroblastic morphology and proliferate like immortal cells. They contain tetraploid to hexaploid porcine chromosomes accompanied by hypo-diploid murine chromosomes. Interestingly, many hybrid cell lines also express high levels of tissue-nonspecific alkaline phosphatase, which is considered to be a marker of undifferentiated embryonic stem cells. All tested hybrid cell lines retain osteogenic differentiation, a few of them also retain adipogenic potential, but none retain chondrogenic differentiation. Conditioned media from hybrid cells enhance the proliferation of both early-passage and late-passage porcine MSC, indicating that the hybrid cells secrete diffusible growth stimulatory factors. Murine F7 cells thus have the unique property of generating immortal cell hybrids containing unusually high numbers of chromosomes derived from normal cells. These hybrid cells can be employed in various studies to improve our understanding of regenerative biology. This is the first report, to our knowledge, describing the generation of experimentally induced cell hybrids by using normal primary MSC.

75 FR 8085 - National Institutes of Health Guidelines for Human Stem Cell Research

Federal Register 2010, 2011, 2012, 2013, 2014

2010-02-23

... Health Guidelines for Human Stem Cell Research SUMMARY: The National Institutes of Health (NIH) is requesting public comment on a revision to the definition of human embryonic stem cells (hESCs) in the ``National Institutes of Health Guidelines for Human Stem Cell Research'' (Guidelines). On July 7, 2009, NIH...

75 FR 13137 - National Institutes of Health Guidelines for Human Stem Cell Research

Federal Register 2010, 2011, 2012, 2013, 2014

2010-03-18

... Health Guidelines for Human Stem Cell Research SUMMARY: The National Institutes of Health (NIH) is extending the public comment period on a revision to the definition of human embryonic stem cells (hESCs) in the ``National Institutes of Health Guidelines for Human Stem Cell Research'' (Guidelines). Due to a...

A legal defense for compensating research egg donors.

PubMed

Crockin, Susan L

2010-02-05

Given the continued need for human eggs for hESCs, this article analyzes and refutes the legal theories against compensating research egg donors, contrasts the legal histories of compensating reproductive donors and human subjects with noncompensation for ESC donors, and suggests that limited compensation is legally defensible. Copyright 2010 Elsevier Inc. All rights reserved.

How Is European Governance Configuring the EHEA?

ERIC Educational Resources Information Center

Magalhães, António; Veiga, Amélia; Sousa, Sofia; Ribeiro, Filipa

2012-01-01

This article focuses on the interaction between the European dimension driven by the creation of the European Higher Education Area (EHEA) and the development of national reforms to fulfil that objective. On the basis of data gathered in eight countries involved in EuroHESC project TRUE (Transforming European Universities), the curricular and the…

Human pluripotent stem cell-derived cardiomyocytes for heart regeneration, drug discovery and disease modeling: from the genetic, epigenetic, and tissue modeling perspectives.

PubMed

Chow, Maggie; Boheler, Kenneth R; Li, Ronald A

2013-08-14

Heart diseases remain a major cause of mortality and morbidity worldwide. However, terminally differentiated human adult cardiomyocytes (CMs) possess a very limited innate ability to regenerate. Directed differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) into CMs has enabled clinicians and researchers to pursue the novel therapeutic paradigm of cell-based cardiac regeneration. In addition to tissue engineering and transplantation studies, the need for functional CMs has also prompted researchers to explore molecular pathways and develop strategies to improve the quality, purity and quantity of hESC-derived and iPSC-derived CMs. In this review, we describe various approaches in directed CM differentiation and driven maturation, and discuss potential limitations associated with hESCs and iPSCs, with an emphasis on the role of epigenetic regulation and chromatin remodeling, in the context of the potential and challenges of using hESC-CMs and iPSC-CMs for drug discovery and toxicity screening, disease modeling, and clinical applications.

Modeling conduction in host-graft interactions between stem cell grafts and cardiomyocytes.

PubMed

Chen, Michael Q; Yu, Jin; Whittington, R Hollis; Wu, Joseph C; Kovacs, Gregory T A; Giovangrandi, Laurent

2009-01-01

Cell therapy has recently made great strides towards aiding heart failure. However, while transplanted cells may electromechanically integrate into host tissue, there may not be a uniform propagation of a depolarization wave between the heterogeneous tissue boundaries. A model using microelectrode array technology that maps the electrical interactions between host and graft tissues in co-culture is presented and sheds light on the effects of having a mismatch of conduction properties at the boundary. Skeletal myoblasts co-cultured with cardiomyocytes demonstrated that conduction velocity significantly decreases at the boundary despite electromechanical coupling. In an attempt to improve the uniformity of conduction with host cells, differentiating human embryonic stem cells (hESC) were used in co-culture. Over the course of four to seven days, synchronous electrical activity was observed at the hESC boundary, implying differentiation and integration. Activity did not extend far past the boundary, and conduction velocity was significantly greater than that of the host tissue, implying the need for other external measures to properly match the conduction properties between host and graft tissue.

Human primordial germ cell formation is diminished by exposure to environmental toxicants acting through the AHR signaling pathway.

PubMed

Kee, Kehkooi; Flores, Martha; Cedars, Marcelle I; Reijo Pera, Renee A

2010-09-01

Historically, effects of environmental toxicants on human development have been deduced via epidemiological studies because direct experimental analysis has not been possible. However, in recent years, the derivation of human pluripotent stem cells has provided a potential experimental system to directly probe human development. Here, we used human embryonic stem cells (hESCs) to study the effect of environmental toxicants on human germ cell development, with a focus on differentiation of the founding population of primordial germ cells (PGCs), which will go on to form the oocytes of the adult. We demonstrate that human PGC numbers are specifically reduced by exposure to polycyclic aromatic hydrocarbons (PAHs), a group of toxicants common in air pollutants released from gasoline combustion or tobacco smoke. Further, we demonstrate that the adverse effects of PAH exposure are mediated through the aromatic hydrocarbon receptor (AHR) and BAX pathway. This study demonstrates the utility of hESCs as a model system for direct examination of the molecular and genetic pathways of environmental toxicants on human germ cell development.

MV-NIS or Investigator's Choice Chemotherapy in Treating Patients With Ovarian, Fallopian, or Peritoneal Cancer

ClinicalTrials.gov

2018-04-27

Fallopian Tube Transitional Cell Carcinoma; Malignant Ovarian Clear Cell Tumor; Malignant Ovarian Endometrioid Tumor; Malignant Ovarian Serous Tumor; Ovarian Seromucinous Carcinoma; Ovarian Transitional Cell Carcinoma; Primary Peritoneal Serous Adenocarcinoma; Recurrent Fallopian Tube Carcinoma; Recurrent Ovarian Carcinoma; Recurrent Primary Peritoneal Carcinoma; Undifferentiated Fallopian Tube Carcinoma; Undifferentiated Ovarian Carcinoma

Undifferentiated Gender Role Orientation, Drinking Motives, and Increased Alcohol Use in Men and Women.

PubMed

Fugitt, Jessica L; Ham, Lindsay S; Bridges, Ana J

2017-05-12

Alcohol misuse has historically affected men more than women. However, the differences in drinking behaviors across sex have steadily decreased over time and accumulating research suggests that gender role orientation, or culturally scripted gender-specific characteristics, and negative reinforcement drinking motives may better explain risk for alcohol use and related problems than sex. The current study tested a mediational model of the undifferentiated orientation (low masculinity and low femininity), an oft neglected orientation despite evidence that it could carry much weight in drinking behaviors, versus the other three gender role orientations, coping and conformity drinking motives, and hazardous alcohol use. Participants were 426 current drinkers over age 21 (41% men; 77.8% Caucasian; M age = 34.5, range = 21-73) residing across the United States who completed an online survey. Structural equation modeling analyses suggested that individuals with an undifferentiated orientation (n = 99), compared to masculine (high masculinity, low femininity; n = 102), feminine (high femininity, low masculinity; n = 113), or androgynous (high masculinity, high femininity; n = 112) orientations, reported higher coping drinking motives, which were positively associated with levels of hazardous alcohol use. Although analyses suggested that undifferentiated individuals reported drinking for conformity motives more often than masculine and androgynous individuals, conformity motives were not associated with increased use. Conclusions/Importance: An undifferentiated gender role orientation may contribute a unique risk for alcohol use and related problems by increasing frequency of drinking to cope, a motive specifically associated with hazardous use trajectories.

Fusion with stem cell makes the hepatocellular carcinoma cells similar to liver tumor-initiating cells.

PubMed

Wang, Ran; Chen, Shuxun; Li, Changxian; Ng, Kevin Tak Pan; Kong, Chi-wing; Cheng, Jinping; Cheng, Shuk Han; Li, Ronald A; Lo, Chung Mau; Man, Kwan; Sun, Dong

2016-02-04

Cell fusion is a fast and highly efficient technique for cells to acquire new properties. The fusion of somatic cells with stem cells can reprogram somatic cells to a pluripotent state. Our research on the fusion of stem cells and cancer cells demonstrates that the fused cells can exhibit stemness and cancer cell-like characteristics. Thus, tumor-initiating cell-like cells are generated. We employed laser-induced single-cell fusion technique to fuse the hepatocellular carcinoma cells and human embryonic stem cells (hESC). Real-time RT-PCR, flow cytometry and in vivo tumorigenicity assay were adopted to identify the gene expression difference. We successfully produced a fused cell line that coalesces the gene expression information of hepatocellular carcinoma cells and stem cells. Experimental results showed that the fused cells expressed cancer and stemness markers as well as exhibited increased resistance to drug treatment and enhanced tumorigenesis. Fusion with stem cells transforms liver cancer cells into tumor initiating-like cells. Results indicate that fusion between cancer cell and stem cell may generate tumor initiating-like cells.

VIP induces the decidualization program and conditions the immunoregulation of the implantation process.

PubMed

Grasso, Esteban; Gori, Soledad; Paparini, Daniel; Soczewski, Elizabeth; Fernández, Laura; Gallino, Lucila; Salamone, Gabriela; Martinez, Gustavo; Irigoyen, Marcela; Ruhlmann, Claudio; Pérez Leirós, Claudia; Ramhorst, Rosanna

2018-01-15

The decidualization process involves phenotype and functional changes on endometrial cells and the modulation of mediators with immunoregulatory properties as the vasoactive intestinal peptide (VIP). We investigate VIP contribution to the decidualization program and to immunoregulation throughout the human embryo implantation process. The decidualization of Human endometrial stromal cell line (HESC) with Medroxyprogesterone-dibutyryl-cAMP increased VIP/VPAC-receptors system. In fact, VIP could induce decidualization increasing differentiation markers (IGFBP1, PRL, KLF13/KLF9 ratio, CXCL12, CXCL8 and CCL2) and allowing Blastocyst-like spheroids (BLS) invasion in an in vitro model of embryo implantation. Focus on the tolerogenic effects, decidualized cells induced a semi-mature profile on maternal dendritic cells; restrained CD4 + cells recruitment while increased regulatory T-cells recruitment. Interestingly, the human blastocyst conditioned media from developmentally impaired embryos diminished the invasion and T-regulatory cells recruitment in these settings. These evidences suggest that VIP contributes to the implantation process inducing decidualization, allowing BLS invasion and favoring a tolerogenic micro-environment. Copyright © 2017 Elsevier B.V. All rights reserved.

Fucoidan promotes early step of cardiac differentiation from human embryonic stem cells and long-term maintenance of beating areas.

PubMed

Hamidi, Sofiane; Letourneur, Didier; Aid-Launais, Rachida; Di Stefano, Antonio; Vainchenker, William; Norol, Françoise; Le Visage, Catherine

2014-04-01

Somatic stem cells require specific niches and three-dimensional scaffolds provide ways to mimic this microenvironment. Here, we studied a scaffold based on Fucoidan, a sulfated polysaccharide known to influence morphogen gradients during embryonic development, to support human embryonic stem cells (hESCs) differentiation toward the cardiac lineage. A macroporous (pore 200 μm) Fucoidan scaffold was selected to support hESCs attachment and proliferation. Using a protocol based on the cardiogenic morphogen bone morphogenic protein 2 (BMP2) and transforming growth factor (TGFβ) followed by tumor necrosis factor (TNFα), an effector of cardiopoietic priming, we examined the cardiac differentiation in the scaffold compared to culture dishes and embryoid bodies (EBs). At day 8, Fucoidan scaffolds supported a significantly higher expression of the 3 genes encoding for transcription factors marking the early step of embryonic cardiac differentiation NKX2.5 (p<0.05), MEF2C (p<0.01), and GATA4 (p<0.01), confirmed by flow cytometry analysis for MEF2C and NKX2.5. The ability of Fucoidan scaffolds to locally concentrate and slowly release TGFβ and TNFα was confirmed by Luminex technology. We also found that Fucoidan scaffolds supported the late stage of embryonic cardiac differentiation marked by a significantly higher atrial natriuretic factor (ANF) expression (p<0.001), although only rare beating areas were observed. We postulated that absence of mechanical stress in the soft hydrogel impaired sarcomere formation, as confirmed by molecular analysis of the cardiac muscle myosin MYH6 and immunohistological staining of sarcomeric α-actinin. Nevertheless, Fucoidan scaffolds contributed to the development of thin filaments connecting beating areas through promotion of smooth muscle cells, thus enabling maintenance of beating areas for up to 6 months. In conclusion, Fucoidan scaffolds appear as a very promising biomaterial to control cardiac differentiation from hESCs that could be further combined with mechanical stress to promote sarcomere formation at terminal stages of differentiation.

Human embryonic stem cell-derived neural crest cells capable of expressing markers of osteochondral or meningeal-choroid plexus differentiation.

PubMed

Sternberg, Hal; Jiang, Jianjie; Sim, Pamela; Kidd, Jennifer; Janus, Jeffrey; Rinon, Ariel; Edgar, Ron; Shitrit, Alina; Larocca, David; Chapman, Karen B; Binette, Francois; West, Michael D

2014-01-01

The transcriptome and fate potential of three diverse human embryonic stem cell-derived clonal embryonic progenitor cell lines with markers of cephalic neural crest are compared when differentiated in the presence of combinations of TGFβ3, BMP4, SCF and HyStem-C matrices. The cell lines E69 and T42 were compared with MEL2, using gene expression microarrays, immunocytochemistry and ELISA. In the undifferentiated progenitor state, each line displayed unique markers of cranial neural crest including TFAP2A and CD24; however, none expressed distal HOX genes including HOXA2 or HOXB2, or the mesenchymal stem cell marker CD74. The lines also showed diverse responses when differentiated in the presence of exogenous BMP4, BMP4 and TGFβ3, SCF, and SCF and TGFβ3. The clones E69 and T42 showed a profound capacity for expression of endochondral ossification markers when differentiated in the presence of BMP4 and TGFβ3, choroid plexus markers in the presence of BMP4 alone, and leptomeningeal markers when differentiated in SCF without TGFβ3. The clones E69 and T42 may represent a scalable source of primitive cranial neural crest cells useful in the study of cranial embryology, and potentially cell-based therapy.

Tuft (caveolated) cells in two human colon carcinoma cell lines.

PubMed

Barkla, D H; Whitehead, R H; Foster, H; Tutton, P J

1988-09-01

The presence of an unusual cell type in two human colon carcinoma cell lines is reported. The cells show the same morphology as "tuft" (caveolated) cells present in normal gastrointestinal epithelium. Tuft cells were seen in cell line LIM 1863 growing in vitro and in human colon carcinoma cell line LIM 2210 growing as subcutaneous solid tumour xenografts in nude mice. Characteristic morphologic features of tuft cells included a wide base, narrow apex and a tuft of long microvilli projecting from the apical surface. The microvilli are attached by a core of long microfilaments passing deep into the apical cytoplasm. Between the microvilli are parallel arrays of vesicles (caveoli) containing flocculent material. Two different but not mutually exclusive explanations for the presence of tuft cells are proposed. The first explanation is that tuft cells came from the resected tumour and have survived by mitotic division during subsequent passages. The second explanation suggests that tuft cells are the progeny of undifferentiated tumour cells. Descriptions of tuft cells in colon carcinomas are uncommon and possible reasons for this are presented. The morphology of tuft cells is consistent with that of a highly differentiated cell specialised for absorption, and these new models provide an opportunity to further investigate the structure and function of tuft cells.

Bevacizumab, Cisplatin, Radiation Therapy, and Fluorouracil in Treating Patients With Stage IIB, Stage III, Stage IVA, or Stage IVB Nasopharyngeal Cancer

ClinicalTrials.gov

2018-01-04

Stage II Nasopharyngeal Keratinizing Squamous Cell Carcinoma AJCC v7; Stage III Nasopharyngeal Keratinizing Squamous Cell Carcinoma AJCC v7; Stage III Nasopharyngeal Undifferentiated Carcinoma AJCC v7; Stage IV Nasopharyngeal Keratinizing Squamous Cell Carcinoma AJCC v7; Stage IV Nasopharyngeal Undifferentiated Carcinoma AJCC v7

A study of murine bone marrow cells cultured in bioreactors which create an environment which simulated microgravity

NASA Technical Reports Server (NTRS)

Lawless, Brother Desales

1990-01-01

Previous research indicated that mouse bone marrow cells could be grown in conditions of simulated microgravity. This environment was created in rotating bioreactor vessels. On three attempts mouse cells were grown successfully in the vessels. The cells reached a stage where the concentrations were doubling daily. Phenotypic analysis using a panel of monoclonal antibodies indicated that the cell were hematopoietic pluripotent stem cells. One unsuccessful attempt was made to reestablish the immune system in immunocompromised mice using these cells. Since last summer, several unsuccessful attempts were made to duplicate these results. It was determined by electron microscopy that the cells successfully grown in 1989 contained virus particles. It was suggested that these virally parasitized cells had been immortalized. The work of this summer is a continuation of efforts to grow mouse bone marrow in these vessels. A number of variations of the protocol were introduced. Certified pathogen free mice were used in the repeat experiments. In some attempts the medium of last summer was used; in others Dexture Culture Medium containing Iscove's Medium supplemented with 20 percent horse serum and 10-6 M hydrocortisone. Efforts this summer were directed solely to repeating the work of last summer. Plans were made for investigations if stem cells were isolated. Immortalization of the undifferentiated stem cell would be attempted by transfection with an oncogenic vector. Selective differentiation would be induced in the stem cell line by growing it with known growth factors and immune response modulators. Interest is in identifying any surface antigens unique to stem cells that would help in their characterization. Another goal was to search for markers on stem cells that would distinguish them from stem cells committed to a particular lineage. If the undifferentiated hematopoietic stem cell was obtained, the pathways that would terminally convert it to myeloid, lyphoid, erythroid, or other cell lines would be studied. Transfection with a known gene would be attempted and then conversion to a terminally identifiable cell.

Migration and Differentiation of GFP-transplanted Bone Marrow-derived Cells into Experimentally Induced Periodontal Polyp in Mice.

PubMed

Matsuda, Saeka; Shoumura, Masahito; Osuga, Naoto; Tsujigiwa, Hidetsugu; Nakano, Keisuke; Okafuji, Norimasa; Ochiai, Takanaga; Hasegawa, Hiromasa; Kawakami, Toshiyuki

2016-01-01

Perforation of floor of the dental pulp is often encountered during root canal treatment in routine clinical practice of dental caries. If perforation were large, granulation tissue would grow to form periodontal polyp. Granulation tissue consists of proliferating cells however their origin is not clear. It was shown that the cells in granulation tissue are mainly from migration of undifferentiated mesenchymal cells of the bone marrow. Hence, this study utilized GFP bone marrow transplantation mouse model. The floor of the pulp chamber in maxillary first molar was perforated using ½ dental round bur. Morphological assessment was carried out by micro CT and microscopy and GFP cell mechanism was further assessed by immunohistochemistry using double fluorescent staining with GFP-S100A4; GFP-Runx2 and GFP-CD31. Results of micro CT revealed alveolar bone resorption and widening of periodontal ligament. Histopathological examination showed proliferation of fibroblasts with some round cells and blood vessels in the granulation tissue. At 2 weeks, the outermost layer of the granulation tissue was lined by squamous cells with distinct intercellular bridges. At 4 weeks, the granulation tissue became larger than the perforation and the outermost layer was lined by relatively typical stratified squamous epithelium. Double immunofluorescent staining of GFP and Runx2 revealed that both proteins were expressed in spindle-shaped cells. Double immunofluorescent staining of GFP and CD31 revealed that both proteins were expressed in vascular endothelial cells in morphologically distinct vessels. The results suggest that fibroblasts, periodontal ligament fibroblasts and blood vessels in granulation tissue were derived from transplanted-bone marrow cells. Thus, essential growth of granulation tissue in periodontal polyp was caused by the migration of undifferentiated mesenchymal cells derived from bone marrow, which differentiated into fibroblasts and later on differentiated into other cells in response to injury.

Undifferentiated carcinoma of the esophagus: a clinicopathological study of 16 cases☆

PubMed Central

Singhi, Aatur D.; Seethala, Raja R.; Nason, Katie; Foxwell, Tyler J.; Roche, Robyn L.; McGrath, Kevin M.; Levy, Ryan M.; Luketich, James D.; Davison, Jon M.

2015-01-01

Summary Undifferentiated carcinoma of the esophagus is a rare histologic variant of esophageal carcinoma. Using criteria based on studies of undifferentiated carcinomas arising at other sites, we have collected 16 cases of resected esophageal undifferentiated carcinomas. Patients ranged in age from 39 to 84 years (mean, 65.5 years) and were predominantly male (94%). The tumors were characterized by an expansile growth pattern of neoplastic cells organized in solid sheets and without significant glandular, squamous, or neuroendocrine differentiation. The neoplastic cells had a syncytial-like appearance, little intervening stroma, and patchy tumor necrosis. In a subset of cases, the tumor cells adopted a sarcomatoid (n = 2), rhabdoid (n = 1), or minor component (<5%) of glandular morphology (n = 3). In 1 case, reactive osteoclast-like giant cells were found interspersed among the neoplastic cells. Lymphovascular invasion, perineural invasion, and lymph node metastases were identified in 88%, 56%, and 81% of cases, respectively. In 12 (75%) specimens, the background esophageal mucosa was notable for Barrett esophagus. Consistent with the epithelial nature of these neoplasms, cytokeratin positivity was identified in all cases. In addition, SALL4 expression was present in 8 (67%) of 12 cases. Follow-up information was available for 15 (94%) of 16 patients, all of whom were deceased. Survival after surgery ranged from 1 to 50 months (mean, 11.9 months). Before death, 67% patients had documented locoregional recurrence and/or distant organ metastases. In summary, esophageal undifferentiated carcinomas are aggressive neoplasms and associated with a high incidence of recurrence and/or metastases and a dismal prognosis. PMID:25582499

Identification of integrin heterodimers functioning on the surface of undifferentiated porcine primed embryonic stem cells.

PubMed

Kim, Hwa-Young; Baek, Song; Han, Na Rae; Lee, Eunsong; Park, Choon-Keun; Lee, Seung Tae

2018-05-29

In vitro expansion of undifferentiated porcine primed embryonic stem (ES) cells is facilitated by use of non-cellular niches that mimic three-dimensional (3D) microenvironments enclosing an inner cell mass of porcine blastocysts. Therefore, we investigated the integrin heterodimers on the surface of undifferentiated porcine primed ES cells for the purpose of developing a non-cellular niche to support in vitro maintenance of the self-renewal ability of porcine primed ES cells. Immunocytochemistry and a fluorescence immunoassay were performed to assess integrin α and β subunit levels, and attachment and antibody inhibition assays were used to evaluate the function of integrin heterodimers. The integrin α 3 , α 5 , α 6 , α 9 , α V , and β 1 subunits, but not the α 1 , α 2 , α 4 , α 7 , and α 8 subunits, were identified on the surface of undifferentiated porcine primed ES cells. Subsequently, significant increase of their adhesion to fibronectin, tenascin C and vitronectin were observed and functional blocking of integrin heterodimer α 5 β 1 , α 9 β 1 , or α V β 1 showed significantly inhibited adhesion to fibronectin, tenascin C, or vitronectin. No integrin α 6 β 1 heterodimer?mediated adhesion to laminin was detected. These results demonstrate that active α 5 β 1 , α 9 β 1 , and α V β 1 integrin heterodimers are present on the surface of undifferentiated porcine primed ES cells, together with inactive integrin α 3 (presumed) and α 6 subunits. This article is protected by copyright. All rights reserved.

Identification and genotyping of human papillomavirus in a Spanish cohort of penile squamous cell carcinomas: correlation with pathologic subtypes, p16(INK4a) expression, and prognosis.

PubMed

Ferrándiz-Pulido, Carla; Masferrer, Emili; de Torres, Ines; Lloveras, Belen; Hernandez-Losa, Javier; Mojal, Sergio; Salvador, Carlos; Morote, Juan; Ramon y Cajal, Santiago; Pujol, Ramon M; Garcia-Patos, Vicente; Toll, Agustin

2013-01-01

Penile squamous cell carcinoma (PSCC) is a tumor with a high metastatic potential. In PSCC the attributable fraction to human papillomavirus (HPV) is not well established. We sought to provide novel data about the prevalence of HPV in a large series of penile intraepithelial neoplasia (PeIN) and invasive PSCC, correlating the results with the histologic subtype, p16(INK4a) immunostaining, and prognosis. A total of 82 PSCC were included in the study, 69 invasive and 13 PeIN. HPV detection was performed by polymerase chain reaction with SPF-10 broad-spectrum primers followed by DNA enzyme immunoassay and genotyping with a reverse hybridization line probe assay. P16(INK4a) immunohistochemical expression on tissue microarrays was also analyzed. HPV DNA was identified in 31 of 77 (40.2%) PSCC (22 of 67 invasive and 9 of 10 PeIN). In 25 of 31 (80.6%) cases HPV-16 was identified. HPV detection was significantly associated with some histologic subtypes: most basaloid and warty tumors were high-risk HPV (hrHPV) positive, whereas only 15% of usual PSCC were hr-HPV positive. All hrHPV-positive PSCC had an adjacent undifferentiated PeIN. Strong p16(INK4a) immunostaining correlated with hrHPV infection. Most undifferentiated PeIN showed p16(INK4a) immunohistochemical overexpression. Both hrHPV-positive and p16(INK4a)-positive tumors showed a better overall survival without reaching statistical significance. This was a retrospective study. Our results suggest that most hrHPV-positive PSCC develop from undifferentiated hrHPV-positive PeIN. P16(INK4a) immunostaining may be useful in identifying both etiologically related hrHPV-positive tumors and those with better outcome. The routine use of p16(INK4a) staining should be incorporated in histologic evaluation of PSCC. Copyright © 2012 American Academy of Dermatology, Inc. Published by Mosby, Inc. All rights reserved.

Pembrolizumab, Bevacizumab, and Cyclophosphamide in Treating Patients With Recurrent Ovarian, Fallopian Tube, or Primary Peritoneal Cancer

ClinicalTrials.gov

2018-05-03

Fallopian Tube Clear Cell Adenocarcinoma; Fallopian Tube Endometrioid Adenocarcinoma; Fallopian Tube Mucinous Adenocarcinoma; Fallopian Tube Serous Adenocarcinoma; Ovarian Clear Cell Adenocarcinoma; Ovarian Endometrioid Adenocarcinoma; Ovarian Mucinous Adenocarcinoma; Ovarian Serous Adenocarcinoma; Primary Peritoneal Serous Adenocarcinoma; Recurrent Fallopian Tube Carcinoma; Recurrent Ovarian Carcinoma; Recurrent Primary Peritoneal Carcinoma; Undifferentiated Fallopian Tube Carcinoma; Undifferentiated Ovarian Carcinoma

Triple-phase helical computed tomography in dogs with solid splenic masses

PubMed Central

KUTARA, Kenji; SEKI, Mamiko; ISHIGAKI, Kumiko; TESHIMA, Kenji; ISHIKAWA, Chieko; KAGAWA, Yumiko; EDAMURA, Kazuya; NAKAYAMA, Tomohiro; ASANO, Kazushi

2017-01-01

We investigated the utility of triple-phase helical computed tomography (CT) in differentiating between benign and malignant splenic masses in dogs. Forty-two dogs with primary splenic masses underwent triple-phase helical CT scanning (before administration of contrast, and in the arterial phase, portal venous phase, and delayed phase) prior to splenectomy. Tissue specimens were sent for pathological diagnosis; these included hematomas (n=14), nodular hyperplasias (n=12), hemangiosarcomas (n=11), and undifferentiated sarcomas (n=5). The CT findings were compared with the histological findings. Nodular hyperplasia significantly displayed a homogeneous normal enhancement pattern in all phases. Hemangiosarcoma displayed 2 significant contrast-enhancement patterns, including a homogeneous pattern of poor enhancement in all phases, and a heterogeneous remarkable enhancement pattern in the arterial and portal venous phases. Hematoma and undifferentiated sarcoma displayed a heterogeneous normal enhancement pattern in all phases. The contrast-enhanced volumetric ratios of hematoma tended to be greater than those of undifferentiated sarcoma. Our study demonstrated that the characteristic findings on triple-phase helical CT could be useful for the preoperative differentiation of hematoma, nodular hyperplasia, hemangiosarcoma, and undifferentiated sarcoma in dogs. Triple-phase helical CT may be a useful diagnostic tool in dogs with splenic masses. PMID:28993600

Paclitaxel Albumin-Stabilized Nanoparticle Formulation and Bevacizumab in Treating Patients With Stage IV Melanoma That Cannot Be Removed by Surgery or Gynecological Cancers

ClinicalTrials.gov

2018-02-05

Cervical Adenosarcoma; Cervical Adenosquamous Carcinoma; Cervical Carcinosarcoma; Cervical Squamous Cell Carcinoma, Not Otherwise Specified; Endometrial Clear Cell Adenocarcinoma; Endometrial Endometrioid Adenocarcinoma; Endometrial Mixed Adenocarcinoma; Endometrial Mucinous Adenocarcinoma; Endometrial Squamous Cell Carcinoma; Endometrial Transitional Cell Carcinoma; Endometrial Undifferentiated Carcinoma; Fallopian Tube Adenocarcinoma; Fallopian Tube Clear Cell Adenocarcinoma; Fallopian Tube Mucinous Adenocarcinoma; Fallopian Tube Serous Adenocarcinoma; Fallopian Tube Transitional Cell Carcinoma; Malignant Ovarian Epithelial Tumor; Malignant Peritoneal Neoplasm; Ovarian Carcinosarcoma; Ovarian Clear Cell Adenocarcinoma; Ovarian Endometrioid Adenocarcinoma; Ovarian Mucinous Adenocarcinoma; Ovarian Serous Adenocarcinoma; Ovarian Transitional Cell Carcinoma; Primary Peritoneal Serous Adenocarcinoma; Recurrent Fallopian Tube Carcinoma; Recurrent Melanoma; Recurrent Ovarian Carcinoma; Recurrent Primary Peritoneal Carcinoma; Stage IV Skin Melanoma; Undifferentiated Fallopian Tube Carcinoma; Undifferentiated Ovarian Carcinoma; Uterine Corpus Carcinosarcoma

Megakaryoblastic leukemia in a dog.

PubMed

Pucheu-Haston, C M; Camus, A; Taboada, J; Gaunt, S D; Snider, T G; Lopez, M K

1995-07-15

A 7-year-old spayed Louisiana Catahoula Leopard dog was examined to determine the cause of shifting forelimb lameness, anorexia, and lethargy. The dog was pyrectic and had splenomegaly, thrombocytopenia, and nonregenerative anemia. Examination of a bone marrow aspirate revealed hypocellularity with normal maturation of erythroid and granulocytic cell lines; however, approximately half of the cells were large undifferentiated blast cells. These cells were identified as megakaryoblasts, using immunohistochemical techniques to detect reactivity for Factor VIII-related antigen and platelet glycoprotein IIIa. Necropsy revealed diffuse neoplastic involvement of the spleen, liver, lungs, bone marrow, and lymph nodes. Cellular infiltrate was characterized by a mixture of megakaryoblasts and typical megakaryocytes. Megakaryoblastic leukemia (M7) is the designation proposed by the Animal Leukemia Study Group for myeloproliferative neoplasms of megakaryocytic lineage.

Optimising parameters for the differentiation of SH-SY5Y cells to study cell adhesion and cell migration.

PubMed

Dwane, Susan; Durack, Edel; Kiely, Patrick A

2013-09-11

Cell migration is a fundamental biological process and has an important role in the developing brain by regulating a highly specific pattern of connections between nerve cells. Cell migration is required for axonal guidance and neurite outgrowth and involves a series of highly co-ordinated and overlapping signalling pathways. The non-receptor tyrosine kinase, Focal Adhesion Kinase (FAK) has an essential role in development and is the most highly expressed kinase in the developing CNS. FAK activity is essential for neuronal cell adhesion and migration. The objective of this study was to optimise a protocol for the differentiation of the neuroblastoma cell line, SH-SY5Y. We determined the optimal extracellular matrix proteins and growth factor combinations required for the optimal differentiation of SH-SY5Y cells into neuronal-like cells and determined those conditions that induce the expression of FAK. It was confirmed that the cells were morphologically and biochemically differentiated when compared to undifferentiated cells. This is in direct contrast to commonly used differentiation methods that induce morphological differentiation but not biochemical differentiation. We conclude that we have optimised a protocol for the differentiation of SH-SY5Y cells that results in a cell population that is both morphologically and biochemically distinct from undifferentiated SH-SY5Y cells and has a distinct adhesion and spreading pattern and display extensive neurite outgrowth. This protocol will provide a neuronal model system for studying FAK activity during cell adhesion and migration events.

Infection and Propagation of Human Rhinovirus C in Human Airway Epithelial Cells

PubMed Central

Hao, Weidong; Bernard, Katie; Patel, Nita; Ulbrandt, Nancy; Feng, Hui; Svabek, Catherine; Wilson, Susan; Stracener, Christina; Wang, Kathy; Suzich, JoAnn; Blair, Wade

2012-01-01

Human rhinovirus species C (HRV-C) was recently discovered using molecular diagnostic techniques and is associated with lower respiratory tract disease, particularly in children. HRV-C cannot be propagated in immortalized cell lines, and currently sinus organ culture is the only system described that is permissive to HRV-C infection ex vivo. However, the utility of organ culture for studying HRV-C biology is limited. Here, we report that a previously described HRV-C derived from an infectious cDNA, HRV-C15, infects and propagates in fully differentiated human airway epithelial cells but not in undifferentiated cells. We demonstrate that this differentiated epithelial cell culture system supports infection and replication of a second virus generated from a cDNA clone, HRV-C11. We show that HRV-C15 virions preferentially bind fully differentiated airway epithelial cells, suggesting that the block to replication in undifferentiated cells is at the step of viral entry. Consistent with previous reports, HRV-C15 utilizes a cellular receptor other than ICAM-1 or LDLR for infection of differentiated epithelial cells. Furthermore, we demonstrate that HRV-C15 replication can be inhibited by an HRV 3C protease inhibitor (rupintrivir) but not an HRV capsid inhibitor previously under clinical development (pleconaril). The HRV-C cell culture system described here provides a powerful tool for studying the biology of HRV-C and the discovery and development of HRV-C inhibitors. PMID:23035218

Cryopreservation of putative pre-pubertal bovine spermatogonial stem cells by slow freezing.

PubMed

Kim, Ki-Jung; Lee, Yong-An; Kim, Bang-Jin; Kim, Yong-Hee; Kim, Byung-Gak; Kang, Hyun-Gu; Jung, Sang-Eun; Choi, Sun-Ho; Schmidt, Jonathan A; Ryu, Buom-Yong

2015-04-01

Development of techniques for the preservation of mammalian spermatogonial stem cells (SSCs) is a critical step in commercial application of SSC based technologies, including species preservation, amplification of agriculturally valuable germ lines, and human fertility preservations. The objective of this study was to develop an efficient cryopreservation protocol for preservation of bovine SSCs using a slow freezing technique. To maximize the efficiency of SSC cryopreservation, the effects of various methods (tissue vs. cell freezing) and cryoprotective agents (trehalose, sucrose, and polyethylene glycol [PEG]) were tested. Following thawing, cells were enriched for undifferentiated spermatogonia by differential plating and evaluated for recovery rate, proliferation capacity, and apoptosis. Additionally, putative stem cell activity was assessed using SSC xenotransplantation. The recovery rate, and proliferation capacity of undifferentiated spermatogonia were significantly greater for germ cells frozen using tissue freezing methods compared to cell freezing methods. Cryopreservation in the presence of 200 mM trehalose resulted in significantly greater recovery rate, proliferation capacity, and apoptosis of germ cells compared to control. Furthermore, cryopreservation using the tissue freezing method in the presence of 200 mM trehalose resulted in the production of colonies of donor-derived germ cells after xenotransplantation into recipient mouse testes, indicating putative stem cell function. Collectively, these data indicate that cryopreservation using tissue freezing methods in the presence of 200 mM trehalose is an efficient cryopreservation protocol for bovine SSCs. Copyright © 2015 Elsevier Inc. All rights reserved.

Effect of in vitro chondrogenic differentiation of autologous mesenchymal stem cells on cartilage and subchondral cancellous bone repair in osteoarthritis of temporomandibular joint.

PubMed

Chen, K; Man, C; Zhang, B; Hu, J; Zhu, S S

2013-02-01

This study investigated the effects of in vitro chondrogenic differentiated mesenchymal stem cells (MSCs) on cartilage and subchondral cancellous bone in temporomandibular joint osteoarthritis (TMJOA). Four weeks after induction of osteoarthritis (OA), the joints received hylartin solution, non-chondrogenic MSCs or in vitro chondrogenic differentiated MSCs. The changes in cartilage and subchondral cancellous bone were evaluated by histology, reverse transcription polymerase chain reaction and micro-computed tomography (CT). Implanted cells were tracked using Adeno-LacZ labelling. The differentiated MSC-treated group had better histology than the MSC-treated group at 4 and 12 weeks, but no difference at 24 weeks. Increased mRNA expression of collegan II, aggeran, Sox9 and decreased matrix metalloproteinase 13 (MMP13) were observed in differentiated MSC-treated groups compared to the undifferentiated MSC-treated group at 4 weeks. The differentiated MSC-treated group had decreased bone volume fraction, trabecular thickness and bone surface density, and increased trabecular spacing in the subchondral cancellous bone than the undifferentiated MSC-treated group. Transplanted cells were observed at cartilage, subchondral bone, and the synovial membrane lining at 4 weeks. Intra-articular injection of MSCs could delay the progression of TMJOA, and in vitro chondrogenic induction of MSCs could enhance the therapeutic effects. This provides new insights into the role of MSCs in cell-based therapies for TMJOA. Copyright © 2012 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

Pegylated Liposomal Doxorubicin Hydrochloride With Atezolizumab and/or Bevacizumab in Treating Patients With Recurrent Ovarian, Fallopian Tube, or Primary Peritoneal Cancer

ClinicalTrials.gov

2018-06-20

Fallopian Tube Clear Cell Adenocarcinoma; Fallopian Tube Endometrioid Adenocarcinoma; High Grade Fallopian Tube Serous Adenocarcinoma; High Grade Ovarian Serous Adenocarcinoma; Ovarian Clear Cell Adenocarcinoma; Ovarian Endometrioid Adenocarcinoma; Ovarian Seromucinous Carcinoma; Primary Peritoneal High Grade Serous Adenocarcinoma; Recurrent Fallopian Tube Carcinoma; Recurrent Ovarian Carcinoma; Recurrent Primary Peritoneal Carcinoma; Undifferentiated Fallopian Tube Carcinoma; Undifferentiated Ovarian Carcinoma

Maturation of Human Embryonic Stem Cell–Derived Pancreatic Progenitors Into Functional Islets Capable of Treating Pre-existing Diabetes in Mice

PubMed Central

Rezania, Alireza; Bruin, Jennifer E.; Riedel, Michael J.; Mojibian, Majid; Asadi, Ali; Xu, Jean; Gauvin, Rebecca; Narayan, Kavitha; Karanu, Francis; O’Neil, John J.; Ao, Ziliang; Warnock, Garth L.

2012-01-01

Diabetes is a chronic debilitating disease that results from insufficient production of insulin from pancreatic β-cells. Islet cell replacement can effectively treat diabetes but is currently severely limited by the reliance upon cadaveric donor tissue. We have developed a protocol to efficiently differentiate commercially available human embryonic stem cells (hESCs) in vitro into a highly enriched PDX1+ pancreatic progenitor cell population that further develops in vivo to mature pancreatic endocrine cells. Immature pancreatic precursor cells were transplanted into immunodeficient mice with streptozotocin-induced diabetes, and glycemia was initially controlled with exogenous insulin. As graft-derived insulin levels increased over time, diabetic mice were weaned from exogenous insulin and human C-peptide secretion was eventually regulated by meal and glucose challenges. Similar differentiation of pancreatic precursor cells was observed after transplant in immunodeficient rats. Throughout the in vivo maturation period hESC-derived endocrine cells exhibited gene and protein expression profiles that were remarkably similar to the developing human fetal pancreas. Our findings support the feasibility of using differentiated hESCs as an alternative to cadaveric islets for treating patients with diabetes. PMID:22740171

Metabolome Profiling of Partial and Fully Reprogrammed Induced Pluripotent Stem Cells.

PubMed

Park, Soon-Jung; Lee, Sang A; Prasain, Nutan; Bae, Daekyeong; Kang, Hyunsu; Ha, Taewon; Kim, Jong Soo; Hong, Ki-Sung; Mantel, Charlie; Moon, Sung-Hwan; Broxmeyer, Hal E; Lee, Man Ryul

2017-05-15

Acquisition of proper metabolomic fate is required to convert somatic cells toward fully reprogrammed pluripotent stem cells. The majority of induced pluripotent stem cells (iPSCs) are partially reprogrammed and have a transcriptome different from that of the pluripotent stem cells. The metabolomic profile and mitochondrial metabolic functions required to achieve full reprogramming of somatic cells to iPSC status have not yet been elucidated. Clarification of the metabolites underlying reprogramming mechanisms should enable further optimization to enhance the efficiency of obtaining fully reprogrammed iPSCs. In this study, we characterized the metabolites of human fully reprogrammed iPSCs, partially reprogrammed iPSCs, and embryonic stem cells (ESCs). Using capillary electrophoresis time-of-flight mass spectrometry-based metabolomics, we found that 89% of analyzed metabolites were similarly expressed in fully reprogrammed iPSCs and human ESCs (hESCs), whereas partially reprogrammed iPSCs shared only 74% similarly expressed metabolites with hESCs. Metabolomic profiling analysis suggested that converting mitochondrial respiration to glycolytic flux is critical for reprogramming of somatic cells into fully reprogrammed iPSCs. This characterization of metabolic reprogramming in iPSCs may enable the development of new reprogramming parameters for enhancing the generation of fully reprogrammed human iPSCs.

A quality-by-design approach to risk reduction and optimization for human embryonic stem cell cryopreservation processes.

PubMed

Mitchell, Peter D; Ratcliffe, Elizabeth; Hourd, Paul; Williams, David J; Thomas, Robert J

2014-12-01

It is well documented that cryopreservation and resuscitation of human embryonic stem cells (hESCs) is complex and ill-defined, and often suffers poor cell recovery and increased levels of undesirable cell differentiation. In this study we have applied Quality-by-Design (QbD) concepts to the critical processes of slow-freeze cryopreservation and resuscitation of hESC colony cultures. Optimized subprocesses were linked together to deliver a controlled complete process. We have demonstrated a rapid, high-throughput, and stable system for measurement of cell adherence and viability as robust markers of in-process and postrecovery cell state. We observed that measurement of adherence and viability of adhered cells at 1 h postseeding was predictive of cell proliferative ability up to 96 h in this system. Application of factorial design defined the operating spaces for cryopreservation and resuscitation, critically linking the performance of these two processes. Optimization of both processes resulted in enhanced reattachment and post-thaw viability, resulting in substantially greater recovery of cryopreserved, pluripotent cell colonies. This study demonstrates the importance of QbD concepts and tools for rapid, robust, and low-risk process design that can inform manufacturing controls and logistics.

Deliberative Democracy and stem cell research in New York State: the good, the bad, and the ugly.

PubMed

Sulmasy, Daniel P

2009-03-01

Many states in the U.S. have adopted policies regarding human embryonic stem cell (hESC) research in the last few years. Some have arrived at these policies through legislative debate, some by referendum, and some by executive order. New York has chosen a unique structure for addressing policy decisions regarding this morally controversial issue by creating the Empire State Stem Cell Board with two Committees--an Ethics Committee and a Funding Committee. This essay explores the pros and cons of various policy arrangements for making public policy decisions about morally controversial issues in bioethics (as well as other issues) through the lens of Deliberative Democracy, focusing on the principles of reciprocity, publicity, and accountability. Although New York's unique mechanism potentially offers an opportunity to make policy decisions regarding a morally controversial subject like hESC research in accord with the principles of Deliberative Democracy, this essay demonstrates its failure to do so in actual fact. A few relatively simple changes could make New York's program a real model for putting Deliberative Democracy into practice in making policy decisions regarding controversial bioethical issues.

In vivo selection of human embryonic stem cell-derived cells expressing methotrexate-resistant dihydrofolate reductase

PubMed Central

Gori, Jennifer L.; Tian, Xinghui; Swanson, Debra; Gunther, Roland; Shultz, Leonard D.; McIvor, R. Scott; Kaufman, Dan S.

2009-01-01

SUMMARY Human embryonic stem cells (hESCs) provide a novel source of hematopoietic and other cell populations suitable for gene therapy applications. Preclinical studies to evaluate engraftment of hESC-derived hematopoietic cells transplanted into immunodeficient mice demonstrate only limited repopulation. Expression of a drug resistance gene, such as Tyr22-dihydrofolate reductase (Tyr22-DHFR), coupled to methotrexate (MTX) chemotherapy has the potential to selectively increase engraftment of gene-modified hESC-derived cells in mouse xenografts. Here, we describe the generation of Tyr22-DHFR – GFP expressing hESCs that maintain pluripotency, produce teratomas and can differentiate into MTXr-hemato-endothelial cells. We demonstrate that MTX administered to nonobese diabetic/severe combined immunodeficient/IL-2Rγcnull (NSG) mice after injection of Tyr22-DHFR-derived cells significantly increases human CD34+ and CD45+ cell engraftment in the bone marrow (BM) and peripheral blood of transplanted MTX-treated mice. These results demonstrate that MTX treatment supports selective, long-term engraftment of Tyr22-DHFR-cells in vivo, and provides a novel approach for combined human cell and gene therapy. PMID:19829316

Different regulation of aryl hydrocarbon receptor-regulated genes in response to dioxin in undifferentiated and neuronally differentiated human neuroblastoma SH-SY5Y cells.

PubMed

Imran, Saima; Ferretti, Patrizia; Vrzal, Radim

2015-01-01

Some environmental pollutants derived from industrial processes have been suggested to be responsible for neurological impairment in children, especially in heavily polluted areas. Since these compounds are usually activators of aryl hydrocarbon receptor (AhR), it would be important to better understand the molecular pathways downstream of AhR leading to neural deficits. To this purpose, appropriate in vitro human neural model is much needed. Here we have investigated whether undifferentiated and neuronally differentiated human neuroblastoma cells, SH-SY5Y cells, can provide a suitable model for monitoring AhR activity induced by environmental pollutants, focusing on 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD), a known activator of AhR. Further characterization of differentiated SH-SY5Y showed an increase in AhRR (aryl hydrocarbon receptor repressor), no change in ARNT1 (AhR nuclear translocator 1), and a decrease in ARNT2 expression with differentiation; in contrast, AhR was undetectable in both undifferentiated and differentiated cells. Nonetheless, treatment of parental as well as differentiated SH-SY5Y cells with TCDD resulted in the induction of AhR-regulated genes, CYP1A1 and CYP1B1; AhRR expression was also affected, but to a much smaller extent. These results indicate that undifferentiated SH-SY5Y are less sensitive to TCDD than neuronally differentiated ones, suggesting a higher resistance of the undifferentiated tumor cells to toxic insults. They also suggest that TCDD in these cells may not act via direct activation of AhR that is undetectable in SH-SY5Y as well as in differentiated neurons. Hence, these cells do not provide an appropriate model for studying ligand-mediated activation of AhR.

NUTM1 Gene Fusions Characterize a Subset of Undifferentiated Soft Tissue and Visceral Tumors.

PubMed

Dickson, Brendan C; Sung, Yun-Shao; Rosenblum, Marc K; Reuter, Victor E; Harb, Mohammed; Wunder, Jay S; Swanson, David; Antonescu, Cristina R

2018-05-01

NUT midline carcinoma is an aggressive tumor that occurs mainly in the head and neck and, less frequently, the mediastinum and lung. Following identification of an index case of a NUTM1 fusion positive undifferentiated soft tissue tumor, we interrogated additional cases of primary undifferentiated soft tissue and visceral tumors for NUTM1 abnormalities. Targeted next-generation sequencing was performed on RNA extracted from formalin-fixed paraffin-embedded tissue, and results validated by fluorescence in situ hybridization using custom bacterial artificial chromosome probes. Six patients were identified: mean age of 42 years (range, 3 to 71 y); equal sex distribution; and, tumors involved the extremity soft tissues (N=2), kidney (N=2), stomach, and brain. On systemic work-up at presentation all patients lacked a distant primary tumor. Morphologically, the tumors were heterogenous, with undifferentiated round-epithelioid-rhabdoid cells arranged in solid sheets, nests, and cords. Mitotic activity was generally brisk. Four cases expressed pancytokeratin, but in only 2 cases was this diffuse. Next-generation sequencing demonstrated the following fusions: BRD4-NUTM1 (3 cases), BRD3-NUTM1, MXD1-NUTM1, and BCORL1-NUTM1. Independent testing by fluorescence in situ hybridization confirmed the presence of NUTM1 and partner gene rearrangement. This study establishes that NUT-associated tumors transgress the midline and account for a subset of primitive neoplasms occurring in soft tissue and viscera. Tumors harboring NUTM1 gene fusions are presumably underrecognized, and the extent to which they account for undifferentiated mesenchymal, neuroendocrine, and/or epithelial neoplasms is unclear. Moreover, the relationship, if any, between NUT-associated tumors in soft tissue and/or viscera, and conventional NUT carcinoma, remains to be elucidated.

Nivolumab and Ipilimumab in Treating Patients With Rare Tumors

ClinicalTrials.gov

2018-06-27

Acinar Cell Carcinoma; Adenoid Cystic Carcinoma; Adrenal Cortex Carcinoma; Adrenal Gland Pheochromocytoma; Anal Canal Neuroendocrine Carcinoma; Anal Canal Undifferentiated Carcinoma; Appendix Mucinous Adenocarcinoma; Bartholin Gland Transitional Cell Carcinoma; Bladder Adenocarcinoma; Cervical Adenocarcinoma; Cholangiocarcinoma; Chordoma; Colorectal Squamous Cell Carcinoma; Desmoid-Type Fibromatosis; Endometrial Transitional Cell Carcinoma; Endometrioid Adenocarcinoma; Esophageal Neuroendocrine Carcinoma; Esophageal Undifferentiated Carcinoma; Extrahepatic Bile Duct Carcinoma; Fallopian Tube Adenocarcinoma; Fallopian Tube Transitional Cell Carcinoma; Fibromyxoid Tumor; Gastric Neuroendocrine Carcinoma; Gastric Squamous Cell Carcinoma; Gastrointestinal Stromal Tumor; Giant Cell Carcinoma; Intestinal Neuroendocrine Carcinoma; Intrahepatic Cholangiocarcinoma; Lung Carcinoid Tumor; Lung Sarcomatoid Carcinoma; Major Salivary Gland Carcinoma; Malignant Odontogenic Neoplasm; Malignant Peripheral Nerve Sheath Tumor; Malignant Testicular Sex Cord-Stromal Tumor; Metaplastic Breast Carcinoma; Metastatic Malignant Neoplasm of Unknown Primary Origin; Minimally Invasive Lung Adenocarcinoma; Mixed Mesodermal (Mullerian) Tumor; Mucinous Adenocarcinoma; Mucinous Cystadenocarcinoma; Nasal Cavity Adenocarcinoma; Nasal Cavity Carcinoma; Nasopharyngeal Carcinoma; Nasopharyngeal Papillary Adenocarcinoma; Nasopharyngeal Undifferentiated Carcinoma; Oral Cavity Carcinoma; Oropharyngeal Undifferentiated Carcinoma; Ovarian Adenocarcinoma; Ovarian Germ Cell Tumor; Ovarian Mucinous Adenocarcinoma; Ovarian Squamous Cell Carcinoma; Ovarian Transitional Cell Carcinoma; Pancreatic Acinar Cell Carcinoma; Pancreatic Neuroendocrine Carcinoma; Paraganglioma; Paranasal Sinus Adenocarcinoma; Paranasal Sinus Carcinoma; Parathyroid Gland Carcinoma; Pituitary Gland Carcinoma; Placental Choriocarcinoma; Placental-Site Gestational Trophoblastic Tumor; Primary Peritoneal High Grade Serous Adenocarcinoma; Pseudomyxoma Peritonei; Rare Disorder; Scrotal Squamous Cell Carcinoma; Seminal Vesicle Adenocarcinoma; Seminoma; Serous Cystadenocarcinoma; Small Intestinal Adenocarcinoma; Small Intestinal Squamous Cell Carcinoma; Spindle Cell Neoplasm; Squamous Cell Carcinoma of the Penis; Teratoma With Malignant Transformation; Testicular Non-Seminomatous Germ Cell Tumor; Thyroid Gland Carcinoma; Tracheal Carcinoma; Transitional Cell Carcinoma; Undifferentiated Gastric Carcinoma; Ureter Adenocarcinoma; Ureter Squamous Cell Carcinoma; Urethral Adenocarcinoma; Urethral Squamous Cell Carcinoma; Vaginal Adenocarcinoma; Vaginal Squamous Cell Carcinoma, Not Otherwise Specified; Vulvar Carcinoma

BCOR-CCNB3 Fusions Are Frequent in Undifferentiated Sarcomas of Male Children

PubMed Central

Peters, Tricia L.; Kumar, Vijetha; Polikepahad, Sumanth; Lin, Frank Y.; Sarabia, Stephen F.; Liang, Yu; Wang, Wei-Lien; Lazar, Alexander J.; Doddapaneni, Harsha Vardhan; Chao, Hsu; Muzny, Donna M.; Wheeler, David A.; Okcu, M. Fatih; Plon, Sharon E.; Hicks, M. John; López-Terrada, Dolores; Parsons, D. Williams; Roy, Angshumoy

2014-01-01

The BCOR-CCNB3 fusion gene, resulting from a chromosome X paracentric inversion, was recently described in translocation-negative ‘Ewing-like’ sarcomas arising in bone and soft tissue. Genetic subclassification of undifferentiated unclassified sarcomas may potentially offer markers for reproducible diagnosis and substrates for therapy. Using whole transcriptome paired end RNA sequencing (RNA-seq) we unexpectedly identified BCOR-CCNB3 fusion transcripts in an undifferentiated spindle cell sarcoma. RNA-seq results were confirmed through direct RT-PCR of tumor RNA and cloning of the genomic breakpoints from tumor DNA. Five additional undifferentiated sarcomas with BCOR-CCNB3 fusions were identified in a series of 42 pediatric and adult unclassified sarcomas. Genomic breakpoint analysis demonstrated unique breakpoint locations in each case at the DNA level even though the resulting fusion mRNA was identical in all cases. All patients with BCOR-CCNB3 sarcoma were males diagnosed in mid-childhood (7-13 years of age). Tumors were equally distributed between axial and extra-axial locations. Five of the six tumors were soft tissue lesions with either predominant spindle cell morphology or spindle cell areas interspersed with ovoid to round cells. CCNB3 immunohistochemistry showed strong nuclear positivity in 5 tumors prior to oncologic therapy, but was patchy to negative in post-treatment tumor samples. An RT-PCR assay developed to detect the fusion transcript in archival formalin-fixed tissue was positive in all 6 cases, with high sensitivity and specificity in both pre- and post-treated samples. This study adds to recent reports on the clinicopathologic spectrum of BCOR-CCNB3 fusion-positive sarcomas, a newly-emerging entity within the undifferentiated unclassified sarcoma category, and describes a simple RT-PCR assay that in conjunction with CCNB3 immunohistochemistry can be useful in diagnosing these tumors. PMID:25360585

Down-regulated RPS3a/nbl expression during retinoid-induced differentiation of HL-60 cells: a close association with diminished susceptibility to actinomycin D-stimulated apoptosis.

PubMed

Russell, L; Naora, H; Naora, H

2000-04-01

The efficacy of anticancer agents significantly depends on the differential susceptibility of undifferentiated cancer cells and differentiated normal cells to undergo apoptosis. We previously found that enhanced expression of RPS3a/nbl, which apparently encodes a ribosomal protein, seems to prime cells for apoptosis, while suppressing such enhanced expression triggers cell death. The present study found that HL-60 cells induced to differentiate by all-trans retinoic acid did not undergo apoptosis following treatment with actinomycin D whereas undifferentiated HL-60 cells were highly apoptosis-susceptible, confirming earlier suggestions that differentiated cells have diminished apoptosis-susceptibility. Undifferentiated HL-60 cells highly expressed RPS3a/nbl whereas all-trans retinoic acid -induced differentiated cells exhibited markedly reduced levels, suggesting that apoptosis-resistance of differentiated cells could be due to low RPS3a/nbl expression. Down-regulation of enhanced RPS3a/nbl expression was also observed in cells induced to differentiate with the retinoid 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-napthalenyl)-1- propenyl]benzoic acid without any significant induction of cell death. While down-regulation of RPS3a/nbl expression during differentiation did not apparently induce apoptosis, RPS3a/nbl antisense oligomers triggered death of undifferentiated HL-60 cells, but not of retinoid-induced differentiated cells. It therefore seems that while down-regulation of enhanced RPS3a/nbl expression can induce apoptosis in undifferentiated cells, down-regulation of enhanced RPS3a/nbl expression during differentiation occurs independently of apoptosis, and could be regarded as reverting the primed condition to the unprimed (low RPS3a/nbl) state.

mTORC1 is essential for leukemia propagation but not stem cell self-renewal

PubMed Central

Hoshii, Takayuki; Tadokoro, Yuko; Naka, Kazuhito; Ooshio, Takako; Muraguchi, Teruyuki; Sugiyama, Naoyuki; Soga, Tomoyoshi; Araki, Kimi; Yamamura, Ken-ichi; Hirao, Atsushi

2012-01-01

Although dysregulation of mTOR complex 1 (mTORC1) promotes leukemogenesis, how mTORC1 affects established leukemia is unclear. We investigated the role of mTORC1 in mouse hematopoiesis using a mouse model of conditional deletion of Raptor, an essential component of mTORC1. Raptor deficiency impaired granulocyte and B cell development but did not alter survival or proliferation of hematopoietic progenitor cells. In a mouse model of acute myeloid leukemia (AML), Raptor deficiency significantly suppressed leukemia progression by causing apoptosis of differentiated, but not undifferentiated, leukemia cells. mTORC1 did not control cell cycle or cell growth in undifferentiated AML cells in vivo. Transplantation of Raptor-deficient undifferentiated AML cells in a limiting dilution revealed that mTORC1 is essential for leukemia initiation. Strikingly, a subset of AML cells with undifferentiated phenotypes survived long-term in the absence of mTORC1 activity. We further demonstrated that the reactivation of mTORC1 in those cells restored their leukemia-initiating capacity. Thus, AML cells lacking mTORC1 activity can self-renew as AML stem cells. Our findings provide mechanistic insight into how residual tumor cells circumvent anticancer therapies and drive tumor recurrence. PMID:22622041

PI3 K/Akt/mTOR-mediated translational control regulates proliferation and differentiation of lineage-restricted RoSH stem cell lines

PubMed Central

Que, Jianwen; Lian, Qizhou; El Oakley, Reida M; Lim, Bing; Lim, Sai-Kiang

2007-01-01

Background We have previously derived highly similar lineage-restricted stem cell lines, RoSH and E-RoSH cell lines from mouse embryos and CD9hi SSEA-1- differentiated mouse embryonic stem cells, respectively. These cell lines are not pluripotent and differentiate readily into endothelial cells in vitro and in vivo. Results We investigated the signaling pathway that maintains proliferation of these cells in an undifferentiated state, and demonstrate that PI3 K/Akt/mTOR, but not Raf/MEK/Erk, signaling in these cells was active during proliferation and was downregulated during endothelial differentiation. Inhibition of PI3 K/Akt/mTOR signaling, but not Raf/MEK/Erk, reduced proliferation and induced expression of endothelial specific proteins. During differentiation or inhibition of PI3 K/Akt/mTOR signaling, cyclinD2 transcript abundance in ribosome-enriched RNA but not in total RNA was reduced with a corresponding reduction in protein level. In contrast, transcript abundance of endothelial-specific genes e.g. Kdr, Tek and Pdgfrα in ribosome-enriched RNA fraction was not reduced and their protein levels were increased. Together these observations suggested that translational control mediated by PI3K/Akt/mTOR signaling was critical in regulating proliferation and endothelial differentiation of lineage-restricted RoSH-like stem cell lines. Conclusion This study highlights translation regulation as a critical regulatory mechanism during proliferation and differentiation in stem cells. PMID:17892597

Tuft (caveolated) cells in two human colon carcinoma cell lines.

PubMed Central

Barkla, D. H.; Whitehead, R. H.; Foster, H.; Tutton, P. J.

1988-01-01

The presence of an unusual cell type in two human colon carcinoma cell lines is reported. The cells show the same morphology as "tuft" (caveolated) cells present in normal gastrointestinal epithelium. Tuft cells were seen in cell line LIM 1863 growing in vitro and in human colon carcinoma cell line LIM 2210 growing as subcutaneous solid tumour xenografts in nude mice. Characteristic morphologic features of tuft cells included a wide base, narrow apex and a tuft of long microvilli projecting from the apical surface. The microvilli are attached by a core of long microfilaments passing deep into the apical cytoplasm. Between the microvilli are parallel arrays of vesicles (caveoli) containing flocculent material. Two different but not mutually exclusive explanations for the presence of tuft cells are proposed. The first explanation is that tuft cells came from the resected tumour and have survived by mitotic division during subsequent passages. The second explanation suggests that tuft cells are the progeny of undifferentiated tumour cells. Descriptions of tuft cells in colon carcinomas are uncommon and possible reasons for this are presented. The morphology of tuft cells is consistent with that of a highly differentiated cell specialised for absorption, and these new models provide an opportunity to further investigate the structure and function of tuft cells. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:3414781

Human monoclonal antibodies reactive with human myelomonocytic leukemia cells.

PubMed

Posner, M R; Santos, D J; Elboim, H S; Tumber, M B; Frackelton, A R

1989-04-01

Peripheral blood mononuclear cells from a patient with chronic myelogenous leukemia (CML), in remission, were depleted of CD8-positive T-cells and cultured with Epstein-Barr virus. Four of 20 cultures (20%) secreted human IgG antibodies selectively reactive with the cell surfaces of certain human leukemia cell lines. Three polyclonal, Epstein-Barr virus-transformed, B-cell lines were expanded and fused with the human-mouse myeloma analogue HMMA2.11TG/O. Antibody from secreting clones HL 1.2 (IgG1), HL 2.1 (IgG3), and HL 3.1 (IgG1) have been characterized. All three react with HL-60 (promyelocytic), RWLeu4 (CML promyelocytic), and U937 (monocytic), but not with KG-1 (myeloblastic) or K562 (CML erythroid). There is no reactivity with T-cell lines, Burkitt's cell lines, pre-B-leukemia cell lines, or an undifferentiated CML cell line, BV173. Leukemic cells from two of seven patients with acute myelogenous leukemia and one of five with acute lymphocytic leukemia react with all three antibodies. Normal lymphocytes, monocytes, polymorphonuclear cells, red blood cells, bone marrow cells, and platelets do not react. Samples from patients with other diverse hematopoietic malignancies showed no reactivity. Immunoprecipitations suggest that the reactive antigen(s) is a lactoperoxidase iodinatable series of cell surface proteins with molecular weights of 42,000-54,000 and a noniodinatable protein with a molecular weight of 82,000. Based on these data these human monoclonal antibodies appear to react with myelomonocytic leukemic cells and may detect a leukemia-specific antigen or a highly restricted differentiation antigen.

A Chimeric HS4-SAR Insulator (IS2) That Prevents Silencing and Enhances Expression of Lentiviral Vectors in Pluripotent Stem Cells

PubMed Central

Gutierrez-Guerrero, Alejandra; Cobo, Marién; Muñoz, Pilar

2014-01-01

Chromatin insulators, such as the chicken β-globin locus control region hypersensitive site 4 (HS4), and scaffold/matrix attachment regions (SARs/MARs) have been incorporated separately or in combination into retroviral vectors (RVs) in order to increase transgene expression levels, avoid silencing and reduce expression variability. However, their incorporation into RVs either produces a reduction on titer and/or expression levels or do not have sufficient effect on stem cells. In order to develop an improved insulator we decided to combine SAR elements with HS4 insulators. We designed several synthetic shorter SAR elements containing 4 or 5 MAR/SARs recognition signatures (MRS) and studied their effects on a lentiviral vector (LV) expressing eGFP through the SFFV promoter (SE). A 388 bp SAR element containing 5 MRS, named SAR2, was as efficient or superior to the other SARs analyzed. SAR2 enhanced transgene expression and reduced silencing and variability on human embryonic stem cells (hESCs). We next compared the effect of different HS4-based insulators, the HS4-Core (250 bp), the HS4-Ext (400 bp) and the HS4-650 (650 bp). All HS4 elements reduced silencing and expression variability but they also had a negative effect on transgene expression levels and titer. In general, the HS4-650 element had a better overall effect. Based on these data we developed a chimeric insulator, IS2, combining the SAR2 and the HS4-650. When incorporated into the 3′ LTR of the SE LV, the IS2 element was able to enhance expression, avoid silencing and reduce variability of expression on hESCs. Importantly, these effects were maintained after differentiation of the transduced hESCs toward the hematopoietic linage. Neither the HS4-650 nor the SAR2 elements had these effects. The IS2 element is therefore a novel insulator that confers expression stability and enhances expression of LVs on stem cells. PMID:24400083

Progestins Upregulate FKBP51 Expression in Human Endometrial Stromal Cells to Induce Functional Progesterone and Glucocorticoid Withdrawal: Implications for Contraceptive- Associated Abnormal Uterine Bleeding.

PubMed

Guzeloglu Kayisli, Ozlem; Kayisli, Umit A; Basar, Murat; Semerci, Nihan; Schatz, Frederick; Lockwood, Charles J

2015-01-01

Use of long-acting progestin only contraceptives (LAPCs) offers a discrete and highly effective family planning method. Abnormal uterine bleeding (AUB) is the major side effect of, and cause for, discontinuation of LAPCs. The endometria of LAPC-treated women display abnormally enlarged, fragile blood vessels, decreased endometrial blood flow and oxidative stress. To understanding to mechanisms underlying AUB, we propose to identify LAPC-modulated unique gene cluster(s) in human endometrial stromal cells (HESCs). Protein and RNA isolated from cultured HESCs treated 7 days with estradiol (E2) or E2+ medroxyprogesterone acetate (MPA) or E2+ etonogestrel (ETO) or E2+ progesterone (P4) were analyzed by quantitative Real-time (q)-PCR and immunoblotting. HSCORES were determined for immunostained-paired endometria of pre-and 3 months post-Depot MPA (DMPA) treated women and ovariectomized guinea pigs (GPs) treated with placebo or E2 or MPA or E2+MPA for 21 days. In HESCs, whole genome analysis identified a 67 gene group regulated by all three progestins, whereas a 235 gene group was regulated by E2+ETO and E2+MPA, but not E2+P4. Ingenuity pathway analysis identified glucocorticoid receptor (GR) activation as one of upstream regulators of the 235 MPA and ETO-specific genes. Among these, microarray results demonstrated significant enhancement of FKBP51, a repressor of PR/GR transcriptional activity, by both MPA and ETO. q-PCR and immunoblot analysis confirmed the microarray results. In endometria of post-DMPA versus pre-DMPA administered women, FKBP51 expression was significantly increased in endometrial stromal and glandular cells. In GPs, E2+MPA or MPA significantly increased FKBP51 immunoreactivity in endometrial stromal and glandular cells versus placebo- and E2-administered groups. MPA or ETO administration activates GR signaling and increases endometrial FKBP51 expression, which could be one of the mechanisms causing AUB by inhibiting PR and GR-mediated transcription. The resultant PR and/or GR-mediated functional withdrawal may contribute to associated endometrial inflammation, aberrant angiogenesis, and bleeding.

Trisomy 4 in a case of acute undifferentiated myeloblastic leukemia with hand-mirror cells.

PubMed

Kao, Y S; McCormick, C; Vial, R

1990-04-01

A case of acute undifferentiated myelocytic leukemic with trisomy 4 is described. The patient is a 61-year-old woman who developed leukemia 4 1/2 years after receiving radiation therapy for uterine carcinoma. Many leukemic cells exhibited hand-mirror configuration after the bone marrow aspirate was left at room temperature overnight. The relationship between trisomy 4 and hand-mirror cells in acute myelocytic leukemia is unknown.

Olaparib or Cediranib Maleate and Olaparib Compared With Standard Platinum-Based Chemotherapy in Treating Patients With Recurrent Platinum-Sensitive Ovarian, Fallopian Tube, or Primary Peritoneal Cancer

ClinicalTrials.gov

2018-06-11

BRCA Rearrangement; Deleterious BRCA1 Gene Mutation; Deleterious BRCA2 Gene Mutation; Fallopian Tube Clear Cell Adenocarcinoma; Fallopian Tube Transitional Cell Carcinoma; Ovarian Clear Cell Adenocarcinoma; Ovarian Endometrioid Tumor; Ovarian Seromucinous Carcinoma; Ovarian Serous Tumor; Ovarian Transitional Cell Carcinoma; Recurrent Fallopian Tube Carcinoma; Recurrent Ovarian Carcinoma; Recurrent Primary Peritoneal Carcinoma; Undifferentiated Fallopian Tube Carcinoma; Undifferentiated Ovarian Carcinoma

Development of an Aquatic Bioassay Using the Medaka (Oryzias latipes) to Assess Human Health Risks: Tumor Immunodiagnosis.

DTIC Science & Technology

1992-04-27

Skeletal muscle 5 *Undifferentiated sarcoma Peripheral nerve 3 *Undifferentiated sarcoma Peritoneum 1 Hepatocellular carcinoma Liver 2...KD) Neurofilament (3 proteins 68, 150, Detects neuronal cell origin and 200 KD) Other: Alpha-l antitrypsin Common marker for hepatocellular carcinoma Alpha... hepatocellular carcinoma from cholangiocellular carcinoma (8). 14 4 FIGURE 1. Avldln-Blotin-PeroxidSse Complex Tecbnlqu6. :4odif led from A.K.Bhan

Undifferentiated Neuroblastoma Cells Are More Sensitive to Photogenerated Oxidative Stress Than Differentiated Cells.

PubMed

Lee, Chu-I; Perng, Jing-Huei; Chen, Huang-Yo; Hong, Yi-Ren; Wang, Jyh-Jye

2015-09-01

Neuroblastoma is one of the most aggressive cancers and has a complex form of differentiation. We hypothesized that advanced cellular differentiation may alter the susceptibility of neuroblastoma to photodynamic treatment (PDT) and confer selective survival advantage. We demonstrated that hematoporphyrin uptake by undifferentiated SH-SY5Y cells was lower than that of differentiated counterparts, yet the former were more susceptible to PDT-induced oxidative stress killing. Photogenerated reactive oxygen species (ROS) in undifferentiated cells efficiently stimulated cell cycle arrest at G2/M phase, mitochondrial apoptotic pathway activation, the sustained phosphorylation of Akt/GSK-3β and ERK. Differentiated cells with more resistance to PDT exhibited a ROS-independent and a prolonged activation of ERK. Both SH-SY5Y cells exposed to PDT exhibited ROS-independent p38 and JNK activation. These results may have important implications for neuroblastoma patients undergoing photodynamic therapy. © 2015 Wiley Periodicals, Inc.

Mixed emotional and physical symptoms in general practice: what diagnoses do GPs use to describe them?

PubMed

Stone, Louise

2015-04-01

To determine what diagnostic terms are utilized by general practitioners (GPs) when seeing patients with mixed emotional and physical symptoms. Prototype cases of depression, anxiety, hypochondriasis, somatization and undifferentiated somatoform disorders were sourced from the psychiatric literature and the author's clinical practice. These were presented, in paper form, to a sample of GPs and GP registrars who were asked to provide a written diagnosis. Fifty-two questionnaires were returned (30% response rate). The depression and anxiety cases were identified correctly by most participants. There was moderate identification of the hypochondriasis and somatization disorder cases, and poor identification of the undifferentiated somatoform case. Somatization and undifferentiated somatoform disorders were infrequently recognized as diagnostic categories by the GPs in this study. Future research into the language and diagnostic reasoning utilized by GPs may help develop better diagnostic classification systems for use in primary care in this important area of practice.

β2-Microglobulin as a potential factor for the expansion of mesenchymal stem cells

PubMed Central

Zhu, Ying; Su, Yongping; Cheng, Tianmin; Chung, Leland W. K.

2010-01-01

Multipotent mesenchymal stem cells (MSCs) hold great promise in regenerative medicine, but one of the biggest challenges facing for their application is the ex vivo expansion to obtain enough undifferentiated cells. Fetal bovine serum (FBS), which can elicit possible contaminations of prion, virus, zoonosis or immunological reaction against xenogenic serum antigens, still remains essential to the culture formulations. There is an urgent need to identify potential factors for the undifferentiated expansion of MSCs to reduce the use of FBS or eventually replace it. A previously recognized housekeeping gene, β2-microglobulin (β2M), is demonstrated to act as a novel growth factor to stimulate the undifferentiated ex vivo expansion and preserve the pluripotency of adult MSCs from various sources. The use of β2M might have promising implications for future clinical application of MSCs. PMID:19466557

An Unusual Case of Alternating Ventricular Morphology on the 12-Lead Electrocardiogram.

PubMed

Sammon, Maura; Dawood, Alveena; Beaudoin, Scott; Harrigan, Richard A

2017-03-01

One of the principal tasks of an emergency physician is identifying potentially life-threatening conditions in the undifferentiated patient; cardiac dysrhythmia is an example of such a condition. A systematic approach to a patient with atypical dysrhythmia enables proper identification of such-life threatening conditions. We describe a 31-year-old man presenting to the emergency department with an undifferentiated dysrhythmia after naloxone reversal of an opiate overdose. A systematic approach to the electrocardiogram led to the rare diagnosis of Wolff-Parkinson-White (WPW) alternans. We review the differential diagnosis of this dysrhythmia and the initial evaluation of a patient with the WPW pattern present on their electrocardiogram. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: Emergency physicians should be prepared to use a systematic approach to an undifferentiated dysrhythmia to identify potentially life-threatening conditions. Copyright © 2016 Elsevier Inc. All rights reserved.

PC1/3 Deficiency Impacts Pro-opiomelanocortin Processing in Human Embryonic Stem Cell-Derived Hypothalamic Neurons.

PubMed

Wang, Liheng; Sui, Lina; Panigrahi, Sunil K; Meece, Kana; Xin, Yurong; Kim, Jinrang; Gromada, Jesper; Doege, Claudia A; Wardlaw, Sharon L; Egli, Dieter; Leibel, Rudolph L

2017-02-14

We recently developed a technique for generating hypothalamic neurons from human pluripotent stem cells. Here, as proof of principle, we examine the use of these cells in modeling of a monogenic form of severe obesity: PCSK1 deficiency. The cognate enzyme, PC1/3, processes many prohormones in neuroendocrine and other tissues. We generated PCSK1 (PC1/3)-deficient human embryonic stem cell (hESC) lines using both short hairpin RNA and CRISPR-Cas9, and investigated pro-opiomelanocortin (POMC) processing using hESC-differentiated hypothalamic neurons. The increased levels of unprocessed POMC and the decreased ratios (relative to POMC) of processed POMC-derived peptides in both PCSK1 knockdown and knockout hESC-derived neurons phenocopied POMC processing reported in PC1/3-null mice and PC1/3-deficient patients. PC1/3 deficiency was associated with increased expression of melanocortin receptors and PRCP (prolylcarboxypeptidase, a catabolic enzyme for α-melanocyte stimulating hormone (αMSH)), and reduced adrenocorticotropic hormone secretion. We conclude that the obesity accompanying PCSK1 deficiency may not be primarily due to αMSH deficiency. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Inducible overexpression of RUNX1b/c in human embryonic stem cells blocks early hematopoiesis from mesoderm.

PubMed

Chen, B; Teng, Jiawen; Liu, Hongwei; Pan, X; Zhou, Y; Huang, Shu; Lai, Mowen; Bian, Guohui; Mao, Bin; Sun, Wencui; Zhou, Qiongxiu; Yang, Shengyong; Nakahata, Tatsutoshi; Ma, Feng

2017-08-01

RUNX1 is absolutely required for definitive hematopoiesis, but the function of RUNX1b/c, two isoforms of human RUNX1, is unclear. We established inducible RUNX1b/c-overexpressing human embryonic stem cell (hESC) lines, in which RUNX1b/c overexpression prevented the emergence of CD34+ cells from early stage, thereby drastically reducing the production of hematopoietic stem/progenitor cells. Simultaneously, the expression of hematopoiesis-related factors was downregulated. However, such blockage effect disappeared from day 6 in hESC/AGM-S3 cell co-cultures, proving that the blockage occurred before the generation of hemogenic endothelial cells. This blockage was partially rescued by RepSox, an inhibitor of the transforming growth factor (TGF)-β signaling pathway, indicating a close relationship between RUNX1b/c and TGF-β pathway. Our results suggest a unique inhibitory function of RUNX1b/c in the development of early hematopoiesis and may aid further understanding of its biological function in normal and diseased models. © The Author (2017). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.

A Qualitative Analysis of Imitation Performances of Preschoolers With Down Syndrome.

PubMed

Vanvuchelen, Marleen

2016-05-01

A number of studies suggest that imitation is a characteristic strength in children with Down Syndrome (DS). The present study aims to discover whether imitation performances are qualitatively phenotypical in DS. Eight preschoolers with DS were matched on chronological, mental, language and imitation age with 8 preschoolers with intellectual disability of undifferentiated etiology (ID-UND). Imitation performances on the Preschool Imitation and Praxis Scale were videotaped for blind scoring on 30 possible errors. Children with DS made fewer production errors (synkinesias, OR 0.3 [0.1-0.7]), but more conceptual errors (substitution, OR 2.5 [1.6-3.9]) compared to children with ID-UND. This finding is in line with the view of a cognitive phenotype in DS, which is characterized by preserved visuospatial and impaired language abilities.

Rabbit embryonic stem cell lines derived from fertilized, parthenogenetic or somatic cell nuclear transfer embryos

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fang, Zhen F.; Gai, Hui; Huang, You Z.

2006-11-01

Embryonic stem cells were isolated from rabbit blastocysts derived from fertilization (conventional rbES cells), parthenogenesis (pES cells) and nuclear transfer (ntES cells), and propagated in a serum-free culture system. Rabbit ES (rbES) cells proliferated for a prolonged time in an undifferentiated state and maintained a normal karyotype. These cells grew in a monolayer with a high nuclear/cytoplasm ratio and contained a high level of alkaline phosphate activity. In addition, rbES cells expressed the pluripotent marker Oct-4, as well as EBAF2, FGF4, TDGF1, but not antigens recognized by antibodies against SSEA-1, SSEA-3, SSEA-4, TRA-1-10 and TRA-1-81. All 3 types of ESmore » cells formed embryoid bodies and generated teratoma that contained tissue types of all three germ layers. rbES cells exhibited a high cloning efficiency, were genetically modified readily and were used as nuclear donors to generate a viable rabbit through somatic cell nuclear transfer. In combination with genetic engineering, the ES cell technology should facilitate the creation of new rabbit lines.« less

The Human Cytomegalovirus Lytic Cycle Is Induced by 1,25-Dihydroxyvitamin D3 in Peripheral Blood Monocytes and in the THP-1 Monocytic Cell Line

PubMed Central

Wu, Shu-En; Miller, William E.

2015-01-01

Human cytomegalovirus (HCMV) resides in a latent form in hematopoietic progenitors and undifferentiated cells within the myeloid lineage. Maturation and differentiation along the myeloid lineage triggers lytic replication. Here, we used peripheral blood monocytes and the monocytic cell line THP-1 to investigate the effects of 1,25-dihydroxyvitamin D3 on HCMV replication. Interestingly, 1,25-dihydroxyvitamin D3 induces lytic replication marked by upregulation of HCMV gene expression and production of infectious virus. Moreover, we demonstrate that the effects of 1,25-dihydroxyvitamin D3 correlate with maturation/differentiation of the monocytes and not by directly stimulating the MIEP. These results are somewhat surprising as 1,25-dihydroxyvitamin D3 typically boosts immunity to bacteria and viruses rather than driving the infectious life cycle as it does for HCMV. Defining the signaling pathways kindled by 1,25-dihydroxyvitamin D3 will lead to a better understanding of the underlying molecular mechanisms that determine the fate of HCMV once it infects cells in the myeloid lineage. PMID:25965798