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.

SCOUP: a probabilistic model based on the Ornstein-Uhlenbeck process to analyze single-cell expression data during differentiation.

PubMed

Matsumoto, Hirotaka; Kiryu, Hisanori

2016-06-08

Single-cell technologies make it possible to quantify the comprehensive states of individual cells, and have the power to shed light on cellular differentiation in particular. Although several methods have been developed to fully analyze the single-cell expression data, there is still room for improvement in the analysis of differentiation. In this paper, we propose a novel method SCOUP to elucidate differentiation process. Unlike previous dimension reduction-based approaches, SCOUP describes the dynamics of gene expression throughout differentiation directly, including the degree of differentiation of a cell (in pseudo-time) and cell fate. SCOUP is superior to previous methods with respect to pseudo-time estimation, especially for single-cell RNA-seq. SCOUP also successfully estimates cell lineage more accurately than previous method, especially for cells at an early stage of bifurcation. In addition, SCOUP can be applied to various downstream analyses. As an example, we propose a novel correlation calculation method for elucidating regulatory relationships among genes. We apply this method to a single-cell RNA-seq data and detect a candidate of key regulator for differentiation and clusters in a correlation network which are not detected with conventional correlation analysis. We develop a stochastic process-based method SCOUP to analyze single-cell expression data throughout differentiation. SCOUP can estimate pseudo-time and cell lineage more accurately than previous methods. We also propose a novel correlation calculation method based on SCOUP. SCOUP is a promising approach for further single-cell analysis and available at https://github.com/hmatsu1226/SCOUP.

Delay in Apoptosome Formation Attenuates Apoptosis in Mouse Embryonic Stem Cell Differentiation

PubMed Central

Akbari-Birgani, Shiva; Hosseinkhani, Saman; Mollamohamadi, Sepideh; Baharvand, Hossein

2014-01-01

Differentiation is an inseparable process of development in multicellular organisms. Mouse embryonic stem cells (mESCs) represent a valuable research tool to conduct in vitro studies of cell differentiation. Apoptosis as a well known cell death mechanism shows some common features with cell differentiation, which has caused a number of ambiguities in the field. The research question here is how cells could differentiate these two processes from each other. We have investigated the role of the mitochondrial apoptotic pathway and cell energy level during differentiation of mESCs into the cardiomyocytes and their apoptosis. p53 expression, cytochrome c release, apoptosome formation, and caspase-3/7 activation are observed upon induction of both apoptosis and differentiation. However, remarkable differences are detected in time of cytochrome c appearance, apoptosome formation, and caspase activity upon induction of both processes. In apoptosis, apoptosome formation and caspase activity were observed rapidly following the cytochrome c release. Unlike apoptosis, the release of cytochrome c upon differentiation took more time, and the maximum caspase activity was also postponed for 24 h. This delay suggests that there is a regulatory mechanism during differentiation of mESCs into cardiomyocytes. The highest ATP content of cells was observed immediately after cytochrome c release 6 h after apoptosis induction and then decreased, but it was gradually increased up to 48 h after differentiation. These observations suggest that a delay in the release of cytochrome c or delay in ATP increase attenuate apoptosome formation, and caspase activation thereby discriminates apoptosis from differentiation in mESCs. PMID:24755221

Induction of mice adult bone marrow mesenchymal stem cells into functional motor neuron-like cells.

PubMed

Abdullah, Rafal H; Yaseen, Nahi Y; Salih, Shahlaa M; Al-Juboory, Ahmad Adnan; Hassan, Ayman; Al-Shammari, Ahmed Majeed

2016-11-01

The differentiation of mesenchymal stem cells (MSC) into acetylcholine secreted motor neuron-like cells, followed by elongation of the cell axon, is a promising treatment for spinal cord injury and motor neuron cell dysfunction in mammals. Differentiation is induced through a pre-induction step using Beta- mercaptoethanol (BME) followed by four days of induction with retinoic acid and sonic hedgehog. This process results in a very efficient differentiation of BM-MSCs into motor neuron-like cells. Immunocytochemistry showed that these treated cells had specific motor neural markers: microtubule associated protein-2 and acetylcholine transferase. The ability of these cells to function as motor neuron cells was assessed by measuring acetylcholine levels in a culture media during differentiation. High-performance liquid chromatography (HPLC) showed that the differentiated cells were functional. Motor neuron axon elongation was then induced by adding different concentrations of a nerve growth factor (NGF) to the differentiation media. Using a collagen matrix to mimic the natural condition of neural cells in a three-dimensional model showed that the MSCs were successfully differentiated into motor neuron-like cells. This process can efficiently differentiate MSCs into functional motor neurons that can be used for autologous nervous system therapy and especially for treating spinal cord injuries. Copyright © 2016 Elsevier B.V. All rights reserved.

JAG1-Mediated Notch Signaling Regulates Secretory Cell Differentiation of the Human Airway Epithelium.

PubMed

Gomi, Kazunori; Staudt, Michelle R; Salit, Jacqueline; Kaner, Robert J; Heldrich, Jonna; Rogalski, Allison M; Arbelaez, Vanessa; Crystal, Ronald G; Walters, Matthew S

2016-08-01

Basal cells (BC) are the stem/progenitor cells of the human airway epithelium capable of differentiating into secretory and ciliated cells. Notch signaling activation increases BC differentiation into secretory cells, but the role of individual Notch ligands in regulating this process in the human airway epithelium is largely unknown. The objective of this study was to define the role of the Notch ligand JAG1 in regulating human BC differentiation. JAG1 over-expression in BC increased secretory cell differentiation, with no effect on ciliated cell differentiation. Conversely, knockdown of JAG1 decreased expression of secretory cell genes. These data demonstrate JAG1-mediated Notch signaling regulates differentiation of BC into secretory cells.

Production of erythrocytes from directly isolated or Delta1 Notch ligand expanded CD34+ hematopoietic progenitor cells: process characterization, monitoring and implications for manufacture.

PubMed

Glen, Katie E; Workman, Victoria L; Ahmed, Forhad; Ratcliffe, Elizabeth; Stacey, Adrian J; Thomas, Robert J

2013-09-01

Economic ex vivo manufacture of erythrocytes at 10(12) cell doses requires an efficiently controlled bio-process capable of extensive proliferation and high terminal density. High-resolution characterization of the process would identify production strategies for increased efficiency, monitoring and control. CD34(+) cord blood cells or equivalent cells that had been pre-expanded for 7 days with Delta1 Notch ligand were placed in erythroid expansion and differentiation conditions in a micro-scale ambr suspension bioreactor. Multiple culture parameters were varied, and phenotype markers and metabolites measured to identify conserved trends and robust monitoring markers. The cells exhibited a bi-modal erythroid differentiation pattern with an erythroid marker peak after 2 weeks and 3 weeks of culture; differentiation was comparatively weighted toward the second peak in Delta1 pre-expanded cells. Both differentiation events were strengthened by omission of stem cell factor and dexamethasone. The cumulative cell proliferation and death, or directly measured CD45 expression, enabled monitoring of proliferative rate of the cells. The metabolic activities of the cultures (glucose, glutamine and ammonia consumption or production) were highly variable but exhibited systematic change synchronized with the change in differentiation state. Erythroid differentiation chronology is partly determined by the heterogeneous CD34(+) progenitor compartment with implications for input control; Delta1 ligand-mediated progenitor culture can alter differentiation profile with control benefits for engineering production strategy. Differentiation correlated changes in cytokine response, markers and metabolic state will enable scientifically designed monitoring and timing of manufacturing process steps. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Metabolic pathways in T cell activation and lineage differentiation.

PubMed

Almeida, Luís; Lochner, Matthias; Berod, Luciana; Sparwasser, Tim

2016-10-01

Recent advances in the field of immunometabolism support the concept that fundamental processes in T cell biology, such as TCR-mediated activation and T helper lineage differentiation, are closely linked to changes in the cellular metabolic programs. Although the major task of the intermediate metabolism is to provide the cell with a constant supply of energy and molecular precursors for the production of biomolecules, the dynamic regulation of metabolic pathways also plays an active role in shaping T cell responses. Key metabolic processes such as glycolysis, fatty acid and mitochondrial metabolism are now recognized as crucial players in T cell activation and differentiation, and their modulation can differentially affect the development of T helper cell lineages. In this review, we describe the diverse metabolic processes that T cells engage during their life cycle from naïve towards effector and memory T cells. We consider in particular how the cellular metabolism may actively support the function of T cells in their different states. Moreover, we discuss how molecular regulators such as mTOR or AMPK link environmental changes to adaptations in the cellular metabolism and elucidate the consequences on T cell differentiation and function. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

In vitro long-term development of cultured inner ear stem cells of newborn rat.

PubMed

Carricondo, Francisco; Iglesias, Mari Cruz; Rodríguez, Fernando; Poch-Broto, Joaquin; Gil-Loyzaga, Pablo

2010-10-01

The adult mammalian auditory receptor lacks any ability to repair and/or regenerate after injury. However, the late developing cochlea still contains some stem-cell-like elements that might be used to regenerate damaged neurons and/or cells of the organ of Corti. Before their use in any application, stem cell numbers need to be amplified because they are usually rare in late developing and adult tissues. The numerous re-explant cultures required for the progressive amplification process can result in a spontaneous differentiation process. This aspect has been implicated in the tumorigenicity of stem cells when transplanted into a tissue. The aim of this study has been to determine whether cochlear stem cells can proliferate and differentiate spontaneously in long-term cultures without the addition of any factor that might influence these processes. Cochlear stem cells, which express nestin protein, were cultured in monolayers and fed with DMEM containing 5% FBS. They quickly organized themselves into typical spheres exhibiting a high proliferation rate, self-renewal property, and differentiation ability. Secondary cultures of these stem cell spheres spontaneously differentiated into neuroectodermal-like cells. The expression of nestin, glial-fibrillary-acidic protein, vimentin, and neurofilaments was evaluated to identify early differentiation. Nestin expression appeared in primary and secondary cultures. Other markers were also identified in differentiating cells. Further research might demonstrate the spontaneous differentiation of cochlear stem cells and their teratogenic probability when they are used for transplantation.

Characterization of Apoptosis Signaling Cascades During the Differentiation Process of Human Neural ReNcell VM Progenitor Cells In Vitro.

PubMed

Jaeger, Alexandra; Fröhlich, Michael; Klum, Susanne; Lantow, Margareta; Viergutz, Torsten; Weiss, Dieter G; Kriehuber, Ralf

2015-11-01

Apoptosis is an essential physiological process accompanying the development of the central nervous system and human neurogenesis. However, the time scale and the underlying molecular mechanisms are yet poorly understood. Due to this fact, we investigated the functionality and general inducibility of apoptosis in the human neural ReNcell VM progenitor cell line during differentiation and also after exposure to staurosporine (STS) and ultraviolet B (UVB) irradiation. Transmission light microscopy, flow cytometry, and Western-/Immunoblot analysis were performed to compare proliferating and differentiating, in addition to STS- and UVB-treated cells. In particular, from 24 to 72 h post-initiation of differentiation, G0/G1 cell cycle arrest, increased loss of apoptotic cells, activation of pro-apoptotic BAX, Caspase-3, and cleavage of its substrate PARP were observed during cell differentiation and, to a higher extent, after treatment with STS and UVB. We conclude that redundant or defective cells are eliminated by apoptosis, while otherwise fully differentiated cells were less responsive to apoptosis induction by STS than proliferating cells, likely as a result of reduced APAF-1 expression, and increased levels of BCL-2. These data provide the evidence that apoptotic mechanisms in the neural ReNcell VM progenitor cell line are not only functional, but also inducible by external stimuli like growth factor withdrawal or treatment with STS and UVB, which marks this cell line as a suitable model to investigate apoptosis signaling pathways in respect to the differentiation processes of human neural progenitor cells in vitro.

Transcriptomic Profiling Discloses Molecular and Cellular Events Related to Neuronal Differentiation in SH-SY5Y Neuroblastoma Cells.

PubMed

Pezzini, Francesco; Bettinetti, Laura; Di Leva, Francesca; Bianchi, Marzia; Zoratti, Elisa; Carrozzo, Rosalba; Santorelli, Filippo M; Delledonne, Massimo; Lalowski, Maciej; Simonati, Alessandro

2017-05-01

Human SH-SY5Y neuroblastoma cells are widely utilized in in vitro studies to dissect out pathogenetic mechanisms of neurodegenerative disorders. These cells are considered as neuronal precursors and differentiate into more mature neuronal phenotypes under selected growth conditions. In this study, in order to decipher the pathways and cellular processes underlying neuroblastoma cell differentiation in vitro, we performed systematic transcriptomic (RNA-seq) and bioinformatic analysis of SH-SY5Y cells differentiated according to a two-step paradigm: retinoic acid treatment followed by enriched neurobasal medium. Categorization of 1989 differentially expressed genes (DEGs) identified in differentiated cells functionally linked them to changes in cell morphology including remodelling of plasma membrane and cytoskeleton, and neuritogenesis. Seventy-three DEGs were assigned to axonal guidance signalling pathway, and the expression of selected gene products such as neurotrophin receptors, the functionally related SLITRK6, and semaphorins, was validated by immunoblotting. Along with these findings, the differentiated cells exhibited an ability to elongate longer axonal process as assessed by the neuronal cytoskeletal markers biochemical characterization and morphometric evaluation. Recognition of molecular events occurring in differentiated SH-SY5Y cells is critical to accurately interpret the cellular responses to specific stimuli in studies on disease pathogenesis.

Insight into the Role of Long Non-coding RNAs During Osteogenesis in Mesenchymal Stem Cells.

PubMed

Huo, Sibei; Zhou, Yachuan; He, Xinyu; Wan, Mian; Du, Wei; Xu, Xin; Ye, Ling; Zhou, Xuedong; Zheng, Liwei

2018-01-01

Long non-coding RNAs (LncRNAs) are non-protein coding transcripts longer than 200 nucleotides in length. Instead of being "transcriptional noise", lncRNAs are emerging as a key modulator in various biological processes and disease development. Mesenchymal stem cells can be isolated from various adult tissues, such as bone marrow and dental tissues. The differentiation processes into multiple lineages, such as osteogenic differentiation, are precisely orchestrated by molecular signals in both genetic and epigenetic ways. Recently, several lines of evidence suggested the role of lncRNAs participating in cell differentiation through the regulation of gene transcriptions. And the involvement of lncRNAs may be associated with initiation and progression of mesenchymal stem cell-related diseases. We aimed at addressing the role of lncRNAs in the regulation of osteogenesis of mesenchymal stem cells derived from bone marrow and dental tissues, and discussing the potential utility of lncRNAs as biomarkers and therapeutic targets for mesenchymal stem cell-related diseases. Numerous lncRNAs were differentially expressed during osteogenesis or odontogenesis of mesenchymal stem cells, and some of them were confirmed to be able to regulate the differentiation processes through the modifications of chromatin, transcriptional and post-transcriptional processes. LncRNAs were also associated with some diseases related with pathologic differentiation of mesenchymal stem cells. LncRNAs involve in the osteogenic differentiation of bone marrow and dental tissuederived mesenchymal stem cells, and they could become promising therapeutic targets and prognosis parameters. However, the mechanisms of the role of lncRNAs are still enigmatic and require further investigation. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

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.

Effect of Wnt-1 inducible signaling pathway protein-2 (WISP-2/CCN5), a downstream protein of Wnt signaling, on adipocyte differentiation

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

Inadera, Hidekuni; Shimomura, Akiko; Tachibana, Shinjiro

2009-02-20

Wnt signaling negatively regulates adipocyte differentiation, and ectopic expression of Wnt-1 in 3T3-L1 cells induces several downstream molecules of Wnt signaling, including Wnt-1 inducible signaling pathway protein (WISP)-2. In this study, we examined the role of WISP-2 in the process of adipocyte differentiation using an in vitro cell culture system. In the differentiation of 3T3-L1 cells, WISP-2 expression was observed in growing cells and declined thereafter. In the mitotic clonal expansion phase of adipocyte differentiation, WISP-2 expression was transiently down-regulated concurrently with up-regulation of CCAAT/enhancer-binding protein {delta} expression. Treatment of 3T3-L1 cells in the differentiation medium with lithium, an activatormore » of Wnt signaling, inhibited the differentiation process with concomitant induction of WISP-2. Treatment of differentiated cells with lithium induced de-differentiation as evidenced by profound reduction of peroxisome proliferator-activator receptor {gamma} expression and concomitant induction of WISP-2. However, de-differentiation of differentiated cells induced by tumor necrosis factor-{alpha} did not induce WISP-2 expression. To directly examine the effect of WISP-2 on adipocyte differentiation, 3T3-L1 cells were infected with a retrovirus carrying WISP-2. Although forced expression of WISP-2 inhibited preadipocyte proliferation, it had no effect on adipocyte differentiation. Thus, although WISP-2 is a downstream protein of Wnt signaling, the role of WISP-2 on adipocyte differentiation may be marginal, at least in this in vitro culture model.« less

NURR1 Downregulation Favors Osteoblastic Differentiation of MSCs

PubMed Central

Di Benedetto, Adriana; Cantore, Stefania; Centonze, Matteo; Grano, Maria; Cavalcanti-Adam, Elisabetta A.

2017-01-01

Mesenchymal stem cells (MSCs) have been identified in human dental tissues. Dental pulp stem cells (DPSCs) were classified within MSC family, are multipotent, can be isolated from adult teeth, and have been shown to differentiate, under particular conditions, into various cell types including osteoblasts. In this work, we investigated how the differentiation process of DPSCs toward osteoblasts is controlled. Recent literature data attributed to the nuclear receptor related 1 (NURR1), a still unclarified role in osteoblast differentiation, while NURR1 is primarily involved in dopaminergic neuron differentiation and activity. Thus, in order to verify if NURR1 had a role in DPSC osteoblastic differentiation, we silenced it during all the processes and compared the expression of the main osteoblastic markers with control cultures. Our results showed that the inhibition of NURR1 significantly increased the expression of osteoblast markers collagen I and alkaline phosphatase. Further, in long time cultures, the mineral matrix deposition was strongly enhanced in NURR1-silenced cultures. These results suggest that NURR1 plays a key role in switching DPSC differentiation toward osteoblasts rather than neuronal or even other cell lines. In conclusion, DPSCs represent a source of osteoblast-like cells and downregulation of NURR1 strongly prompted their differentiation toward the osteoblastogenesis process. PMID:28769982

Up-regulation of Jun/AP-1 during differentiation of N1E-115 neuroblastoma cells.

PubMed

de Groot, R P; Kruijer, W

1991-12-01

Neuroblastoma cell lines isolated from neuroblastoma tumors can be induced to differentiate into neuronal cell types by treatment with chemical agents, such as dimethyl sulfoxide and retinoic acid. The molecular mechanisms underlying this differentiation process, however, are completely obscure. In this paper, we show that neuronal differentiation of mouse N1E-115 neuroblastoma cells by dimethyl sulfoxide is accompanied by a prolonged rise in c-jun, junB, and junD expression and AP-1 activity. Multiple sequence elements in the Jun promoters are involved in this process. Furthermore, we show that c-jun and junD, but not junB, are expressed at high levels in the neuronal cell types obtained after dimethyl sulfoxide treatment. These results suggest an important role for c-jun and junD in neuronal differentiation of N1E-115 cells.

Proteomic changes during intestinal cell maturation in vivo

PubMed Central

Chang, Jinsook; Chance, Mark R.; Nicholas, Courtney; Ahmed, Naseem; Guilmeau, Sandra; Flandez, Marta; Wang, Donghai; Byun, Do-Sun; Nasser, Shannon; Albanese, Joseph M.; Corner, Georgia A.; Heerdt, Barbara G.; Wilson, Andrew J.; Augenlicht, Leonard H.; Mariadason, John M.

2008-01-01

Intestinal epithelial cells undergo progressive cell maturation as they migrate along the crypt-villus axis. To determine molecular signatures that define this process, proteins differentially expressed between the crypt and villus were identified by 2D-DIGE and MALDI-MS. Forty-six differentially expressed proteins were identified, several of which were validated by immunohistochemistry. Proteins upregulated in the villus were enriched for those involved in brush border assembly and lipid uptake, established features of differentiated intestinal epithelial cells. Multiple proteins involved in glycolysis were also upregulated in the villus, suggesting increased glycolysis is a feature of intestinal cell differentiation. Conversely, proteins involved in nucleotide metabolism, and protein processing and folding were increased in the crypt, consistent with functions associated with cell proliferation. Three novel paneth cell markers, AGR2, HSPA5 and RRBP1 were also identified. Notably, significant correlation was observed between overall proteomic changes and corresponding gene expression changes along the crypt-villus axis, indicating intestinal cell maturation is primarily regulated at the transcriptional level. This proteomic profiling analysis identified several novel proteins and functional processes differentially induced during intestinal cell maturation in vivo. Integration of proteomic, immunohistochemical, and parallel gene expression datasets demonstrate the coordinated manner in which intestinal cell maturation is regulated. PMID:18824147

CTCF-Mediated and Pax6-Associated Gene Expression in Corneal Epithelial Cell-Specific Differentiation

PubMed Central

Tsui, Shanli; Wang, Jie; Wang, Ling; Dai, Wei; Lu, Luo

2016-01-01

Background The purpose of the study is to elicit the epigenetic mechanism involving CCCTC binding factor (CTCF)-mediated chromatin remodeling that regulates PAX6 gene interaction with differentiation-associated genes to control corneal epithelial differentiation. Methods Cell cycle progression and specific keratin expressions were measured to monitor changes of differentiation-induced primary human limbal stem/progenitor (HLS/P), human corneal epithelial (HCE) and human telomerase-immortalized corneal epithelial (HTCE) cells. PAX6-interactive and differentiation-associated genes in chromatin remodeling mediated by the epigenetic factor CTCF were detected by circular chromosome conformation capture (4C) and ChIP (Chromatin immunoprecipitation)-on-chip approaches, and verified by FISH (Fluorescent in situ hybridization). Furthermore, CTCF activities were altered by CTCF-shRNA to study the effect of CTCF on mediating interaction of Pax6 and differentiation-associated genes in corneal epithelial cell fate. Results Our results demonstrated that differentiation-induced human corneal epithelial cells expressed typical corneal epithelial characteristics including morphological changes, increased keratin12 expression and G0/G1 accumulations. Expressions of CTCF and PAX6 were suppressed and elevated following the process of differentiation, respectively. During corneal epithelial cell differentiation, differentiation-induced RCN1 and ADAM17 were found interacting with PAX6 in the process of CTCF-mediated chromatin remodeling detected by 4C and verified by ChIP-on-chip and FISH. Diminished CTCF mRNA with CTCF-shRNA in HTCE cells weakened the interaction of PAX6 gene in controlling RCN1/ADAM17 and enhanced early onset of the genes in cell differentiation. Conclusion Our results explain how epigenetic factor CTCF-mediated chromatin remodeling regulates interactions between eye-specific PAX6 and those genes that are induced/associated with cell differentiation to modulate corneal epithelial cell-specific differentiation. PMID:27583466

Gene expression profile in human induced pluripotent stem cells: Chondrogenic differentiation in vitro, part A

PubMed Central

Suchorska, Wiktoria Maria; Augustyniak, Ewelina; Richter, Magdalena; Trzeciak, Tomasz

2017-01-01

Human induced pluripotent stem cells (hiPSCs) offer promise in regenerative medicine, however more data are required to improve understanding of key aspects of the cell differentiation process, including how specific chondrogenic processes affect the gene expression profile of chondrocyte-like cells and the relative value of cell differentiation markers. The main aims of the present study were as follows: To determine the gene expression profile of chondrogenic-like cells derived from hiPSCs cultured in mediums conditioned with HC-402-05a cells or supplemented with transforming growth factor β3 (TGF-β3), and to assess the relative utility of the most commonly used chondrogenic markers as indicators of cell differentiation. These issues are relevant with regard to the use of human fibroblasts in the reprogramming process to obtain hiPSCs. Human fibroblasts are derived from the mesoderm and thus share a wide range of properties with chondrocytes, which also originate from the mesenchyme. Thus, the exclusion of dedifferentiation instead of chondrogenic differentiation is crucial. The hiPSCs were obtained from human primary dermal fibroblasts during a reprogramming process. Two methods, both involving embryoid bodies (EB), were used to obtain chondrocytes from the hiPSCs: EBs formed in a chondrogenic medium supplemented with TGF-β3 (10 ng/ml) and EBs formed in a medium conditioned with growth factors from HC-402-05a cells. Based on immunofluorescence and reverse transcription-quantiative polymerase chain reaction analysis, the results indicated that hiPSCs have the capacity for effective chondrogenic differentiation, in particular cells differentiated in the HC-402-05a-conditioned medium, which present morphological features and markers that are characteristic of mature human chondrocytes. By contrast, cells differentiated in the presence of TGF-β3 may demonstrate hypertrophic characteristics. Several genes [paired box 9, sex determining region Y-box (SOX) 5, SOX6, SOX9 and cartilage oligomeric matrix protein] were demonstrated to be good markers of early hiPSC chondrogenic differentiation: Insulin-like growth factor 1, Tenascin-C, and β-catenin were less valuable. These observations provide valuable data on the use of hiPSCs in cartilage tissue regeneration. PMID:28447755

Poised Regeneration of Zebrafish Melanocytes Involves Direct Differentiation and Concurrent Replenishment of Tissue-Resident Progenitor Cells.

PubMed

Iyengar, Sharanya; Kasheta, Melissa; Ceol, Craig J

2015-06-22

Efficient regeneration following injury is critical for maintaining tissue function and enabling organismal survival. Cells reconstituting damaged tissue are often generated from resident stem or progenitor cells or from cells that have dedifferentiated and become proliferative. While lineage-tracing studies have defined cellular sources of regeneration in many tissues, the process by which these cells execute the regenerative process is largely obscure. Here, we have identified tissue-resident progenitor cells that mediate regeneration of zebrafish stripe melanocytes and defined how these cells reconstitute pigmentation. Nearly all regeneration melanocytes arise through direct differentiation of progenitor cells. Wnt signaling is activated prior to differentiation, and inhibition of Wnt signaling impairs regeneration. Additional progenitors divide symmetrically to sustain the pool of progenitor cells. Combining direct differentiation with symmetric progenitor divisions may serve as a means to rapidly repair injured tissue while preserving the capacity to regenerate. Copyright © 2015 Elsevier Inc. All rights reserved.

Terminal Differentiation of Adult Hippocampal Progenitor Cells Is a Step Functionally Dissociable from Proliferation and Is Controlled by Tis21, Id3 and NeuroD2

PubMed Central

Micheli, Laura; Ceccarelli, Manuela; Gioia, Roberta; D’Andrea, Giorgio; Farioli-Vecchioli, Stefano; Costanzi, Marco; Saraulli, Daniele; Cestari, Vincenzo; Tirone, Felice

2017-01-01

Cell proliferation and differentiation are interdependent processes. Here, we have asked to what extent the two processes of neural progenitor cell amplification and differentiation are functionally separated. Thus, we analyzed whether it is possible to rescue a defect of terminal differentiation in progenitor cells of the dentate gyrus, where new neurons are generated throughout life, by inducing their proliferation and/or their differentiation with different stimuli appropriately timed. As a model we used the Tis21 knockout mouse, whose dentate gyrus neurons, as demonstrated by us and others, have an intrinsic defect of terminal differentiation. We first tested the effect of two proliferative as well as differentiative neurogenic stimuli, one pharmacological (fluoxetine), the other cognitive (the Morris water maze (MWM) training). Both effectively enhanced the number of new dentate gyrus neurons produced, and fluoxetine also reduced the S-phase length of Tis21 knockout dentate gyrus progenitor cells and increased the rate of differentiation of control cells, but neither factor enhanced the defective rate of differentiation. In contrast, the defect of terminal differentiation was fully rescued by in vivo infection of proliferating dentate gyrus progenitor cells with retroviruses either silencing Id3, an inhibitor of neural differentiation, or expressing NeuroD2, a proneural gene expressed in terminally differentiated dentate gyrus neurons. This is the first demonstration that NeuroD2 or the silencing of Id3 can activate the differentiation of dentate gyrus neurons, complementing a defect of differentiation. It also highlights how the rate of differentiation of dentate gyrus neurons is regulated genetically at several levels and that a neurogenic stimulus for amplification of neural stem/progenitor cells may not be sufficient in itself to modify this rate. PMID:28740463

Live Cell Imaging of the Nascent Inactive X Chromosome during the Early Differentiation Process of Naive ES Cells towards Epiblast Stem Cells

PubMed Central

Guyochin, Aurélia; Maenner, Sylvain; Chu, Erin Tsi-Jia; Hentati, Asma; Attia, Mikael; Avner, Philip; Clerc, Philippe

2014-01-01

Random X-chromosome inactivation ensures dosage compensation in mammals through the transcriptional silencing of one of the two X chromosomes present in each female cell. Silencing is initiated in the differentiating epiblast of the mouse female embryos through coating of the nascent inactive X chromosome by the non-coding RNA Xist, which subsequently recruits the Polycomb Complex PRC2 leading to histone H3-K27 methylation. Here we examined in mouse ES cells the early steps of the transition from naive ES cells towards epiblast stem cells as a model for inducing X chromosome inactivation in vitro. We show that these conditions efficiently induce random XCI. Importantly, in a transient phase of this differentiation pathway, both X chromosomes are coated with Xist RNA in up to 15% of the XX cells. In an attempt to determine the dynamics of this process, we designed a strategy aimed at visualizing the nascent inactive X-chromosome in live cells. We generated transgenic female XX ES cells expressing the PRC2 component Ezh2 fused to the fluorescent protein Venus. The fluorescent fusion protein was expressed at sub-physiological levels and located in nuclei of ES cells. Upon differentiation of ES cell towards epiblast stem cell fate, Venus-fluorescent territories appearing in interphase nuclei were identified as nascent inactive X chromosomes by their association with Xist RNA. Imaging of Ezh2-Venus for up to 24 hours during the differentiation process showed survival of some cells with two fluorescent domains and a surprising dynamics of the fluorescent territories across cell division and in the course of the differentiation process. Our data reveal a strategy for visualizing the nascent inactive X chromosome and suggests the possibility for a large plasticity of the nascent inactive X chromosome. PMID:25546018

Effects of inorganic lead on the differentiation and growth of cultured hippocampal and neuroblastoma cells.

PubMed

Audesirk, T; Audesirk, G; Ferguson, C; Shugarts, D

1991-01-01

Lead exposure has devastating effects on the developing nervous system, and has been implicated in variety of behavioral and cognitive deficits as well as neural morphological abnormalities. Since lead impacts many calcium-dependent processes, one likely mechanism of lead toxicity is its disruption of calcium dependent processes, among which is neuronal differentiation. We investigated the effects of inorganic lead on survival and several parameters of differentiation of cultured neurons. Three different cell types were used: Rat hippocampal neurons (a primary CNS cell type), B50 rat neuroblastoma cells (a transformed CNS-derived cell line), and N1E-115 mouse neuroblastoma cells (a transformed peripherally-derived cell line). Lead concentrations ranged from low nM to 1 mM. Lead effects differed considerably among the three cell types, with B50 cells least affected. Lead effects were generally multimodal, with fewest effects observed at intermediate concentrations. Lead inhibited neurite initiation in hippocampal neurons, but stimulated initiation in N1E-115 cells. In those cells that differentiated, lead increased dendrite numbers in hippocampal neurons and neurite numbers in N1E-115 cells. Lead exposure increased both the length and the degree of branching of axons in hippocampal neurons and the length of neurites in N1E-115 cells. We hypothesize that lead impacts multiple regulatory processes that influence neuron survival and differentiation, and that its effects show differing dose-dependencies. The differing responses of the different cell types to lead suggests that differentiation may be regulated in different ways by the three types of cells. Alternatively, or additionally, the cell types may differ in their ability to compensate for, sequester, or expel lead.

Small GTPase Tc10 and its homologue RhoT induce N-WASP-mediated long process formation and neurite outgrowth.

PubMed

Abe, Tomoyuki; Kato, Masayoshi; Miki, Hiroaki; Takenawa, Tadaomi; Endo, Takeshi

2003-01-01

Rho family small GTPases regulate multiple cellular functions through reorganization of the actin cytoskeleton. Among them, Cdc42 and Tc10 induce filopodia or peripheral processes in cultured cells. We have identified a member of the family, designated as RhoT, which is closely related to Tc10. Tc10 was highly expressed in muscular tissues and brain and remarkably induced during differentiation of C2 skeletal muscle cells and neuronal differentiation of PC12 and N1E-115 cells. On the other hand, RhoT was predominantly expressed in heart and uterus and induced during neuronal differentiation of N1E-115 cells. Tc10 exogenously expressed in fibroblasts generated actin-filament-containing peripheral processes longer than the Cdc42-formed filopodia, whereas RhoT produced much longer and thicker processes containing actin filaments. Furthermore, both Tc10 and RhoT induced neurite outgrowth in PC12 and N1E-115 cells, but Cdc42 did not do this by itself. Tc10 and RhoT as well as Cdc42 bound to the N-terminal CRIB-motif-containing portion of N-WASP and activated N-WASP to induce Arp2/3-complex-mediated actin polymerization. The formation of peripheral processes and neurites by Tc10 and RhoT was prevented by the coexpression of dominant-negative mutants of N-WASP. Thus, N-WASP is essential for the process formation and neurite outgrowth induced by Tc10 and RhoT. Neuronal differentiation of PC12 and N1E-115 cells induced by dibutyryl cyclic AMP and by serum starvation, respectively, was prevented by dominant-negative Cdc42, Tc10 and RhoT. Taken together, all these Rho family proteins are required for neuronal differentiation, but they exert their functions differentially in process formation and neurite extension. Consequently, N-WASP activated by these small GTPases mediates neuronal differentiation in addition to its recently identified role in glucose uptake.

From embryonic stem cells to functioning germ cells: science, clinical and ethical perspectives.

PubMed

Kiatpongsan, Sorapop

2007-10-01

Embryonic stem cells have been well recognized as cells having a versatile potential to differentiate into all types of cells in the body including germ cells. There are many research studies focusing on the differentiation processes and protocols to derive various types of somatic cells from embryonic stem cells. However, germ cells have unique differentiation process and developmental pathway compared with somatic cells. Consequently, they will require different differentiation protocols and special culture techniques. More understanding and established in vitro systems for gametogenesis will greatly contribute to further progression of knowledge and technology in germ cell biology, reproductive biology and reproductive medicine. Moreover if oocytes can be efficiently produced in vitro, this will play an important role on progression in nuclear transfer and nuclear reprogramming technology. The present article will provide concise review on past important discoveries, current ongoing studies and future views of this challenging research area. An ethical perspective has also been proposed to give comprehensive summary and viewpoint for future clinical application.

Selection and dynamics of embryonic stem cell integration into early mouse embryos

PubMed Central

Alexandrova, Stoyana; Kalkan, Tuzer; Humphreys, Peter; Riddell, Andrew; Scognamiglio, Roberta; Trumpp, Andreas; Nichols, Jennifer

2016-01-01

The process by which pluripotent cells incorporate into host embryos is of interest to investigate cell potency and cell fate decisions. Previous studies suggest that only a minority of the embryonic stem cell (ESC) inoculum contributes to the adult chimaera. How incoming cells are chosen for integration or elimination remains unclear. By comparing a heterogeneous mix of undifferentiated and differentiating ESCs (serum/LIF) with more homogeneous undifferentiated culture (2i/LIF), we examine the role of cellular heterogeneity in this process. Time-lapse ex vivo imaging revealed a drastic elimination of serum/LIF ESCs during early development in comparison with 2i/LIF ESCs. Using a fluorescent reporter for naive pluripotency (Rex1-GFP), we established that the acutely eliminated serum/LIF ESCs had started to differentiate. The rejected cells were apparently killed by apoptosis. We conclude that a selection process exists by which unwanted differentiating cells are eliminated from the embryo. However, occasional Rex1− cells were able to integrate. Upregulation of Rex1 occurred in a proportion of these cells, reflecting the potential of the embryonic environment to expedite diversion from differentiation priming to enhance the developing embryonic epiblast. PMID:26586221

[Proliferation and morphological differentiation of neurblastoma cells in cultured under the effect of avermectins].

PubMed

Miakisheva, S N; Kostenko, M A; Driniaev, V A; Mosin, V A

2001-01-01

The effect of natural avermectin complex (Aversectin C) and Abamectin on the processes of proliferation and morphological differentiation of the neural cells was studied using N1E-115 murine neuroblastoma cells (clone C-1300) as a model. Aversectin C in concentrations 10(-7)-10(-8) was shown to induce morphological differentiation of cultured nervous cells. Treatment with Abamectin resulted in the changes of proliferation pattern of the cells. Morphological differentiation of the cultured nervous cells treated with Aversectin C was associated with electrophysiological one.

Immobilization of concanavalin A receptors during differentiation of neuroblastoma cells.

PubMed

Fishman, M C; Dragsten, P R; Spector, I

1981-04-30

Neuroblastoma cells serve as a useful model of neuronal development because compounds such as dimethyl sulphoxide (DMSO) and dibutyryl cyclic AMP cause them to undergo a process of controlled differentiation in tissue culture, during which they can extend long processes, develop characteristic excitability mechanisms, synthesize neurotransmitters and form synapses. We have used the technique of fluorescence photobleaching recovery to study the lateral mobility of cell-surface constituents during the differentiation of neuroblastoma clone N1E-115 cells. The concanavalin A (Con A) binding sites appear as discrete patches distributed over the entire cell surface and exhibit lateral mobility in undifferentiated cells comparable with that of surface glycoproteins of other cells. After induction of differentiation, however, the vast majority of Con A binding sites become immobilized, and we present data which suggest that the mechanism of this immobilization may involve linkage to the internal actin network.

Differentiating Mouse Embryonic Stem Cells into Embryoid Bodies by Hanging-Drop Cultures.

PubMed

Behringer, Richard; Gertsenstein, Marina; Nagy, Kristina Vintersten; Nagy, Andras

2016-12-01

Embryonic stem (ES) cells can develop into many types of differentiated tissues if they are placed into a differentiating environment. This can occur in vivo when the ES cells are injected into or aggregated with an embryo, or in vitro if their culture conditions are modified to induce differentiation. There are an increasing number of differentiating culture conditions that can bias the differentiation of ES cells into desired cell types. Determining the mechanisms that control ES cell differentiation into therapeutically important cell types is a quickly growing area of research. Knowledge gained from these studies may eventually lead to the use of stem cells to repair specific damaged tissues. Many times ES cell differentiation proceeds through an intermediate stage called the embryoid body (EB). EBs are round structures composed of ES cells that have undergone some of the initial stages of differentiation. EBs can then be manipulated further to generate more specific cell types. This protocol describes a method to differentiate ES cells into EBs. It produces EBs of comparable size. This aspect is important because the differentiation processes taking place inside an EB are influenced by its size. © 2016 Cold Spring Harbor Laboratory Press.

Differentiation potentials of perivascular cells in the bone tissue remodeling zones under microgravity

NASA Astrophysics Data System (ADS)

Rodionova, Natalia; Katkova, Olena

Adaptive remodeling processes in the skeleton bones occur in the close topographical interconnection with blood capillaries followed by perivascular cells. Radioautographic studies with 3H- thymidine (Kimmel D.B., Fee W.S., 1980; Rodionova N.V., 1989, 2006) has shown that in osteogenesis zones there is sequential differentiation process of the perivascular cells into osteogenic ones. Using electron microscopy and cytochemistry we studied perivsacular cells in metaphysis of the rats femoral bones under conditions of modeling microgravity (28 days duration) and in femoral bones metaphyses of rats flown on board of the space laboratory (Spacelab - 2) It was revealed that population of the perivascular cells is not homogeneous in adaptive zones of the remodeling in both control and test groups (lowering support loading). This population comprises adjacent to endothelium little differentiated forms and isolated cells with differentiation features (specific volume of rough endoplasmic reticulum in cytoplasm is increased). Majority of the perivascular cells in the control group reveals reaction to alkaline phosphatase (marker of the osteogenic differentiation). In little differentiated cells this reaction is registered in nucleolus, nucleous and cytoplasm. In differentiating cells activity of the alkaline phosphatase is also detected on the outer surface of the cellular membrane. Unlike the control group in the bones of animals under microgravitaty reaction to the alkaline phosphatase is registered not for all cells of perivascular population. Part of the differentiating perivascular cells does not contain a product of the reaction. There is also visible trend of individual alkaline phosphatase containing perivascular cells amounts decrease (i.e. osteogenic cells-precursors). Under microgravity some little differentiated perivascular cells reveal destruction signs. Found decrease trend of the alkaline phosphatase containing cells (i.e. osteogenic cells) number in perivascular cells population. It is one of the mechanisms of the osteogenic process intensity decrease in bones due to lowering support loading on the bone skeleton. In particular this is confirmed by the fact that in the zones of adaptive remodeling we found fibroblasts and fibrosis zones - areas filled with non mineralized collagen fibrils on the bones surfaces. Hence it should be considered that lowering (removal) support loading slows down (or blocks) osteogenic differentiation of the perivascular cells part and stimulates differentiation of the fibroblast cells. Obtained data considered as one of the cellular mechanisms of the adaptive reactions development in spongy bone under microgravity which could lead to the bone mass loss.

Materials for Neural Differentiation, Trans-Differentiation, and Modeling of Neurological Disease.

PubMed

Gong, Lulu; Cao, Lining; Shen, Zhenmin; Shao, Li; Gao, Shaorong; Zhang, Chao; Lu, Jianfeng; Li, Weida

2018-04-01

Neuron regeneration from pluripotent stem cells (PSCs) differentiation or somatic cells trans-differentiation is a promising approach for cell replacement in neurodegenerative diseases and provides a powerful tool for investigating neural development, modeling neurological diseases, and uncovering the mechanisms that underlie diseases. Advancing the materials that are applied in neural differentiation and trans-differentiation promotes the safety, efficiency, and efficacy of neuron regeneration. In the neural differentiation process, matrix materials, either natural or synthetic, not only provide a structural and biochemical support for the monolayer or three-dimensional (3D) cultured cells but also assist in cell adhesion and cell-to-cell communication. They play important roles in directing the differentiation of PSCs into neural cells and modeling neurological diseases. For the trans-differentiation of neural cells, several materials have been used to make the conversion feasible for future therapy. Here, the most current applications of materials for neural differentiation for PSCs, neuronal trans-differentiation, and neurological disease modeling is summarized and discussed. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Gene amplification during myogenic differentiation

PubMed Central

Fischer, Ulrike; Ludwig, Nicole; Raslan, Abdulrahman; Meier, Carola; Meese, Eckart

2016-01-01

Gene amplifications are mostly an attribute of tumor cells and drug resistant cells. Recently, we provided evidence for gene amplifications during differentiation of human and mouse neural progenitor cells. Here, we report gene amplifications in differentiating mouse myoblasts (C2C12 cells) covering a period of 7 days including pre-fusion, fusion and post-fusion stages. After differentiation induction we found an increase in copy numbers of CDK4 gene at day 3, of NUP133 at days 4 and 7, and of MYO18B at day 4. The amplification process was accompanied by gamma-H2AX foci that are indicative of double stand breaks. Amplifications during the differentiating process were also found in primary human myoblasts with the gene CDK4 and NUP133 amplified both in human and mouse myoblasts. Amplifications of NUP133 and CDK4 were also identified in vivo on mouse transversal cryosections at stage E11.5. In the course of myoblast differentiation, we found amplifications in cytoplasm indicative of removal of amplified sequences from the nucleus. The data provide further evidence that amplification is a fundamental mechanism contributing to the differentiation process in mammalians. PMID:26760505

Silk scaffolds with tunable mechanical capability for cell differentiation

PubMed Central

Bai, Shumeng; Han, Hongyan; Huang, Xiaowei; Xu, Weian; Kaplan, David L.; Zhu, Hesun; Lu, Qiang

2015-01-01

Bombyx mori silk fibroin is a promising biomaterial for tissue regeneration and is usually considered an “inert” material with respect to actively regulating cell differentiation due to few specific cell signaling peptide domains in the primary sequence and the generally stiffer mechanical properties due to crystalline content formed in processing. In the present study, silk fibroin porous 3D scaffolds with nanostructures and tunable stiffness were generated via a silk fibroin nanofiber-assisted lyophilization process. The silk fibroin nanofibers with high β-sheet content were added into the silk fibroin solutions to modulate the self-assembly, and to directly induce water-insoluble scaffold formation after lyophilization. Unlike previously reported silk fibroin scaffold formation processes, these new scaffolds had lower overall β-sheet content and softer mechanical properties for improved cell compatibility. The scaffold stiffness could be further tuned to match soft tissue mechanical properties, which resulted in different differentiation outcomes with rat bone marrow-derived mesenchymal stem cells towards myogenic and endothelial cells, respectively. Therefore, these silk fibroin scaffolds regulate cell differentiation outcomes due to their mechanical features. PMID:25858557

Centriole, differentiation, and senescence.

PubMed

Tkemaladze, J; Chichinadze, K

2010-01-01

Irreversible differentiation (change of morphogenetic status) and programmed death (apoptosis) are observed only in somatic cells, and cell division is the only way by which the morphogenetic status of the offspring cells may be modified. It is known that there is a fixed limit to the number of possible cell divisions, the so-called Hayflick limit. Existing links between cell division, differentiation, and apoptosis make it possible to conclude that all of these processes could be controlled by a single self-reproducing structure. Potential candidates for this replicable structure in a somatic cell are the chromosomes, mitochondria (both contain DNA), and centrioles. Centrioles (a diplosome, or pair of centrioles) are the most likely unit that can fully regulate the processes of irreversible differentiation, determination, and modification of the morphogenetic status. Centrioles may contain differently encoded RNA molecules stacked in a definite order, and during mitosis, these RNA molecules are released one by one into the cytoplasm. In the presence of reverse transcriptase and endonuclease, processing of this RNA presumably changes the status of repressed and potentially active genes and, subsequently, the morphogenetic status of a cell.

Oligodendroglial p130Cas Is a Target of Fyn Kinase Involved in Process Formation, Cell Migration and Survival

PubMed Central

Gonsior, Constantin; Binamé, Fabien; Frühbeis, Carsten; Bauer, Nina M.; Hoch-Kraft, Peter; Luhmann, Heiko J.; Trotter, Jacqueline; White, Robin

2014-01-01

Oligodendrocytes are the myelinating glial cells of the central nervous system. In the course of brain development, oligodendrocyte precursor cells migrate, scan the environment and differentiate into mature oligodendrocytes with multiple cellular processes which recognize and ensheath neuronal axons. During differentiation, oligodendrocytes undergo dramatic morphological changes requiring cytoskeletal rearrangements which need to be tightly regulated. The non-receptor tyrosine kinase Fyn plays a central role in oligodendrocyte differentiation and myelination. In order to improve our understanding of the role of oligodendroglial Fyn kinase, we have identified Fyn targets in these cells. Purification and mass-spectrometric analysis of tyrosine-phosphorylated proteins in response to overexpressed active Fyn in the oligodendrocyte precursor cell line Oli-neu, yielded the adaptor molecule p130Cas. We analyzed the function of this Fyn target in oligodendroglial cells and observed that reduction of p130Cas levels by siRNA affects process outgrowth, the thickness of cellular processes and migration behavior of Oli-neu cells. Furthermore, long term p130Cas reduction results in decreased cell numbers as a result of increased apoptosis in cultured primary oligodendrocytes. Our data contribute to understanding the molecular events taking place during oligodendrocyte migration and morphological differentiation and have implications for myelin formation. PMID:24586768

Proliferation and osteo/odontogenic differentiation of stem cells from apical papilla regulated by Zinc fingers and homeoboxes 2: An in vitro study.

PubMed

Wan, Fang; Gao, Lifen; Lu, Yating; Ma, Hongxin; Wang, Hongxing; Liang, Xiaohong; Wang, Yan; Ma, Chunhong

2016-01-15

In the process of tooth root development, stem cells from the apical papilla (SCAPs) can differentiate into odontoblasts and form root dentin, however, molecules regulating SCAPs differentiation have not been elucidated. Zinc fingers and homeoboxes 2 (ZHX2) is a novel transcriptional inhibitor. It is reported to modulate the development of nerve cells, liver cells, B cells, red blood cells, and so on. However, the role of ZHX2 in tooth root development remains unclear. In this study, we explored the potential role of ZHX2 in the process of SCAPs differentiation. The results showed that overexpression of ZHX2 upregulated the expression of osteo/odontogenic related genes and ALP activity, inhibited the proliferation of SCAPs. Consistently, ZHX2 knockdown reduced SCAPs mineralization and promoted SCAPs proliferation. These results indicated that ZHX2 plays a critical role in the proliferation and osteo/odontogenic differentiation of SCAPs. Copyright © 2015 Elsevier Inc. All rights reserved.

Integrated Transcriptomic and Epigenomic Analysis of Primary Human Lung Epithelial Cell Differentiation

PubMed Central

Marconett, Crystal N.; Zhou, Beiyun; Rieger, Megan E.; Selamat, Suhaida A.; Dubourd, Mickael; Fang, Xiaohui; Lynch, Sean K.; Stueve, Theresa Ryan; Siegmund, Kimberly D.; Berman, Benjamin P.

2013-01-01

Elucidation of the epigenetic basis for cell-type specific gene regulation is key to gaining a full understanding of how the distinct phenotypes of differentiated cells are achieved and maintained. Here we examined how epigenetic changes are integrated with transcriptional activation to determine cell phenotype during differentiation. We performed epigenomic profiling in conjunction with transcriptomic profiling using in vitro differentiation of human primary alveolar epithelial cells (AEC). This model recapitulates an in vivo process in which AEC transition from one differentiated cell type to another during regeneration following lung injury. Interrogation of histone marks over time revealed enrichment of specific transcription factor binding motifs within regions of changing chromatin structure. Cross-referencing of these motifs with pathways showing transcriptional changes revealed known regulatory pathways of distal alveolar differentiation, such as the WNT and transforming growth factor beta (TGFB) pathways, and putative novel regulators of adult AEC differentiation including hepatocyte nuclear factor 4 alpha (HNF4A), and the retinoid X receptor (RXR) signaling pathways. Inhibition of the RXR pathway confirmed its functional relevance for alveolar differentiation. Our incorporation of epigenetic data allowed specific identification of transcription factors that are potential direct upstream regulators of the differentiation process, demonstrating the power of this approach. Integration of epigenomic data with transcriptomic profiling has broad application for the identification of regulatory pathways in other models of differentiation. PMID:23818859

Early-stage detection of VE-cadherin during endothelial differentiation of human mesenchymal stem cells using SPR biosensor.

PubMed

Fathi, Farzaneh; Rezabakhsh, Aysa; Rahbarghazi, Reza; Rashidi, Mohammad-Reza

2017-10-15

Surface plasmon resonance (SPR) biosensors are most commonly applied for real-time dynamic analysis and measurement of interactions in bio-molecular studies and cell-surface analysis without the need for labeling processes. Up to present, SPR application in stem cell biology and biomedical sciences was underused. Herein, a very simple and sensitive method was developed to evaluate human mesenchymal stem cells trans-differentiation to endothelial lineage of over a period of 14 days based on VE-cadherin biomarker. The SPR signals increased with the increase of the amount of VE-cadherin expression on the cell surface during cell differentiation process. The method was able to detect ≈27 cells permm 2 . No significant effect was observed on the cell viability during the cell attachment to the surface of immune-reactive biochips and during the SPR analysis. Using this highly sensitive SPR method, it was possible to sense the early stage of endothelial differentiation on day 3 in label-free form, whereas flow cytometry and fluorescent microscopy methods were found unable to detect the cell differentiation at the same time. Therefore, the proposed method can rapidly and accurately detect cell differentiation in live cells and label-free manner without any need of cell breakage and has great potential for both diagnostic and experimental approaches. Copyright © 2017. Published by Elsevier B.V.

Induction of temporally dissociated morphological and physiological differentiation of N1E-115 cells.

PubMed

Cosgrove, C; Cobbett, P

1991-07-01

Clonal cells derived from neural tumors have been widely used to study the processes of neuronal differentiation in vitro. The murine neuroblastoma clone N1E-115 has recently been shown to differentiate morphologically in response to removal of serum from the culture medium. In the present study, the nature and time course of electrophysiological differentiation of N1E-115 cells maintained in serum-free medium was examined. Differentiated cells had a higher resting potential and lower input conductance than nondifferentiated cells. Differentiated but not nondifferentiated cells generated current evoked action potentials, and differentiated cells fired spontaneous, repetitive action potentials after 13 days in serum-free medium. The rate of potential change during the depolarizing and repolarizing phases of the action potential became faster as the duration of maintenance of cells in serum-free medium increased. Remarkably, morphological differentiation appeared to be complete after exposure to serum-free medium for 5 days but electrophysiological differentiation was not complete until 13 days in this medium.

Stem Cell Differentiation Stage Factors and Their Role in Triggering Symmetry Breaking Processes during Cancer Development: A Quantum Field Theory Model for Reprogramming Cancer Cells to Healthy Phenotypes.

PubMed

Biava, Pier Mario; Burigana, Fabio; Germano, Roberto; Kurian, Philip; Verzegnassi, Claudio; Vitiello, Giuseppe

2017-09-20

A long history of research has pursued the use of embryonic factors isolated during cell differentiation processes for the express purpose of transforming cancer cells back to healthy phenotypes. Recent results have clarified that the substances present at different stages of cell differentiation-which we call stem cell differentiation stage factors (SCDSFs)-are proteins with low molecular weight and nucleic acids that regulate genomic expression. The present review summarizes how these substances, taken at different stages of cellular maturation, are able to retard proliferation of many human tumor cell lines and thereby reprogram cancer cells to healthy phenotypes. The model presented here is a quantum field theory (QFT) model in which SCDSFs are able to trigger symmetry breaking processes during cancer development. These symmetry breaking processes, which lie at the root of many phenomena in elementary particle physics and condensed matter physics, govern the phase transitions of totipotent cells to higher degrees of diversity and order, resulting in cell differentiation. In cancers, which share many genomic and metabolic similarities with embryonic stem cells, stimulated re-differentiation often signifies the phenotypic reversion back to health and non-proliferation. In addition to acting on key components of the cellular cycle, SCDSFs are able to reprogram cancer cells by delicately influencing the cancer microenvironment, modulating the electrochemistry and thus the collective electrodynamic behaviors between dipole networks in biomacromolecules and the interstitial water field. Coherent effects in biological water, which are derived from a dissipative QFT framework, may offer new diagnostic and therapeutic targets at a systemic level, before tumor instantiation occurs in specific tissues or organs. Thus, by including the environment as an essential component of our model, we may push the prevailing paradigm of mutation-driven oncogenesis toward a closer description of reality. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Induction of endoplasmic reticulum calcium pump expression during early leukemic B cell differentiation.

PubMed

Aït Ghezali, Lamia; Arbabian, Atousa; Roudot, Hervé; Brouland, Jean-Philippe; Baran-Marszak, Fanny; Salvaris, Evelyn; Boyd, Andrew; Drexler, Hans G; Enyedi, Agnes; Letestu, Remi; Varin-Blank, Nadine; Papp, Bela

2017-06-26

Endoplasmic reticulum (ER) calcium storage and release play important roles in B lymphocyte maturation, survival, antigen-dependent cell activation and immunoglobulin synthesis. Calcium is accumulated in the endoplasmic reticulum (ER) by Sarco/Endoplasmic Reticulum Calcium ATPases (SERCA enzymes). Because lymphocyte function is critically dependent on SERCA activity, it is important to understand qualitative and quantitative changes of SERCA protein expression that occur during B lymphoid differentiation and leukemogenesis. In this work we investigated the modulation of SERCA expression during the pharmacologically induced differentiation of leukemic precursor B lymphoblast cell lines that carry the E2A-PBX1 fusion oncoprotein. Changes of SERCA levels during differentiation were determined and compared to those of established early B lymphoid differentiation markers. SERCA expression of the cells was compared to that of mature B cell lines as well, and the effect of the direct inhibition of SERCA-dependent calcium transport on the differentiation process was investigated. We show that E2A-PBX1 + leukemia cells simultaneously express SERCA2 and SERCA3-type calcium pumps; however, their SERCA3 expression is markedly inferior to that of mature B cells. Activation of protein kinase C enzymes by phorbol ester leads to phenotypic differentiation of the cells, and this is accompanied by the induction of SERCA3 expression. Direct pharmacological inhibition of SERCA-dependent calcium transport during phorbol ester treatment interferes with the differentiation process. These data show that the calcium pump composition of the ER is concurrent with increased SERCA3 expression during the differentiation of precursor B acute lymphoblastic leukemia cells, that a cross-talk exists between SERCA function and the control of differentiation, and that SERCA3 may constitute an interesting new marker for the study of early B cell phenotype.

Neural Differentiation of Embryonic Stem Cells In Vitro: A Road Map to Neurogenesis in the Embryo

PubMed Central

Abranches, Elsa; Silva, Margarida; Pradier, Laurent; Schulz, Herbert; Hummel, Oliver; Henrique, Domingos; Bekman, Evguenia

2009-01-01

Background The in vitro generation of neurons from embryonic stem (ES) cells is a promising approach to produce cells suitable for neural tissue repair and cell-based replacement therapies of the nervous system. Available methods to promote ES cell differentiation towards neural lineages attempt to replicate, in different ways, the multistep process of embryonic neural development. However, to achieve this aim in an efficient and reproducible way, a better knowledge of the cellular and molecular events that are involved in the process, from the initial specification of neuroepithelial progenitors to their terminal differentiation into neurons and glial cells, is required. Methodology/Principal Findings In this work, we characterize the main stages and transitions that occur when ES cells are driven into a neural fate, using an adherent monolayer culture system. We established improved conditions to routinely produce highly homogeneous cultures of neuroepithelial progenitors, which organize into neural tube-like rosettes when they acquire competence for neuronal production. Within rosettes, neuroepithelial progenitors display morphological and functional characteristics of their embryonic counterparts, namely, apico-basal polarity, active Notch signalling, and proper timing of production of neurons and glia. In order to characterize the global gene activity correlated with each particular stage of neural development, the full transcriptome of different cell populations that arise during the in vitro differentiation protocol was determined by microarray analysis. By using embryo-oriented criteria to cluster the differentially expressed genes, we define five gene expression signatures that correlate with successive stages in the path from ES cells to neurons. These include a gene signature for a primitive ectoderm-like stage that appears after ES cells enter differentiation, and three gene signatures for subsequent stages of neural progenitor development, from an early stage that follows neural induction to a final stage preceding terminal differentiation. Conclusions/Significance Overall, our work confirms and extends the cellular and molecular parallels between monolayer ES cell neural differentiation and embryonic neural development, revealing in addition novel aspects of the genetic network underlying the multistep process that leads from uncommitted cells to differentiated neurons. PMID:19621087

Oscillatory Protein Expression Dynamics Endows Stem Cells with Robust Differentiation Potential

PubMed Central

Kaneko, Kunihiko

2011-01-01

The lack of understanding of stem cell differentiation and proliferation is a fundamental problem in developmental biology. Although gene regulatory networks (GRNs) for stem cell differentiation have been partially identified, the nature of differentiation dynamics and their regulation leading to robust development remain unclear. Herein, using a dynamical system modeling cell approach, we performed simulations of the developmental process using all possible GRNs with a few genes, and screened GRNs that could generate cell type diversity through cell-cell interactions. We found that model stem cells that both proliferated and differentiated always exhibited oscillatory expression dynamics, and the differentiation frequency of such stem cells was regulated, resulting in a robust number distribution. Moreover, we uncovered the common regulatory motifs for stem cell differentiation, in which a combination of regulatory motifs that generated oscillatory expression dynamics and stabilized distinct cellular states played an essential role. These findings may explain the recently observed heterogeneity and dynamic equilibrium in cellular states of stem cells, and can be used to predict regulatory networks responsible for differentiation in stem cell systems. PMID:22073296

Insulin receptor-mediated signaling via phospholipase C-γ regulates growth and differentiation in Drosophila.

PubMed

Murillo-Maldonado, Juan M; Zeineddine, Fouad Bou; Stock, Rachel; Thackeray, Justin; Riesgo-Escovar, Juan R

2011-01-01

Coordination between growth and patterning/differentiation is critical if appropriate final organ structure and size is to be achieved. Understanding how these two processes are regulated is therefore a fundamental and as yet incompletely answered question. Here we show through genetic analysis that the phospholipase C-γ (PLC-γ) encoded by small wing (sl) acts as such a link between growth and patterning/differentiation by modulating some MAPK outputs once activated by the insulin pathway; particularly, sl promotes growth and suppresses ectopic differentiation in the developing eye and wing, allowing cells to attain a normal size and differentiate properly. sl mutants have previously been shown to have a combination of both growth and patterning/differentiation phenotypes: small wings, ectopic wing veins, and extra R7 photoreceptor cells. We show here that PLC-γ activated by the insulin pathway participates broadly and positively during cell growth modulating EGF pathway activity, whereas in cell differentiation PLC-γ activated by the insulin receptor negatively regulates the EGF pathway. These roles require different SH2 domains of PLC-γ, and act via classic PLC-γ signaling and EGF ligand processing. By means of PLC-γ, the insulin receptor therefore modulates differentiation as well as growth. Overall, our results provide evidence that PLC-γ acts during development at a time when growth ends and differentiation begins, and is important for proper coordination of these two processes.

Quantitative Proteomics Uncovers Novel Factors Involved in Developmental Differentiation of Trypanosoma brucei

PubMed Central

Dejung, Mario; Subota, Ines; Bucerius, Ferdinand; Dindar, Gülcin; Freiwald, Anja; Engstler, Markus; Boshart, Michael; Butter, Falk; Janzen, Christian J.

2016-01-01

Developmental differentiation is a universal biological process that allows cells to adapt to different environments to perform specific functions. African trypanosomes progress through a tightly regulated life cycle in order to survive in different host environments when they shuttle between an insect vector and a vertebrate host. Transcriptomics has been useful to gain insight into RNA changes during stage transitions; however, RNA levels are only a moderate proxy for protein abundance in trypanosomes. We quantified 4270 protein groups during stage differentiation from the mammalian-infective to the insect form and provide classification for their expression profiles during development. Our label-free quantitative proteomics study revealed previously unknown components of the differentiation machinery that are involved in essential biological processes such as signaling, posttranslational protein modifications, trafficking and nuclear transport. Furthermore, guided by our proteomic survey, we identified the cause of the previously observed differentiation impairment in the histone methyltransferase DOT1B knock-out strain as it is required for accurate karyokinesis in the first cell division during differentiation. This epigenetic regulator is likely involved in essential chromatin restructuring during developmental differentiation, which might also be important for differentiation in higher eukaryotic cells. Our proteome dataset will serve as a resource for detailed investigations of cell differentiation to shed more light on the molecular mechanisms of this process in trypanosomes and other eukaryotes. PMID:26910529

Differentiation induction of mouse embryonic stem cells into sinus node-like cells by suramin

PubMed Central

Wiese, Cornelia; Nikolova, Teodora; Zahanich, Ihor; Sulzbacher, Sabine; Fuchs, Joerg; Yamanaka, Satoshi; Graf, Eva; Ravens, Ursula; Boheler, Kenneth R.; Wobus, Anna M.

2015-01-01

Background Embryonic stem (ES) cells differentiate into cardiac phenotypes representing early pacemaker-, atrial-, ventricular-, and sinus node-like cells, however, ES-derived specification into sinus nodal cells is not yet known. By using the naphthylamine derivative of urea, suramin, we were able to follow the process of cardiac specialization into sinus node-like cells. Methods Differentiating mouse ES cells were treated with suramin (500 μM) from day 5 to 7 of embryoid body formation, and cells were analysed for their differentiation potential via morphological analysis, flow cytometry, RT-PCR, immunohistochemistry and patch clamp analysis. Results Application of suramin resulted in an increased number of cardiac cells, but inhibition of neuronal, skeletal muscle and definitive endoderm differentiation. Immediately after suramin treatment, a decreased mesendoderm differentiation was found. Brachyury, FGF10, Wnt8 and Wnt3a transcript levels were significantly down-regulated, followed by a decrease in mesoderm- and cardiac progenitor-specific markers BMP2, GATA4/5, Wnt11, Isl1, Nkx2.5 and Tbx5 immediately after removal of the substance. With continued differentiation, a significant up-regulation of Brachyury, FGF10 and GATA5 transcript levels was observed, whereas Nkx2.5, Isl1, Tbx5, BMP2 and Wnt11 levels were normalized to control levels. At advanced differentiation stages, sinus node-specific HCN4, Tbx2 and Tbx3 transcript levels were significantly up-regulated. Immunofluorescence and patch-clamp analysis confirmed the increased number of sinus node-like cells, and electrophysiological analysis revealed a lower number of atrial- and ventricular-like cardiomyocytes following suramin treatment. Conclusion We conclude that the interference of suramin with the cardiac differentiation process modified mesoderm- and cardiac-specific gene expression resulting in enhanced formation of sinus node-like cells. PMID:19775764

Wnt/β-Catenin Signaling Determines the Vasculogenic Fate of Postnatal Mesenchymal Stem Cells.

PubMed

Zhang, Zhaocheng; Nör, Felipe; Oh, Min; Cucco, Carolina; Shi, Songtao; Nör, Jacques E

2016-06-01

Vasculogenesis is the process of de novo blood vessel formation observed primarily during embryonic development. Emerging evidence suggest that postnatal mesenchymal stem cells are capable of recapitulating vasculogenesis when these cells are engaged in tissue regeneration. However, the mechanisms underlining the vasculogenic differentiation of mesenchymal stem cells remain unclear. Here, we used stem cells from human permanent teeth (dental pulp stem cells [DPSC]) or deciduous teeth (stem cells from human exfoliated deciduous teeth [SHED]) as models of postnatal primary human mesenchymal stem cells to understand mechanisms regulating their vasculogenic fate. GFP-tagged mesenchymal stem cells seeded in human tooth slice/scaffolds and transplanted into immunodeficient mice differentiate into human blood vessels that anastomize with the mouse vasculature. In vitro, vascular endothelial growth factor (VEGF) induced the vasculogenic differentiation of DPSC and SHED via potent activation of Wnt/β-catenin signaling. Further, activation of Wnt signaling is sufficient to induce the vasculogenic differentiation of postnatal mesenchymal stem cells, while Wnt inhibition blocked this process. Notably, β-catenin-silenced DPSC no longer differentiate into endothelial cells in vitro, and showed impaired vasculogenesis in vivo. Collectively, these data demonstrate that VEGF signaling through the canonical Wnt/β-catenin pathway defines the vasculogenic fate of postnatal mesenchymal stem cells. Stem Cells 2016;34:1576-1587. © 2016 AlphaMed Press.

Sumoylation Dynamics During Keratinocyte Differentiation

PubMed Central

Deyrieux, Adeline F.; Rosas-Acosta, Germán; Ozbun, Michelle A.; Wilson, Van G.

2012-01-01

Summary SUMO modification regulates the activity of numerous transcription factors that have a direct role in cell cycle progression, apoptosis, cellular proliferation, and development, but its role in differentiation processes is less clear. Keratinocyte differentiation requires the coordinated activation of a series of transcription factors, and as several critical keratinocyte transcription factors are known to be SUMO substrates, we investigated the role of sumoylation in keratinocyte differentiation. In a human keratinocyte cell line model (HaCaT cells), calcium-induced differentiation led to the transient and coordinated transcriptional activation of the genes encoding critical sumoylation system components, including SAE1, SAE2, Ubc9, SENP1, Miz-1 (PIASxβ), SUMO2, and SUMO3. The increased gene expression resulted in higher levels of the respective proteins and changes in the pattern of sumoylated substrate proteins during the differentiation process. Similar to the HaCaT results, stratified human foreskin keratinocytes showed an upregulation of Ubc9 in the suprabasal layers. Lastly, abrogation of sumoylation by Gam1 expression severely disrupted normal HaCaT differentiation, consistent with an important role for sumoylation in the proper progression of this biological process. PMID:17164289

Changes in the population of perivascular cells in the bone tissue remodeling zones under microgravity

NASA Astrophysics Data System (ADS)

Katkova, Olena; Rodionova, Natalia; Shevel, Ivan

2016-07-01

Microgravity and long-term hypokinesia induce reduction both in bone mass and mineral saturation, which can lead to the development of osteoporosis and osteopenia. (Oganov, 2003). Reorganizations and adaptive remodeling processes in the skeleton bones occur in the topographical interconnection with blood capillaries and perivascular cells. Radioautographic studies with 3H- thymidine (Kimmel, Fee, 1980; Rodionova, 1989, 2006) have shown that in osteogenesis zones there is sequential differentiation process of the perivascular cells into osteogenic. Hence the study of populations of perivascular stromal cells in areas of destructive changes is actual. Perivascular cells from metaphysis of the rat femoral bones under conditions of modeling microgravity were studied using electron microscopy and cytochemistry (hindlimb unloading, 28 days duration) and biosatellite «Bion-M1» (duration of flight from April 19 till May 19, 2013 on C57, black mice). It was revealed that both control and test groups populations of the perivascular cells are not homogeneous in remodeling adaptive zones. These populations comprise of adjacent to endothelium poorly differentiated forms and isolated cells with signs of differentiation (specific increased volume of rough endoplasmic reticulum in cytoplasm). Majority of the perivascular cells in the control group (modeling microgravity) reveals reaction to alkaline phosphatase (marker of the osteogenic differentiation). In poorly differentiated cells this reaction is registered in nucleolus, nucleous and cytoplasm. In differentiating cells activity of the alkaline phosphatase is also detected on the outer surface of the cellular membrane. Unlike the control group in the bones of experimental animals reaction to the alkaline phosphatase is registered not in all cells of perivascular population. Part of the differentiating perivascular cells does not contain a product of the reaction. Under microgravity some poorly differentiated perivascular cells reveal signs of destruction. Thus it was found that number of the alkaline phosphatase containing cells (i.e. osteogenic cells) declines in perivascular cells population. It is one of the mechanisms of the osteogenic process decrease of intensity in bones because of lessening support loading on the bone skeleton. In the adaptive remodeling zones of bone tissue (near the vascular canals) in experiments fibroblasts and fibrosis zones were found - areas filled with non-mineralized collagen fibrils on the bones surfaces. Hence it should be considered that decrease (removal) of support loading slows down osteogenic differentiation of the part of perivascular cells and stimulates differentiation of the fibroblast cells. Obtained data is considered as one of the cellular mechanisms of the adaptive reactions development in spongy bone under microgravity which could lead to the bone mass loss.

miR-203 modulates epithelial differentiation of human embryonic stem cells towards epidermal stratification.

PubMed

Nissan, Xavier; Denis, Jérôme Alexandre; Saidani, Manoubia; Lemaitre, Gilles; Peschanski, Marc; Baldeschi, Christine

2011-08-15

The molecular mechanisms controlling the differentiation of human basal keratinocyte stem cells towards the epidermis are well characterized, whereas the earliest process leading to the specification of embryonic stem cells into keratinocytes is still not well understood. MicroRNAs are regulators of many cellular events, but evidence for microRNA acting on the differentiation of human embryonic stem cells into a specific lineage has been elusive. By using our recent protocol for obtaining functional keratinocytes from hESC, we attempted to analyze the role of microRNAs in the early stages of epidermal differentiation. Thus, we identified a set of 5 microRNAs, namely miR-200a, miR-200b, miR-203, miR-205 and miR-429, that are specifically overexpressed during the early stages of the differentiation process. Interestingly, our functional analyses revealed an instrumental role of miR-203, which had been previously shown to play a key role during the formation of the pluristratified epidermis by basal keratinocyte stem cells, in the early keratinocyte commitment. These results highlight the determinant and unique role of miR-203 during the entire process of epidermal development by extending its spectrum of action from the early commitment of embryonic stem cells to ultimate differentiation of the organ. Copyright © 2011 Elsevier Inc. All rights reserved.

Establishment of human induced pluripotent stem cell lines from normal fibroblast TIG-1.

PubMed

Kumazaki, Tsutomu; Kurata, Sayaka; Matsuo, Taira; Mitsui, Youji; Takahashi, Tomoko

2011-06-01

Normal human cells have a replicative life span and therefore senesce. Usually, normal human cell strains are differentiated cells and reach a terminally differentiated state after a number of cell divisions. At present, definitive differences are not known between replicative senescence and terminal differentiation. TIG-1 is a human fibroblast strain established from fetal lung and has been used extensively in studies of cellular senescence, and numerous data were accumulated at the molecular level. Recently, a method for generating induced pluripotent stem cells (iPSCs) was developed. Using the method, we introduced four reprogramming genes to TIG-1 fibroblasts and succeeded in isolating colonies that had embryonic stem cell (ESC)-like morphologies. They showed alkaline phosphatase activity and expressed ESC markers, as shown by immunostaining of OCT4, SOX2, SSEA4, and TRA-1-81 as well as reverse-transcription polymerase chain reaction (RT-PCR) for OCT4 and NANOG transcripts. Thus, we succeeded in establishing iPSC clones from TIG-1. The iPSC clones could differentiate to cells originated from all three germ-cell layers, as shown by RT-PCR, for messenger RNA (mRNA) expression of α-fetoprotein (endoderm), MSX1 (mesoderm) and microtubule-associated protein 2 (ectoderm), and by immunostaining for α-fetoprotein (endoderm), α-smooth muscle actin (mesoderm), and β-III-tubulin (ectoderm). The iPSCs formed teratoma containing the structures developed from all three germ-cell layers in severe combined immune-deficiency mice. Thus, by comparing the aging process of parental TIG-1 cells and the differentiation process of iPSC-derived fibrocytes to fibroblasts, we can reveal the exact differences in processes between senescence and terminal differentiation.

Dynamics and heterogeneity of a fate determinant during transition towards cell differentiation

DOE PAGES

Pelaez, Nicolas; Gavalda-Miralles, Arnau; Wang, Bao; ...

2015-11-19

Yan is an ETS-domain transcription factor responsible for maintaining Drosophila eye cells in a multipotent state. Yan is at the core of a regulatory network that determines the time and place in which cells transit from multipotency to one of several differentiated lineages. Using a fluorescent reporter for Yan expression, we observed a biphasic distribution of Yan in multipotent cells, with a rapid inductive phase and slow decay phase. Transitions to various differentiated states occurred over the course of this dynamic process, suggesting that Yan expression level does not strongly determine cell potential. Consistent with this conclusion, perturbing Yan expressionmore » by varying gene dosage had no effect on cell fate transitions. However, we observed that as cells transited to differentiation, Yan expression became highly heterogeneous and this heterogeneity was transient. Signals received via the EGF Receptor were necessary for the transience in Yan noise since genetic loss caused sustained noise. As a result, since these signals are essential for eye cells to differentiate, we suggest that dynamic heterogeneity of Yan is a necessary element of the transition process, and cell states are stabilized through noise reduction.« less

Perspective for special Gurdon issue for differentiation: can cell fusion inform nuclear reprogramming?

PubMed

Burns, David; Blau, Helen M

2014-07-01

Nuclear reprogramming was first shown to be possible by Sir John Gurdon over a half century ago. The process has been revolutionized by the production of induced pluripotent cells by overexpression of the four transcription factors discovered by Shinya Yamanaka, which now enables mammalian applications. Yet, reprogramming by a few transcription factors remains incomplete and inefficient, whether to pluripotent or differentiated cells. We propose that a better understanding of mechanistic insights based on developmental principles gained from heterokaryon studies may inform the process of directing cell fate, fundamentally and clinically. Copyright © 2014 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Rethinking differentiation: Stem cells, regeneration, and plasticity

PubMed Central

Alvarado, Alejandro Sánchez; Yamanaka, Shinya

2014-01-01

Cell differentiation is an essential process for the development, growth, reproduction and longevity of all multicellular organisms, and its regulation has been the focus of intense investigation for the past 4 decades. The study of natural and induced stem cells has ushered an age of re-examination of what it means to be a stem or a differentiated cell. Past and recent discoveries in plants and animals, as well as novel experimental manipulations are beginning to erode many of these established concepts, and are forcing a re-evaluation of the experimental systems and paradigms presently being used to explore these and other biological process. PMID:24679530

Response of the flat cochlear epithelium to forced expression of Atoh1.

PubMed

Izumikawa, Masahiko; Batts, Shelley A; Miyazawa, Toru; Swiderski, Donald L; Raphael, Yehoash

2008-06-01

Following hair cell elimination in severely traumatized cochleae, differentiated supporting cells are often replaced by a simple epithelium with cuboidal or flat appearance. Atoh1 (previously Math1) is a basic helix-loop-helix transcription factor critical to hair cell differentiation during mammalian embryogenesis. Forced expression of Atoh1 in the differentiated supporting cell population can induce transdifferentiation leading to hair cell regeneration. Here, we examined the outcome of adenovirus mediated over-expression of Atoh1 in the non-sensory cells of the flat epithelium. We determined that seven days after unilateral elimination of hair cells with neomycin, differentiated supporting cells are absent, replaced by a flat epithelium. Nerve processes were also missing from the auditory epithelium, with the exception of infrequent looping nerve processes above the habenula perforata. We then inoculated an adenovirus vector with Atoh1 insert into the scala media of the deafened cochlea. The inoculation resulted in upregulation of Atoh1 in the flat epithelium. However, two months after the inoculation, Atoh1-treated ears did not exhibit clear signs of hair cell regeneration. Combined with previous data on induction of supporting cell to hair cell transdifferentiation by forced expression of Atoh1, these results suggest that the presence of differentiated supporting cells in the organ of Corti is necessary for transdifferentiation to occur.

Overlapping DNA Methylation Dynamics in Mouse Intestinal Cell Differentiation and Early Stages of Malignant Progression

PubMed Central

Forn, Marta; Díez-Villanueva, Anna; Merlos-Suárez, Anna; Muñoz, Mar; Lois, Sergi; Carriò, Elvira; Jordà, Mireia; Bigas, Anna; Batlle, Eduard; Peinado, Miguel A.

2015-01-01

Mouse models of intestinal crypt cell differentiation and tumorigenesis have been used to characterize the molecular mechanisms underlying both processes. DNA methylation is a key epigenetic mark and plays an important role in cell identity and differentiation programs and cancer. To get insights into the dynamics of cell differentiation and malignant transformation we have compared the DNA methylation profiles along the mouse small intestine crypt and early stages of tumorigenesis. Genome-scale analysis of DNA methylation together with microarray gene expression have been applied to compare intestinal crypt stem cells (EphB2high), differentiated cells (EphB2negative), ApcMin/+ adenomas and the corresponding non-tumor adjacent tissue, together with small and large intestine samples and the colon cancer cell line CT26. Compared with late stages, small intestine crypt differentiation and early stages of tumorigenesis display few and relatively small changes in DNA methylation. Hypermethylated loci are largely shared by the two processes and affect the proximities of promoter and enhancer regions, with enrichment in genes associated with the intestinal stem cell signature and the PRC2 complex. The hypermethylation is progressive, with minute levels in differentiated cells, as compared with intestinal stem cells, and reaching full methylation in advanced stages. Hypomethylation shows different signatures in differentiation and cancer and is already present in the non-tumor tissue adjacent to the adenomas in ApcMin/+ mice, but at lower levels than advanced cancers. This study provides a reference framework to decipher the mechanisms driving mouse intestinal tumorigenesis and also the human counterpart. PMID:25933092

Exploring the Mechanisms of Differentiation, Dedifferentiation, Reprogramming and Transdifferentiation

PubMed Central

Xu, Li; Zhang, Kun; Wang, Jin

2014-01-01

We explored the underlying mechanisms of differentiation, dedifferentiation, reprogramming and transdifferentiation (cell type switchings) from landscape and flux perspectives. Lineage reprogramming is a new regenerative method to convert a matured cell into another cell including direct transdifferentiation without undergoing a pluripotent cell state and indirect transdifferentiation with an initial dedifferentiation-reversion (reprogramming) to a pluripotent cell state. Each cell type is quantified by a distinct valley on the potential landscape with higher probability. We investigated three driving forces for cell fate decision making: stochastic fluctuations, gene regulation and induction, which can lead to cell type switchings. We showed that under the driving forces the direct transdifferentiation process proceeds from a differentiated cell valley to another differentiated cell valley through either a distinct stable intermediate state or a certain series of unstable indeterminate states. The dedifferentiation process proceeds through a pluripotent cell state. Barrier height and the corresponding escape time from the valley on the landscape can be used to quantify the stability and efficiency of cell type switchings. We also uncovered the mechanisms of the underlying processes by quantifying the dominant biological paths of cell type switchings on the potential landscape. The dynamics of cell type switchings are determined by both landscape gradient and flux. The flux can lead to the deviations of the dominant biological paths for cell type switchings from the naively expected landscape gradient path. As a result, the corresponding dominant paths of cell type switchings are irreversible. We also classified the mechanisms of cell fate development from our landscape theory: super-critical pitchfork bifurcation, sub-critical pitchfork bifurcation, sub-critical pitchfork with two saddle-node bifurcation, and saddle-node bifurcation. Our model showed good agreements with the experiments. It provides a general framework to explore the mechanisms of differentiation, dedifferentiation, reprogramming and transdifferentiation. PMID:25133589

Control of growth and squamous differentiation in normal human bronchial epithelial cells by chemical and biological modifiers and transferred genes.

PubMed Central

Pfeifer, A M; Lechner, J F; Masui, T; Reddel, R R; Mark, G E; Harris, C C

1989-01-01

The majority of human lung cancers arise from bronchial epithelial cells. The normal pseudostratified bronchial epithelium is composed of basal, mucous, and ciliated cells. This multi-differentiated epithelium usually responds to xenobiotics and physical injury by undergoing basal cell hyperplasia, mucous cell hyperplasia, and squamous metaplasia. One step of the multistage process of carcinogenesis is thought to involve aberrations in control of the squamous metaplastic processes. Decreased responsiveness to regulators of terminal squamous differentiation may confer a selective clonal expansion advantage to an initiated cell. We studied the effects of endogenous [e.g., transforming growth factor beta 1 (TGF-beta 1) and serum] and exogenous [e.g., 12-O-tetradecanoyl-13-phorbol-acetate (TPA), tobacco smoke condensate, and aldehydes] modifiers of normal human bronchial epithelial (NHBE) cell in a serum-free culture system. NHBE cells are growth inhibited by all of these compounds and induced to undergo squamous differentiation by TGF-beta 1 or TPA. In contrast, lung carcinoma cell lines are relatively resistant to inducers of terminal squamous differentiation which may provide them with a selective growth advantage. Chemical agents and activated protooncogenes (ras,raf,myc) altered the response to endogenous and exogenous inducers of squamous differentiation and caused extended cellular lifespan, aneuploidy, and/or tumorigenicity. The data suggest a close relationship between dysregulation of terminal differentiation pathways and neoplastic transformation of human bronchial epithelial cells. PMID:2538323

Expression of Ulex europaeus agglutinin I lectin-binding sites in squamous cell carcinomas and their absence in basal cell carcinomas. Indicator of tumor type and differentiation.

PubMed

Heng, M C; Fallon-Friedlander, S; Bennett, R

1992-06-01

Lectins bind tightly to carbohydrate moieties on cell surfaces. Alterations in lectin binding have been reported to accompany epidermal cell differentiation, marking alterations in membrane sugars during this process. The presence of UEA I (Ulex europaeus agglutinin I) L-fucose-specific lectin-binding sites has been used as a marker for terminally differentiated (committed) keratinocytes. In this article, we report the presence of UEA-I-binding sites on squamous keratinocytes of well-differentiated squamous cell carcinomas, with patchy loss of UEA I positivity on poorly differentiated cells of squamous cell carcinomas, suggesting a possible use for this technique in the rapid assessment of less differentiated areas within the squamous cell tumor. The absence of UEA-I-binding sites on basal cell carcinomas may be related to an inability of cells comprising this tumor to convert the L-D-pyranosyl moiety on basal cells to the L-fucose moiety, resulting in an inability of basal cell carcinoma cell to undergo terminal differentiation into a committed keratinocyte.

Bach2 is involved in neuronal differentiation of N1E-115 neuroblastoma cells.

PubMed

Shim, Ki Shuk; Rosner, Margit; Freilinger, Angelika; Lubec, Gert; Hengstschläger, Markus

2006-07-15

Bach1 and Bach2 are evolutionarily related members of the BTB-basic region leucine zipper transcription factor family. We found that Bach2 downregulates cell proliferation of N1E-115 cells and negatively affects their potential to differentiate. Nuclear localization of the cyclin-dependent kinase inhibitor p21 is known to arrest cell cycle progression, and cytoplasmic p21 has been shown to promote neuronal differentiation of N1E-115 cells. We found that ectopic Bach2 causes upregulation of p21 expression in the nucleus and in the cytoplasm in undifferentiated N1E-115 cells. In differentiated cells, Bach2 specifically triggers upregulation of cytoplasmic p21. Our data suggest that Bach2 expression could represent a switch during the process of neuronal differentiation. Bach2 is not expressed in neuronal precursor cells. It would have negative effects on proliferation and differentiation of these cells. In differentiated neuronal cells Bach2 expression is upregulated, which could allow Bach2 to function as a gatekeeper of the differentiated status.

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

Shim, Ki Shuk; Department of Neonatology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna; Rosner, Margit

Bach1 and Bach2 are evolutionarily related members of the BTB-basic region leucine zipper transcription factor family. We found that Bach2 downregulates cell proliferation of N1E-115 cells and negatively affects their potential to differentiate. Nuclear localization of the cyclin-dependent kinase inhibitor p21 is known to arrest cell cycle progression, and cytoplasmic p21 has been shown to promote neuronal differentiation of N1E-115 cells. We found that ectopic Bach2 causes upregulation of p21 expression in the nucleus and in the cytoplasm in undifferentiated N1E-115 cells. In differentiated cells, Bach2 specifically triggers upregulation of cytoplasmic p21. Our data suggest that Bach2 expression could representmore » a switch during the process of neuronal differentiation. Bach2 is not expressed in neuronal precursor cells. It would have negative effects on proliferation and differentiation of these cells. In differentiated neuronal cells Bach2 expression is upregulated, which could allow Bach2 to function as a gatekeeper of the differentiated status.« less

Voltage-Gated K+ Channel, Kv3.3 Is Involved in Hemin-Induced K562 Differentiation

PubMed Central

Song, Min Seok; Choi, Seon Young; Ryu, Pan Dong; Lee, So Yeong

2016-01-01

Voltage-gated K+ (Kv) channels are well known to be involved in cell proliferation. However, even though cell proliferation is closely related to cell differentiation, the relationship between Kv channels and cell differentiation remains poorly investigated. This study demonstrates that Kv3.3 is involved in K562 cell erythroid differentiation. Down-regulation of Kv3.3 using siRNA-Kv3.3 increased hemin-induced K562 erythroid differentiation through decreased activation of signal molecules such as p38, cAMP response element-binding protein, and c-fos. Down-regulation of Kv3.3 also enhanced cell adhesion by increasing integrin β3 and this effect was amplified when the cells were cultured with fibronectin. The Kv channels, or at least Kv3.3, appear to be associated with cell differentiation; therefore, understanding the mechanisms of Kv channel regulation of cell differentiation would provide important information regarding vital cellular processes. PMID:26849432

A lysigenic programmed cell death-dependent process shapes schizogenously formed aerenchyma in the stems of the waterweed Egeria densa

PubMed Central

Bartoli, G.; Forino, L. M. C.; Durante, M.; Tagliasacchi, A. M.

2015-01-01

Background and Aims Plant adaptation to submergence can include the formation of prominent aerenchyma to facilitate gas exchange. The aim of this study was to characterize the differentiation of the constitutive aerenchyma in the stem of the aquatic macrophyte Egeria densa (Hydrocharitaceae) and to verify if any form of cell death might be involved. Methods Plants were collected from a pool in a botanical garden. Aerenchyma differentiation and apoptotic hallmarks were investigated by light microscopy and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay coupled with genomic DNA extraction and gel electrophoresis (DNA laddering assay). Cell viability and the occurrence of peroxides and nitric oxide (NO) were determined histochemically using specific fluorogenic probes. Key Results Aerenchyma differentiation started from a hexagonally packed pre-aerenchymatic tissue and, following a basipetal and centripetal developmental pattern, produced a honeycomb arrangement. After an early schizogenous differentiation process, a late lysigenous programmed cell death- (PCD) dependent mechanism occurred. This was characterized by a number of typical apoptotic hallmarks, including DNA fragmentation, chromatin condensation, apoptotic-like bodies, partial cell wall lysis and plasmolysis. In addition, local increases in H2O2 and NO were observed and quantified. Conclusions The differentiation of cortical aerenchyma in the stem of E. densa is a complex process, consisting of a combination of an early schizogenous differentiation mechanism and a late lysigenous PCD-dependent process. The PCD remodels the architecture of the gas spaces previously formed schizogenously, and also results in a reduction of O2-consuming cells and in recycling of material derived from the lysigenic dismantling of the cells. PMID:26002256

A lysigenic programmed cell death-dependent process shapes schizogenously formed aerenchyma in the stems of the waterweed Egeria densa.

PubMed

Bartoli, G; Forino, L M C; Durante, M; Tagliasacchi, A M

2015-07-01

Plant adaptation to submergence can include the formation of prominent aerenchyma to facilitate gas exchange. The aim of this study was to characterize the differentiation of the constitutive aerenchyma in the stem of the aquatic macrophyte Egeria densa (Hydrocharitaceae) and to verify if any form of cell death might be involved. Plants were collected from a pool in a botanical garden. Aerenchyma differentiation and apoptotic hallmarks were investigated by light microscopy and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay coupled with genomic DNA extraction and gel electrophoresis (DNA laddering assay). Cell viability and the occurrence of peroxides and nitric oxide (NO) were determined histochemically using specific fluorogenic probes. Aerenchyma differentiation started from a hexagonally packed pre-aerenchymatic tissue and, following a basipetal and centripetal developmental pattern, produced a honeycomb arrangement. After an early schizogenous differentiation process, a late lysigenous programmed cell death- (PCD) dependent mechanism occurred. This was characterized by a number of typical apoptotic hallmarks, including DNA fragmentation, chromatin condensation, apoptotic-like bodies, partial cell wall lysis and plasmolysis. In addition, local increases in H2O2 and NO were observed and quantified. The differentiation of cortical aerenchyma in the stem of E. densa is a complex process, consisting of a combination of an early schizogenous differentiation mechanism and a late lysigenous PCD-dependent process. The PCD remodels the architecture of the gas spaces previously formed schizogenously, and also results in a reduction of O2-consuming cells and in recycling of material derived from the lysigenic dismantling of the cells. © The Author 2015. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Integrating human stem cell expansion and neuronal differentiation in bioreactors

PubMed Central

Serra, Margarida; Brito, Catarina; Costa, Eunice M; Sousa, Marcos FQ; Alves, Paula M

2009-01-01

Background Human stem cells are cellular resources with outstanding potential for cell therapy. However, for the fulfillment of this application, major challenges remain to be met. Of paramount importance is the development of robust systems for in vitro stem cell expansion and differentiation. In this work, we successfully developed an efficient scalable bioprocess for the fast production of human neurons. Results The expansion of undifferentiated human embryonal carcinoma stem cells (NTera2/cl.D1 cell line) as 3D-aggregates was firstly optimized in spinner vessel. The media exchange operation mode with an inoculum concentration of 4 × 105 cell/mL was the most efficient strategy tested, with a 4.6-fold increase in cell concentration achieved in 5 days. These results were validated in a bioreactor where similar profile and metabolic performance were obtained. Furthermore, characterization of the expanded population by immunofluorescence microscopy and flow cytometry showed that NT2 cells maintained their stem cell characteristics along the bioreactor culture time. Finally, the neuronal differentiation step was integrated in the bioreactor process, by addition of retinoic acid when cells were in the middle of the exponential phase. Neurosphere composition was monitored and neuronal differentiation efficiency evaluated along the culture time. The results show that, for bioreactor cultures, we were able to increase significantly the neuronal differentiation efficiency by 10-fold while reducing drastically, by 30%, the time required for the differentiation process. Conclusion The culture systems developed herein are robust and represent one-step-forward towards the development of integrated bioprocesses, bridging stem cell expansion and differentiation in fully controlled bioreactors. PMID:19772662

Differences in irradiation susceptibility and turnover between mucosal and connective tissue-type mast cells of mice

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

Fukuzumi, T.; Waki, N.; Kanakura, Y.

Although precursors of mast cells are derived from the bone marrow, phenotypes of mast cells are influenced by the tissues in which final differentiation occurs. Connective tissue-type mast cells (CTMC) and mucosal mast cells (MMC) are different in morphological, biochemical, immunological, and functional criteria. The purpose of the present study was to obtain information about the differentiation process of MMC. First, we compared changes in irradiation susceptibility in mice during the differentiation process of CTMC and MMC. The decrease in irradiation susceptibility was remarkable in the CTMC differentiation process, but it was moderate in that of MMC. Some morphologically identifiablemore » CTMC in the peritoneal cavity had proliferative potential and were highly radioresistant, whereas such a radioresistant population of MMC was not detectable in the gastric mucosa. Second, we estimated the turnover of CTMC and MMC by determining the proportion of mast cells that were labeled with continuously administered bromodeoxyuridine. The turnover of MMC was significantly faster than that of CTMC. The absence of the radioresistant mast cell population in the gastric mucosa appeared to be related to the short life span of MMC.« less

Metabolic phenotyping of a model of adipocyte differentiation

PubMed Central

Roberts, Lee D.; Virtue, Sam; Vidal-Puig, Antonio; Nicholls, Andrew W.

2009-01-01

The 3T3-L1 murine cell line is a robust and widely used model for the study of adipogenesis and processes occurring in mature adipocytes. The fibroblastic like cells can be induced by hormones to differentiate into mature adipocytes. In this study, the metabolic phenotype associated with differentiation of the 3T3-L1 cell line has been studied using gas chromatography-mass spectrometry, 1H nuclear magnetic resonance spectroscopy, liquid chromatography-mass spectrometry, direct infusion-mass spectrometry, and 13C substrate labeling in conjunction with multivariate statistics. The changes in metabolite concentrations at distinct periods during differentiation have been defined including alterations in the TCA cycle, glycolysis, the production of odd chain fatty acids by α-oxidation, fatty acid synthesis, fatty acid desaturation, polyamine biosynthesis, and trans-esterification to produce complex lipids. The metabolic changes induced during differentiation of the 3T3-L1 cell line were then compared with the metabolic differences between pre- and postdifferentiation primary adipocytes. These metabolic alterations reflect the changing role of the 3T3-L1 cells during differentiation, as well as possibly providing metabolic triggers to stimulate the processes which occur during differentiation. PMID:19602617

Brg1 coordinates multiple processes during retinogenesis and is a tumor suppressor in retinoblastoma

DOE PAGES

Aldiri, Issam; Ajioka, Itsuki; Xu, Beisi; ...

2015-12-01

Retinal development requires precise temporal and spatial coordination of cell cycle exit, cell fate specification, cell migration and differentiation. When this process is disrupted, retinoblastoma, a developmental tumor of the retina, can form. Epigenetic modulators are central to precisely coordinating developmental events, and many epigenetic processes have been implicated in cancer. Studying epigenetic mechanisms in development is challenging because they often regulate multiple cellular processes; therefore, elucidating the primary molecular mechanisms involved can be difficult. Here we explore the role of Brg1 (Smarca4) in retinal development and retinoblastoma in mice using molecular and cellular approaches. Brg1 was found to regulatemore » retinal size by controlling cell cycle length, cell cycle exit and cell survival during development. Brg1 was not required for cell fate specification but was required for photoreceptor differentiation and cell adhesion/polarity programs that contribute to proper retinal lamination during development. The combination of defective cell differentiation and lamination led to retinal degeneration in Brg1-deficient retinae. Despite the hypocellularity, premature cell cycle exit, increased cell death and extended cell cycle length, retinal progenitor cells persisted in Brg1-deficient retinae, making them more susceptible to retinoblastoma. In conclusion, ChIP-Seq analysis suggests that Brg1 might regulate gene expression through multiple mechanisms.« less

Brg1 coordinates multiple processes during retinogenesis and is a tumor suppressor in retinoblastoma

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

Aldiri, Issam; Ajioka, Itsuki; Xu, Beisi

Retinal development requires precise temporal and spatial coordination of cell cycle exit, cell fate specification, cell migration and differentiation. When this process is disrupted, retinoblastoma, a developmental tumor of the retina, can form. Epigenetic modulators are central to precisely coordinating developmental events, and many epigenetic processes have been implicated in cancer. Studying epigenetic mechanisms in development is challenging because they often regulate multiple cellular processes; therefore, elucidating the primary molecular mechanisms involved can be difficult. Here we explore the role of Brg1 (Smarca4) in retinal development and retinoblastoma in mice using molecular and cellular approaches. Brg1 was found to regulatemore » retinal size by controlling cell cycle length, cell cycle exit and cell survival during development. Brg1 was not required for cell fate specification but was required for photoreceptor differentiation and cell adhesion/polarity programs that contribute to proper retinal lamination during development. The combination of defective cell differentiation and lamination led to retinal degeneration in Brg1-deficient retinae. Despite the hypocellularity, premature cell cycle exit, increased cell death and extended cell cycle length, retinal progenitor cells persisted in Brg1-deficient retinae, making them more susceptible to retinoblastoma. In conclusion, ChIP-Seq analysis suggests that Brg1 might regulate gene expression through multiple mechanisms.« less

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.

P-channel differential multiple-time programmable memory cells by laterally coupled floating metal gate fin field-effect transistors

NASA Astrophysics Data System (ADS)

Wang, Tai-Min; Chien, Wei-Yu; Hsu, Chia-Ling; Lin, Chrong Jung; King, Ya-Chin

2018-04-01

In this paper, we present a new differential p-channel multiple-time programmable (MTP) memory cell that is fully compatible with advanced 16 nm CMOS fin field-effect transistors (FinFET) logic processes. This differential MTP cell stores complementary data in floating gates coupled by a slot contact structure, which make different read currents possible on a single cell. In nanoscale CMOS FinFET logic processes, the gate dielectric layer becomes too thin to retain charges inside floating gates for nonvolatile data storage. By using a differential architecture, the sensing window of the cell can be extended and maintained by an advanced blanket boost scheme. The charge retention problem in floating gate cells can be improved by periodic restoring lost charges when significant read window narrowing occurs. In addition to high programming efficiency, this p-channel MTP cells also exhibit good cycling endurance as well as disturbance immunity. The blanket boost scheme can remedy the charge loss problem under thin gate dielectrics.

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.

Osteogenic differentiation capacity of human skeletal muscle-derived progenitor cells.

PubMed

Oishi, Teruyo; Uezumi, Akiyoshi; Kanaji, Arihiko; Yamamoto, Naoki; Yamaguchi, Asami; Yamada, Harumoto; Tsuchida, Kunihiro

2013-01-01

Heterotopic ossification (HO) is defined as the formation of ectopic bone in soft tissue outside the skeletal tissue. HO is thought to result from aberrant differentiation of osteogenic progenitors within skeletal muscle. However, the precise origin of HO is still unclear. Skeletal muscle contains two kinds of progenitor cells, myogenic progenitors and mesenchymal progenitors. Myogenic and mesenchymal progenitors in human skeletal muscle can be identified as CD56(+) and PDGFRα(+) cells, respectively. The purpose of this study was to investigate the osteogenic differentiation potential of human skeletal muscle-derived progenitors. Both CD56(+) cells and PDGFRα(+) cells showed comparable osteogenic differentiation potential in vitro. However, in an in vivo ectopic bone formation model, PDGFRα(+) cells formed bone-like tissue and showed successful engraftment, while CD56(+) cells did not form bone-like tissue and did not adapt to an osteogenic environment. Immunohistological analysis of human HO sample revealed that many PDGFRα(+) cells were localized in proximity to ectopic bone formed in skeletal muscle. MicroRNAs (miRNAs) are known to regulate many biological processes including osteogenic differentiation. We investigated the participation of miRNAs in the osteogenic differentiation of PDGFRα(+) cells by using microarray. We identified miRNAs that had not been known to be involved in osteogenesis but showed dramatic changes during osteogenic differentiation of PDGFRα(+) cells. Upregulation of miR-146b-5p and -424 and downregulation of miR-7 during osteogenic differentiation of PDGFRα(+) cells were confirmed by quantitative real-time RT-PCR. Inhibition of upregulated miRNAs, miR-146b-5p and -424, resulted in the suppression of osteocyte maturation, suggesting that these two miRNAs have the positive role in the osteogenesis of PDGFRα(+) cells. Our results suggest that PDGFRα(+) cells may be the major source of HO and that the newly identified miRNAs may regulate osteogenic differentiation process of PDGFRα(+) cells.

Concise Review: Stem Cells in Osteoimmunology.

PubMed

Fierro, Fernando A; Nolta, Jan A; Adamopoulos, Iannis E

2017-06-01

Bone remodeling is a lifelong process in which mature bone tissue is removed from the skeleton by bone resorption and is replenished by new during ossification or bone formation. The remodeling cycle requires both the differentiation and activation of two cell types with opposing functions; the osteoclast, which orchestrates bone resorption, and the osteoblast, which orchestrates bone formation. The differentiation of these cells from their respective precursors is a process which has been overshadowed by enigma, particularly because the precise osteoclast precursor has not been identified and because the identification of skeletal stem cells, which give rise to osteoblasts, is very recent. Latest advances in the area of stem cell biology have enabled us to gain a better understanding of how these differentiation processes occur in physiological and pathological conditions. In this review we postulate that modulation of stem cells during inflammatory conditions is a necessary prerequisite of bone remodeling and therefore an essential new component to the field of osteoimmunology. In this context, we highlight the role of transcription factor nuclear factor of activated T cells cytoplasmic 1 (NFATc1), because it directly links inflammation with differentiation of osteoclasts and osteoblasts. Stem Cells 2017;35:1461-1467. © 2017 The Authors Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Derivation, expansion and differentiation of induced pluripotent stem cells in continuous suspension cultures

PubMed Central

Fluri, David A.; Tonge, Peter D.; Song, Hannah; Baptista, Ricardo P.; Shakiba, Nika; Shukla, Shreya; Clarke, Geoffrey; Nagy, Andras; Zandstra, Peter W.

2016-01-01

We demonstrate derivation of induced pluripotent stem cells (iPSCs) from terminally differentiated mouse cells in serum- and feeder-free stirred suspension cultures. Temporal analysis of global gene expression revealed high correlations between cells reprogrammed in suspension and cells reprogrammed in adhesion-dependent conditions. Suspension (S) reprogrammed iPSCs (SiPSCs) could be differentiated into all three germ layers in vitro and contributed to chimeric embryos in vivo. SiPSC generation allowed for efficient selection of reprogramming factor expressing cells based on their differential survival and proliferation in suspension. Seamless integration of SiPSC reprogramming and directed differentiation enabled the scalable production of functionally and phenotypically defined cardiac cells in a continuous single cell- and small aggregate-based process. This method is an important step towards the development of a robust PSC generation, expansion and differentiation technology. PMID:22447133

Application of a Parallelizable Perfusion Bioreactor for Physiologic 3D Cell Culture.

PubMed

Egger, Dominik; Spitz, Sarah; Fischer, Monica; Handschuh, Stephan; Glösmann, Martin; Friemert, Benedikt; Egerbacher, Monika; Kasper, Cornelia

2017-01-01

It is crucial but challenging to keep physiologic conditions during the cultivation of 3D cell scaffold constructs for the optimization of 3D cell culture processes. Therefore, we demonstrate the benefits of a recently developed miniaturized perfusion bioreactor together with a specialized incubator system that allows for the cultivation of multiple samples while screening different conditions. Hence, a decellularized bone matrix was tested towards its suitability for 3D osteogenic differentiation under flow perfusion conditions. Subsequently, physiologic shear stress and hydrostatic pressure (HP) conditions were optimized for osteogenic differentiation of human mesenchymal stem cells (MSCs). X-ray computed microtomography and scanning electron microscopy (SEM) revealed a closed cell layer covering the entire matrix. Osteogenic differentiation assessed by alkaline phosphatase activity and SEM was found to be increased in all dynamic conditions. Furthermore, screening of different fluid shear stress (FSS) conditions revealed 1.5 mL/min (equivalent to ∼10 mPa shear stress) to be optimal. However, no distinct effect of HP compared to flow perfusion without HP on osteogenic differentiation was observed. Notably, throughout all experiments, cells cultivated under FSS or HP conditions displayed increased osteogenic differentiation, which underlines the importance of physiologic conditions. In conclusion, the bioreactor system was used for biomaterial testing and to develop and optimize a 3D cell culture process for the osteogenic differentiation of MSCs. Due to its versatility and higher throughput efficiency, we hypothesize that this bioreactor/incubator system will advance the development and optimization of a variety of 3D cell culture processes. © 2017 S. Karger AG, Basel.

Critical role of histone demethylase Jmjd3 in the regulation of CD4+ T cell differentiation

PubMed Central

Ding, Xilai; Chepelev, Iouri; Zhou, Xikun; Zhao, Wei; Wei, Gang; Cui, Jun; Zhao, Keji; Wang, Helen Y.; Wang, Rong-Fu

2014-01-01

Epigenetic factors have been implicated in the regulation of CD4+ T cell differentiation. Jmjd3 plays a role in many biological processes, but its in vivo function in T cell differentiation remains unknown. Here, we report that Jmjd3 ablation promotes CD4+ T cell differentiation into Th2 and Th17 cells in the small intestine and colon, and inhibits T cell differentiation into Th1 cells under different cytokine-polarizing conditions and in a Th1-dependent colitis model. Jmjd3 deficiency also restrains the plasticity of the conversion of Th2, Th17 or Treg cells to Th1 cells. The skewing of T cell differentiation is concomitant with changes in the expression of key transcription factors and cytokines. H3K27me3 and H3K4me3 levels in Jmjd3-deficient cells are correlated with altered gene expression through interactions with specific transcription factors. Our results identify Jmjd3 as an epigenetic factor in T cell differentiation via changes in histone methylation and target gene expression. PMID:25531312

Single-cell RNA sequencing reveals metallothionein heterogeneity during hESC differentiation to definitive endoderm.

PubMed

Lu, Junjie; Baccei, Anna; Lummertz da Rocha, Edroaldo; Guillermier, Christelle; McManus, Sean; Finney, Lydia A; Zhang, Cheng; Steinhauser, Matthew L; Li, Hu; Lerou, Paul H

2018-04-01

Differentiation of human pluripotent stem cells towards definitive endoderm (DE) is the critical first step for generating cells comprising organs such as the gut, liver, pancreas and lung. This in-vitro differentiation process generates a heterogeneous population with a proportion of cells failing to differentiate properly and maintaining expression of pluripotency factors such as Oct4. RNA sequencing of single cells collected at four time points during a 4-day DE differentiation identified high expression of metallothionein genes in the residual Oct4-positive cells that failed to differentiate to DE. Using X-ray fluorescence microscopy and multi-isotope mass spectrometry, we discovered that high intracellular zinc level corresponds with persistent Oct4 expression and failure to differentiate. This study improves our understanding of the cellular heterogeneity during in-vitro directed differentiation and provides a valuable resource to improve DE differentiation efficiency. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

DNA replication fading as proliferating cells advance in their commitment to terminal differentiation.

PubMed

Estefanía, Monturus Ma; Ganier, Olivier; Hernández, Pablo; Schvartzman, Jorge B; Mechali, Marcel; Krimer, Dora B

2012-01-01

Terminal differentiation is the process by which cycling cells stop proliferating to start new specific functions. It involves dramatic changes in chromatin organization as well as gene expression. In the present report we used cell flow cytometry and genome wide DNA combing to investigate DNA replication during murine erythroleukemia-induced terminal cell differentiation. The results obtained indicated that the rate of replication fork movement slows down and the inter-origin distance becomes shorter during the precommitment and commitment periods before cells stop proliferating and accumulate in G1. We propose this is a general feature caused by the progressive heterochromatinization that characterizes terminal cell differentiation.

Cell Aggregation-induced FGF8 Elevation Is Essential for P19 Cell Neural Differentiation

PubMed Central

Wang, Chen; Xia, Caihong; Bian, Wei; Liu, Li; Lin, Wei; Chen, Ye-Guang; Ang, Siew-Lan

2006-01-01

FGF8, a member of the fibroblast growth factor (FGF) family, has been shown to play important roles in different developing systems. Mouse embryonic carcinoma P19 cells could be induced by retinoic acid (RA) to differentiate into neuroectodermal cell lineages, and this process is cell aggregation dependent. In this report, we show that FGF8 expression is transiently up-regulated upon P19 cell aggregation, and the aggregation-dependent FGF8 elevation is pluripotent stem cell related. Overexpressing FGF8 promotes RA-induced monolayer P19 cell neural differentiation. Inhibition of FGF8 expression by RNA interference or blocking FGF signaling by the FGF receptor inhibitor, SU5402, attenuates neural differentiation of the P19 cell. Blocking the bone morphogenetic protein (BMP) pathway by overexpressing Smad6 in P19 cells, we also show that FGF signaling plays a BMP inhibition–independent role in P19 cell neural differentiation. PMID:16641368

Hacking cell differentiation: transcriptional rerouting in reprogramming, lineage infidelity and metaplasia

PubMed Central

Regalo, Gonçalo; Leutz, Achim

2013-01-01

Initiating neoplastic cell transformation events are of paramount importance for the comprehension of regeneration and vanguard oncogenic processes but are difficult to characterize and frequently clinically overlooked. In epithelia, pre-neoplastic transformation stages are often distinguished by the appearance of phenotypic features of another differentiated tissue, termed metaplasia. In haemato/lymphopoietic malignancies, cell lineage ambiguity is increasingly recorded. Both, metaplasia and biphenotypic leukaemia/lymphoma represent examples of dysregulated cell differentiation that reflect a history of trans-differentiation and/or epigenetic reprogramming. Here we compare the similarity between molecular events of experimental cell trans-differentiation as an emerging therapeutic concept, with lineage confusion, as in metaplasia and dysplasia forecasting tumour development. PMID:23828660

Biosynthesis of ribosomal RNA in nucleoli regulates pluripotency and differentiation ability of pluripotent stem cells.

PubMed

Watanabe-Susaki, Kanako; Takada, Hitomi; Enomoto, Kei; Miwata, Kyoko; Ishimine, Hisako; Intoh, Atsushi; Ohtaka, Manami; Nakanishi, Mahito; Sugino, Hiromu; Asashima, Makoto; Kurisaki, Akira

2014-12-01

Pluripotent stem cells have been shown to have unique nuclear properties, for example, hyperdynamic chromatin and large, condensed nucleoli. However, the contribution of the latter unique nucleolar character to pluripotency has not been well understood. Here, we show that fibrillarin (FBL), a critical methyltransferase for ribosomal RNA (rRNA) processing in nucleoli, is one of the proteins highly expressed in pluripotent embryonic stem (ES) cells. Stable expression of FBL in ES cells prolonged the pluripotent state of mouse ES cells cultured in the absence of leukemia inhibitory factor (LIF). Analyses using deletion mutants and a point mutant revealed that the methyltransferase activity of FBL regulates stem cell pluripotency. Knockdown of this gene led to significant delays in rRNA processing, growth inhibition, and apoptosis in mouse ES cells. Interestingly, both partial knockdown of FBL and treatment with actinomycin D, an inhibitor of rRNA synthesis, induced the expression of differentiation markers in the presence of LIF and promoted stem cell differentiation into neuronal lineages. Moreover, we identified p53 signaling as the regulatory pathway for pluripotency and differentiation of ES cells. These results suggest that proper activity of rRNA production in nucleoli is a novel factor for the regulation of pluripotency and differentiation ability of ES cells. © 2014 AlphaMed Press.

Leucine Promotes Proliferation and Differentiation of Primary Preterm Rat Satellite Cells in Part through mTORC1 Signaling Pathway

PubMed Central

Dai, Jie-Min; Yu, Mu-Xue; Shen, Zhen-Yu; Guo, Chu-Yi; Zhuang, Si-Qi; Qiu, Xiao-Shan

2015-01-01

Signaling through the mammalian target of rapamycin (mTOR) in response to leucine modulates many cellular and developmental processes. However, in the context of satellite cell proliferation and differentiation, the role of leucine and mTORC1 is less known. This study investigates the role of leucine in the process of proliferation and differentiation of primary preterm rat satellite cells, and the relationship with mammalian target of rapamycin complex 1 (mTORC1) activation. Dissociation of primary satellite cells occurred with type I collagenase and trypsin, and purification, via different speed adherence methods. Satellite cells with positive expression of Desmin were treated with leucine and rapamycin. We observed that leucine promoted proliferation and differentiation of primary satellite cells and increased the phosphorylation of mTOR. Rapamycin inhibited proliferation and differentiation, as well as decreased the phosphorylation level of mTOR. Furthermore, leucine increased the expression of MyoD and myogenin while the protein level of MyoD decreased due to rapamycin. However, myogenin expressed no affect by rapamycin. In conclusion, leucine may up-regulate the activation of mTORC1 to promote proliferation and differentiation of primary preterm rat satellite cells. We have shown that leucine promoted the differentiation of myotubes in part through the mTORC1-MyoD signal pathway. PMID:26007333

Holistic systems biology approaches to molecular mechanisms of human helper T cell differentiation to functionally distinct subsets.

PubMed

Chen, Z; Lönnberg, T; Lahesmaa, R

2013-08-01

Current knowledge of helper T cell differentiation largely relies on data generated from mouse studies. To develop therapeutical strategies combating human diseases, understanding the molecular mechanisms how human naïve T cells differentiate to functionally distinct T helper (Th) subsets as well as studies on human differentiated Th cell subsets is particularly valuable. Systems biology approaches provide a holistic view of the processes of T helper differentiation, enable discovery of new factors and pathways involved and generation of new hypotheses to be tested to improve our understanding of human Th cell differentiation and immune-mediated diseases. Here, we summarize studies where high-throughput systems biology approaches have been exploited to human primary T cells. These studies reveal new factors and signalling pathways influencing T cell differentiation towards distinct subsets, important for immune regulation. Such information provides new insights into T cell biology and into targeting immune system for therapeutic interventions. © 2013 John Wiley & Sons Ltd.

p62 Promotes Amino Acid Sensitivity of mTOR Pathway and Hepatic Differentiation in Adult Liver Stem/Progenitor Cells.

PubMed

Sugiyama, Masakazu; Yoshizumi, Tomoharu; Yoshida, Yoshihiro; Bekki, Yuki; Matsumoto, Yoshihiro; Yoshiya, Shohei; Toshima, Takeo; Ikegami, Toru; Itoh, Shinji; Harimoto, Norifumi; Okano, Shinji; Soejima, Yuji; Shirabe, Ken; Maehara, Yoshihiko

2017-08-01

Autophagy is a homeostatic process regulating turnover of impaired proteins and organelles, and p62 (sequestosome-1, SQSTM1) functions as the autophagic receptor in this process. p62 also functions as a hub for intracellular signaling such as that in the mammalian target of rapamycin (mTOR) pathway. Liver stem/progenitor cells have the potential to differentiate to form hepatocytes or cholangiocytes. In this study, we examined effects of autophagy, p62, and associated signaling on hepatic differentiation. Adult stem/progenitor cells were isolated from the liver of mice with chemically induced liver injury. Effects of autophagy, p62, and related signaling pathways on hepatic differentiation were investigated by silencing the genes for autophagy protein 5 (ATG5) and/or SQSTM1/p62 using small interfering RNAs. Hepatic differentiation was assessed based on increased albumin and hepatocyte nuclear factor 4α, as hepatocyte markers, and decreased cytokeratin 19 and SOX9, as stem/progenitor cell markers. These markers were measured using quantitative RT-PCR, immunofluorescence, and Western blotting. ATG5 silencing decreased active LC3 and increased p62, indicating inhibition of autophagy. Inhibition of autophagy promoted hepatic differentiation in the stem/progenitor cells. Conversely, SQSTM1/p62 silencing impaired hepatic differentiation. A suggested mechanism for p62-dependent hepatic differentiation in our study was activation of the mTOR pathway by amino acids. Amino acid activation of mTOR signaling was enhanced by ATG5 silencing and suppressed by SQSTM1/p62 silencing. Our findings indicated that promoting amino acid sensitivity of the mTOR pathway is dependent on p62 accumulated by inhibition of autophagy and that this process plays an important role in the hepatic differentiation of stem/progenitor cells. J. Cell. Physiol. 232: 2112-2124, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Investigation for the differentiation process of mouse ES cells by Raman spectroscopy

NASA Astrophysics Data System (ADS)

Yamaguchi, Yoshinori; El-Hagrasy, Maha A.; Shimizu, Eiichi; Saito, Masato; Tamiya, Eiichi

2012-03-01

The arrangement of differentiated pluripotent embryonic stem cells into three-dimensional aggregates, which are known as embryonic bodies, is a main step for progressing the embryonic stem cells differentiation. In this work, embryonic stem cells that were directly produced from the hanging drop step as a three-dimensional structure with no further twodimensional differentiation were diagnosed with Raman spectroscopy as a non-invasive and label-free technique. Raman spectroscopy was employed to discriminate between mouse embryonic bodies of different degrees of maturation. EBs were prepared applying the hanging drop method. The Raman scattering measurements were obtained in vitro with a Nanophoton RAMAN-11 micro-spectrometer (Japan: URL: www.nanophoton.jp equipped with an Olympus XLUM Plan FLN 20X/NA= 1.0 objective lens. Spectral data were smoothed, baseline corrected and normalized to the a welldefined intense 1003 cm-1 band (phenylalanine) which is insensitive to changes in conformation or environment. The differentiation process of embryonic stem cells is initiated by the removal of LIF from culture medium. 1, 7 and 17-dayold embryonic stem cells were collected and investigated by Raman spectroscopy. The main differences involve bands which decreased with maturation such as: 784 cm-1 (U, T, C ring br DNA/RNA, O-P-O str); 1177 cm-1 (cytosine, guanine) and 1578 cm-1 (G, A). It was found that with the progress of differentiation the protein content was amplified. The increase of protein to nucleic acid ratio was also previously observed with the progress of the differentiation process. Raman spectroscopy has the potential to distinguish between the Raman signatures of live embryonic stem cells with different degrees of maturation.

Mitochondrial activity in the regulation of stem cell self-renewal and differentiation.

PubMed

Khacho, Mireille; Slack, Ruth S

2017-12-01

Mitochondria are classically known as the essential energy producers in cells. As such, the activation of mitochondrial metabolism upon cellular differentiation was deemed a necessity to fuel the high metabolic needs of differentiated cells. However, recent studies have revealed a direct role for mitochondrial activity in the regulation of stem cell fate and differentiation. Several components of mitochondrial metabolism and respiration have now been shown to regulate different aspects of stem cell differentiation through signaling, transcriptional, proteomic and epigenetic modulations. In light of these findings mitochondrial metabolism is no longer considered a consequence of cellular differentiation, but rather a key regulatory mechanism of this process. This review will focus on recent progress that defines mitochondria as the epicenters for the regulation of stem cell fate decisions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Isogenic blood-brain barrier models based on patient-derived stem cells display inter-individual differences in cell maturation and functionality.

PubMed

Patel, Ronak; Page, Shyanne; Al-Ahmad, Abraham Jacob

2017-07-01

The blood-brain barrier (BBB) constitutes an important component of the neurovascular unit formed by specialized brain microvascular endothelial cells (BMECs) surrounded by astrocytes, pericytes, and neurons. Recently, isogenic in vitro models of the BBB based on human pluripotent stem cells have been documented, yet the impact of inter-individual variability on the yield and phenotype of such models remains to be documented. In this study, we investigated the impact of inter-individual variability on the yield and phenotype of isogenic models of the BBB, using patient-derived induced pluripotent stem cells (iPSCs). Astrocytes, BMECs, and neurons were differentiated from four asymptomatic patient-derived iPSCs (two males, two females). We differentiated such cells using existing differentiation protocols and quantified expression of cell lineage markers, as well as BBB phenotype, barrier induction, and formation of neurite processes. iPSC-derived BMECs showed barrier properties better than hCMEC/D3 monolayers; however, we noted differences in the expression and activity among iPSC lines. In addition, we noted differences in the differentiation efficiency of these cells into neural stem cells and progenitor cells (as noted by differences in expression of cell lineage markers). Such differences were reflected later in the terminal differentiation, as seen as ability to induce barrier function and to form neurite processes. Although we demonstrated our ability to obtain an isogenic model of the BBB with different patients' iPSCs, we also noted subtle differences in the expression of cell lineage markers and cell maturation processes, suggesting the presence of inter-individual polymorphisms. © 2017 International Society for Neurochemistry.

MicroRNA-432 contributes to dopamine cocktail and retinoic acid induced differentiation of human neuroblastoma cells by targeting NESTIN and RCOR1 genes.

PubMed

Das, Eashita; Bhattacharyya, Nitai Pada

2014-05-02

MicroRNA (miRNA) regulates expression of protein coding genes and has been implicated in diverse cellular processes including neuronal differentiation, cell growth and death. To identify the role of miRNA in neuronal differentiation, SH-SY5Y and IMR-32 cells were treated with dopamine cocktail and retinoic acid to induce differentiation. Detection of miRNAs in differentiated cells revealed that expression of many miRNAs was altered significantly. Among the altered miRNAs, human brain expressed miR-432 induced neurite projections, arrested cells in G0-G1, reduced cell proliferation and could significantly repress NESTIN/NES, RCOR1/COREST and MECP2. Our results reveal that miR-432 regulate neuronal differentiation of human neuroblastoma cells. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Stochasticity and Spatial Interaction Govern Stem Cell Differentiation Dynamics

NASA Astrophysics Data System (ADS)

Smith, Quinton; Stukalin, Evgeny; Kusuma, Sravanti; Gerecht, Sharon; Sun, Sean X.

2015-07-01

Stem cell differentiation underlies many fundamental processes such as development, tissue growth and regeneration, as well as disease progression. Understanding how stem cell differentiation is controlled in mixed cell populations is an important step in developing quantitative models of cell population dynamics. Here we focus on quantifying the role of cell-cell interactions in determining stem cell fate. Toward this, we monitor stem cell differentiation in adherent cultures on micropatterns and collect statistical cell fate data. Results show high cell fate variability and a bimodal probability distribution of stem cell fraction on small (80-140 μm diameter) micropatterns. On larger (225-500 μm diameter) micropatterns, the variability is also high but the distribution of the stem cell fraction becomes unimodal. Using a stochastic model, we analyze the differentiation dynamics and quantitatively determine the differentiation probability as a function of stem cell fraction. Results indicate that stem cells can interact and sense cellular composition in their immediate neighborhood and adjust their differentiation probability accordingly. Blocking epithelial cadherin (E-cadherin) can diminish this cell-cell contact mediated sensing. For larger micropatterns, cell motility adds a spatial dimension to the picture. Taken together, we find stochasticity and cell-cell interactions are important factors in determining cell fate in mixed cell populations.

Diversity in TAF proteomics: consequences for cellular differentiation and migration.

PubMed

Kazantseva, Jekaterina; Palm, Kaia

2014-09-19

Development is a highly controlled process of cell proliferation and differentiation driven by mechanisms of dynamic gene regulation. Specific DNA binding factors for establishing cell- and tissue-specific transcriptional programs have been characterised in different cell and animal models. However, much less is known about the role of "core transcription machinery" during cell differentiation, given that general transcription factors and their spatiotemporally patterned activity govern different aspects of cell function. In this review, we focus on the role of TATA-box associated factor 4 (TAF4) and its functional isoforms generated by alternative splicing in controlling lineage-specific differentiation of normal mesenchymal stem cells and cancer stem cells. In the light of our recent findings, induction, control and maintenance of cell differentiation status implies diversification of the transcription initiation apparatus orchestrated by alternative splicing.

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

Reversible structural alterations of undifferentiated and differentiated human neuroblastoma cells induced by phorbol ester.

PubMed Central

Tint, I S; Bonder, E M; Feder, H H; Reboulleau, C P; Vasiliev, J M; Gelfand, I M

1992-01-01

Morphological alterations in the structure of undifferentiated and morphologically differentiated human neuroblastoma cells induced by phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, were examined by video microscopy and immunomorphology. In undifferentiated cells, PMA induced the formation of motile actin-rich lamellas and of stable cylindrical processes rich in microtubules. Formation of stable processes resulted either from the collapse of lamellas or the movement of the cell body away from the base of a process. In differentiated cells, PMA induced the rapid extension of small lamellas and subsequent formation of short-lived elongated processes from the lateral edges of neurites. Additionally, growth cones exhibited enhanced modulation in shape after PMA treatment. These reversible reorganizations were similar to the actinoplast-tubuloplast transformations exhibited by PMA-treated fibroblasts. We suggest that actinoplast-tubuloplast reorganizations play essential roles in morphogenesis where stable cytoplasmic extensions are induced by external stimuli. In particular, PMA-induced reorganizations of neural cells in culture may be a model for morphological modulations that occur in nerve tissue. Images PMID:1518842

Cancer stem cells and differentiation therapy.

PubMed

Jin, Xiong; Jin, Xun; Kim, Hyunggee

2017-10-01

Cancer stem cells can generate tumors from only a small number of cells, whereas differentiated cancer cells cannot. The prominent feature of cancer stem cells is its ability to self-renew and differentiate into multiple types of cancer cells. Cancer stem cells have several distinct tumorigenic abilities, including stem cell signal transduction, tumorigenicity, metastasis, and resistance to anticancer drugs, which are regulated by genetic or epigenetic changes. Like normal adult stem cells involved in various developmental processes and tissue homeostasis, cancer stem cells maintain their self-renewal capacity by activating multiple stem cell signaling pathways and inhibiting differentiation signaling pathways during cancer initiation and progression. Recently, many studies have focused on targeting cancer stem cells to eradicate malignancies by regulating stem cell signaling pathways, and products of some of these strategies are in preclinical and clinical trials. In this review, we describe the crucial features of cancer stem cells related to tumor relapse and drug resistance, as well as the new therapeutic strategy to target cancer stem cells named "differentiation therapy."

In vivo differentiation of human periodontal ligament cells leads to formation of dental hard tissue.

PubMed

Wolf, M; Lossdörfer, S; Abuduwali, N; Meyer, R; Kebir, S; Götz, W; Jäger, A

2013-11-01

Following trauma, periodontal disease, or orthodontic tooth movement, residual periodontal ligament (PDL) cells at the defect site are considered mandatory for successful regeneration of the injured structures. Recent developments in tissue engineering focus, as one pillar, on the transplantation of PDL cells to support periodontal regeneration processes. Here, we examined the ability of osteogenically predifferentiated PDL cells to undergo further osteoblastic or cementoblastic differentiation and to mineralize their extracellular matrix when transplanted in an in vivo microenvironment. Using collagen sponges as carriers, osteogenically predifferentiated human PDL cells were transplanted subcutaneously into six immunocompromised CD-1® nude mice. Following explantation after 28 days, osteogenic and cementogenic marker protein expression was visualized immunohistochemically. After 28 days, transplanted PDL cells revealed both cellular, cytoplasmatic and extracellular immunoreactivity for the chosen markers alkaline phosphatase, osteopontin, PTH-receptor 1, and osteocalcin. Specific osteogenic and cementoblastic differentiation was demonstrated by RUNX2 and CEMP1 immunoreactivity. Early stages of mineralization were demonstrated by calcium and phosphate staining. Our results reinforce the previously published reports of PDL cell mineralization in vivo and further demonstrate the successful induction of specific osteogenic and cementogenic differentiation of transplanted human PDL cells in vivo. These findings reveal promising possibilities for supporting periodontal remodeling and regeneration processes with PDL cells being potential target cells with which to influence the process of orthodontically induced root resorption.

Modulation of Differentiation Processes in Murine Embryonic Stem Cells Exposed to Parabolic Flight-Induced Acute Hypergravity and Microgravity.

PubMed

Acharya, Aviseka; Brungs, Sonja; Henry, Margit; Rotshteyn, Tamara; Singh Yaduvanshi, Nirmala; Wegener, Lucia; Jentzsch, Simon; Hescheler, Jürgen; Hemmersbach, Ruth; Boeuf, Helene; Sachinidis, Agapios

2018-06-15

Embryonic developmental studies under microgravity conditions in space are very limited. To study the effects of short-term altered gravity on embryonic development processes, we exposed mouse embryonic stem cells (mESCs) to phases of hypergravity and microgravity and studied the differentiation potential of the cells using wide-genome microarray analysis. During the 64th European Space Agency's parabolic flight campaign, mESCs were exposed to 31 parabolas. Each parabola comprised phases lasting 22 s of hypergravity, microgravity, and a repeat of hypergravity. On different parabolas, RNA was isolated for microarray analysis. After exposure to 31 parabolas, mESCs (P31 mESCs) were further differentiated under normal gravity (1 g) conditions for 12 days, producing P31 12-day embryoid bodies (EBs). After analysis of the microarrays, the differentially expressed genes were analyzed using different bioinformatic tools to identify developmental and nondevelopmental biological processes affected by conditions on the parabolic flight experiment. Our results demonstrated that several genes belonging to GOs associated with cell cycle and proliferation were downregulated in undifferentiated mESCs exposed to gravity changes. However, several genes belonging to developmental processes, such as vasculature development, kidney development, skin development, and to the TGF-β signaling pathway, were upregulated. Interestingly, similar enriched and suppressed GOs were obtained in P31 12-day EBs compared with ground control 12-day EBs. Our results show that undifferentiated mESCs exposed to alternate hypergravity and microgravity phases expressed several genes associated with developmental/differentiation and cell cycle processes, suggesting a transition from the undifferentiated pluripotent to a more differentiated stage of mESCs.

A novel differentiation pathway from CD4+ T cells to CD4− T cells for maintaining immune system homeostasis

PubMed Central

Zhao, X; Sun, G; Sun, X; Tian, D; Liu, K; Liu, T; Cong, M; Xu, H; Li, X; Shi, W; Tian, Y; Yao, J; Guo, H; Zhang, D

2016-01-01

CD4+ T lymphocytes are key players in the adaptive immune system and can differentiate into a variety of effector and regulatory T cells. Here, we provide evidence that a novel differentiation pathway of CD4+ T cells shifts the balance from a destructive T-cell response to one that favors regulation in an immune-mediated liver injury model. Peripheral CD4−CD8−NK1.1− double-negative T cells (DNT) was increased following Concanavalin A administration in mice. Adoptive transfer of DNT led to significant protection from hepatocyte necrosis by direct inhibition on the activation of lymphocytes, a process that occurred primarily through the perforin-granzyme B route. These DNT converted from CD4+ rather than CD8+ T cells, a process primarily regulated by OX40. DNT migrated to the liver through the CXCR3-CXCL9/CXCL10 interaction. In conclusion, we elucidated a novel differentiation pathway from activated CD4+ T cells to regulatory DNT cells for maintaining homeostasis of the immune system in vivo, and provided key evidence that utilizing this novel differentiation pathway has potential application in the prevention and treatment of autoimmune diseases. PMID:27077809

Vascular Regeneration in a Basal Chordate Is Due to the Presence of Immobile, Bi-Functional Cells

PubMed Central

Braden, Brian P.; Taketa, Daryl A.; Pierce, James D.; Kassmer, Susannah; Lewis, Daniel D.; De Tomaso, Anthony W.

2014-01-01

The source of tissue turnover during homeostasis or following injury is usually due to proliferation of a small number of resident, lineage-restricted stem cells that have the ability to amplify and differentiate into mature cell types. We are studying vascular regeneration in a chordate model organism, Botryllus schlosseri, and have previously found that following surgical ablation of the extracorporeal vasculature, new tissue will regenerate in a VEGF-dependent process within 48 hrs. Here we use a novel vascular cell lineage tracing methodology to assess regeneration in parabiosed individuals and demonstrate that the source of regenerated vasculature is due to the proliferation of pre-existing vascular resident cells and not a mobile progenitor. We also show that these cells are bi-potential, and can reversibly adopt two fates, that of the newly forming vessels or the differentiated vascular tissue at the terminus of the vasculature, known as ampullae. In addition, we show that pre-existing vascular resident cells differentially express progenitor and differentiated cell markers including the Botryllus homologs of CD133, VEGFR-2, and Cadherin during the regenerative process. PMID:24736432

Retinoic acid induces nuclear accumulation of Raf1 during differentiation of HL-60 cells

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

Smith, James; Bunaciu, Rodica P.; Reiterer, Gudrun

All trans-retinoic acid (RA) is a standard therapeutic agent used in differentiation induction therapy treatment of acute promyelocytic leukemia (APL). RA and its metabolites use a diverse set of signal transduction pathways during the differentiation program. In addition to the direct transcriptional targets of the nuclear RAR and RXR receptors, signals derived from membrane receptors and the Raf-MEK-ERK pathway are required. Raf1 phosphorylation and the prolonged activation of Raf1 persisting during the entire differentiation process are required for RA-dependent differentiation of HL-60 cells. Here we identify a nuclear redistribution of Raf1 during the RA-induced differentiation of HL-60 cells. In addition,more » the nuclear accumulation of Raf1 correlates with an increase in Raf1 phosphorylated at serine 621. The serine 621 phosphorylated Raf1 is predominantly localized in the nucleus. The RA-dependent nuclear accumulation of Raf1 suggests a novel nuclear role for Raf1 during the differentiation process.« less

Role of differential physical properties in emergent behavior of 3D cell co-cultures

NASA Astrophysics Data System (ADS)

Kolbman, Dan; Das, Moumita

2015-03-01

The biophysics of binary cell populations is of great interest in many biological processes, whether the formation of embryos or the initiation of tumors. During these processes, cells are surrounded by other cell types with different physical properties, often with important consequences. For example, recent experiments on a co-culture of breast cancer cells and healthy breast epithelial cells suggest that the mechanical mismatch between the two cell types may contribute to enhanced migration of the cancer cells. Here we explore how the differential physical properties of different cell types may influence cell-cell interaction, aggregation, and migration. To this end, we study a proof of concept model- a three-dimensional binary system of interacting, active, and deformable particles with different physical properties such as elastic stiffness, contractility, and particle-particle adhesion, using Langevin Dynamics simulations. Our results may provide insights into emergent behavior such as segregation and differential migration in cell co-cultures in three dimensions.

Gene expression profiling in multipotent DFAT cells derived from mature adipocytes

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

Ono, Hiromasa; Database Center for Life Science; Oki, Yoshinao

2011-04-15

Highlights: {yields} Adipocyte dedifferentiation is evident in a significant decrease in typical genes. {yields} Cell proliferation is strongly related to adipocyte dedifferentiation. {yields} Dedifferentiated adipocytes express several lineage-specific genes. {yields} Comparative analyses using publicly available datasets boost the interpretation. -- Abstract: Cellular dedifferentiation signifies the withdrawal of cells from a specific differentiated state to a stem cell-like undifferentiated state. However, the mechanism of dedifferentiation remains obscure. Here we performed comparative transcriptome analyses during dedifferentiation in mature adipocytes (MAs) to identify the transcriptional signatures of multipotent dedifferentiated fat (DFAT) cells derived from MAs. Using microarray systems, we explored similarly expressed asmore » well as significantly differentially expressed genes in MAs during dedifferentiation. This analysis revealed significant changes in gene expression during this process, including a significant reduction in expression of genes for lipid metabolism concomitantly with a significant increase in expression of genes for cell movement, cell migration, tissue developmental processes, cell growth, cell proliferation, cell morphogenesis, altered cell shape, and cell differentiation. Our observations indicate that the transcriptional signatures of DFAT cells derived from MAs are summarized in terms of a significant decrease in functional phenotype-related genes and a parallel increase in cell proliferation, altered cell morphology, and regulation of the differentiation of related genes. A better understanding of the mechanisms involved in dedifferentiation may enable scientists to control and possibly alter the plasticity of the differentiated state, which may lead to benefits not only in stem cell research but also in regenerative medicine.« less

Sox10+ adult stem cells contribute to biomaterial encapsulation and microvascularization

PubMed Central

Wang, Dong; Wang, Aijun; Wu, Fan; Qiu, Xuefeng; Li, Ye; Chu, Julia; Huang, Wen-Chin; Xu, Kang; Gong, Xiaohua; Li, Song

2017-01-01

Implanted biomaterials and biomedical devices generally induce foreign body reaction and end up with encapsulation by a dense avascular fibrous layer enriched in extracellular matrix. Fibroblasts/myofibroblasts are thought to be the major cell type involved in encapsulation, but it is unclear whether and how stem cells contribute to this process. Here we show, for the first time, that Sox10+ adult stem cells contribute to both encapsulation and microvessel formation. Sox10+ adult stem cells were found sparsely in the stroma of subcutaneous loose connective tissues. Upon subcutaneous biomaterial implantation, Sox10+ stem cells were activated and recruited to the biomaterial scaffold, and differentiated into fibroblasts and then myofibroblasts. This differentiation process from Sox10+ stem cells to myofibroblasts could be recapitulated in vitro. On the other hand, Sox10+ stem cells could differentiate into perivascular cells to stabilize newly formed microvessels. Sox10+ stem cells and endothelial cells in three-dimensional co-culture self-assembled into microvessels, and platelet-derived growth factor had chemotactic effect on Sox10+ stem cells. Transplanted Sox10+ stem cells differentiated into smooth muscle cells to stabilize functional microvessels. These findings demonstrate the critical role of adult stem cells in tissue remodeling and unravel the complexity of stem cell fate determination. PMID:28071739

Growth and differentiation of embryonic stem cells that lack an intact c-fos gene.

PubMed Central

Field, S J; Johnson, R S; Mortensen, R M; Papaioannou, V E; Spiegelman, B M; Greenberg, M E

1992-01-01

The c-fos protooncogene encodes a transcription factor that is thought to play a critical role in proliferation and differentiation as well as in the physiological response of mature cells to their environment. To test directly the role of c-fos in growth and differentiation, we generated mouse embryonic stem cell lines in which both copies of the c-fos gene were specifically disrupted by homologous recombination. Remarkably, the disruption of both copies of c-fos in these cells has no detectable effect on embryonic stem cell viability, growth rate, or differentiation potential. Embryonic stem cells lacking c-fos can differentiate into a wide range of cell types in tissue culture and also in chimeric mice. We conclude that despite a large body of literature suggesting an important role for c-fos in cell growth and differentiation, in at least some cell types this gene is not essential for these processes. Images PMID:1329091

The epithelial-mesenchymal interactions: insights into physiological and pathological aspects of oral tissues.

PubMed

Santosh, Arvind Babu Rajendra; Jones, Thaon Jon

2014-03-17

In the human biological system, the individual cells divide and form tissues and organs. These tissues are hetero-cellular. Basically any tissue consists of an epithelium and the connective tissue. The latter contains mainly mesenchymally-derived tissues with a diversified cell population. The cell continues to grow and differentiate in a pre-programmed manner using a messenger system. The epithelium and the mesenchymal portion of each tissue have two different origins and perform specific functions, but there is a well-defined interaction mechanism, which mediates between them. Epithelial mesenchymal interactions (EMIs) are part of this mechanism, which can be regarded as a biological conversation between epithelial and mesenchymal cell populations involved in the cellular differentiation of one or both cell populations. EMIs represent a process that is essential for cell growth, cell differentiation and cell multiplication. EMIs are associated with normal physiological processes in the oral cavity, such as odontogenesis, dentino-enamel junction formation, salivary gland development, palatogenesis, and also pathological processes, such as oral cancer. This paper focuses the role EMIs in odontogenesis, salivary gland development, palatogenesis and oral cancer.

Potential role of centrioles in determining the morphogenetic status of animal somatic cells.

PubMed

Tkemaladze, J; Chichinadze, K

2005-05-01

Irreversible differentiation (change of morphogenetic status) and programmed death (apoptosis) are observed only in somatic cells. Cell division is the only way by which the morphogenetic status of the offspring cells may be modified. It is known that there is a fixed limit to the number of possible cell divisions, the so-called 'Hayflick limit'. Existing links between cell division, differentiation and apoptosis make it possible to conclude that all these processes could be controlled by a single self-reproducing structure. Potential candidates for this replicable structure in a somatic cell are chromosomes, mitochondria (both contain DNA), and centrioles. Centrioles (diplosome) are the most likely unit that can fully regulate the processes of irreversible differentiation, determination and modification of the morphogenetic status. It may contain differently encoded RNA molecules stacked in a definite order. During mitosis, these RNA molecules are released one by one into the cytoplasm. In the presence of reverse transcriptase and endonuclease, RNA can be embedded in nuclear DNA. This process presumably changes the status of repressed and potentially active genes and, subsequently, the morphogenetic status of a cell.

Dexamethasone facilitates erythropoiesis in murine embryonic stem cells differentiating into hematopoietic cells in vitro

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

Srivastava, Anand S.; Kaushal, Sharmeela; Mishra, Rangnath

2006-07-28

Differentiating embryonic stem (ES) cells are increasingly emerging as an important source of hematopoietic progenitors with a potential to be useful for both basic and clinical research applications. It has been suggested that dexamethasone facilitates differentiation of ES cells towards erythrocytes but the mechanism responsible for sequential expression of genes regulating this process are not well-understood. Therefore, we in vitro induced differentiation of murine ES cells towards erythropoiesis and studied the sequential expression of a set of genes during the process. We hypothesized that dexamethasone-activates its cognate nuclear receptors inducing up-regulation of erythropoietic genes such as GATA-1, Flk-1, Epo-R, andmore » direct ES cells towards erythropoietic differentiation. ES cells were cultured in primary hematopoietic differentiation media containing methyl-cellulose, IMDM, IL-3, IL-6, and SCF to promote embryoid body (EB) formation. Total RNA of day 3, 5, and 9-old EBs was isolated for gene expression studies using RT-PCR. Cells from day 9 EBs were subjected to secondary differentiation using three different cytokines and growth factors combinations: (1) SCF, EPO, dexamethasone, and IGF; (2) SCF, IL-3, IL-6, and TPO; and (3) SCF IL-3, IL-6, TPO, and EPO. Total RNA from day 12 of secondary differentiated ES cells was isolated to study the gene expression pattern during this process. Our results demonstrate an up-regulation of GATA-1, Flk-1, HoxB-4, Epo-R, and globin genes ({alpha}-globin, {beta}H-1 globin, {beta}-major globin, {epsilon} -globin, and {zeta}-globin) in the 9-day-old EBs, whereas, RNA from 5-day-old EBs showed expression of HoxB-4, {epsilon}-globin, {gamma}-globin, {beta}H1-globin, and Flk-1. Three-day-old EBs showed only HoxB-4 and Flk-1 gene expression and lacked expression of all globin genes. These findings indicate that erythropoiesis-specific genes are activated later in the course of differentiation. Gene expression studies on the ES cells of secondary EB origin cultured in media containing dexamethasone showed a down-regulation of GATA-3 and an up-regulation of GATA-1, Flk-1, and Epo-R in comparison to the two other cytokines and growth factor combinations containing media. The secondary differentiation also showed an enhanced production of erythrocytic precursors in dexamethasone containing media in comparison to that in the control media. Our results indicate that dexamethasone can prove to be an effective agent which can be employed to enhance differentiation towards erythrocytic progenitors from ES cells.« less

Evaluation of biological effects of intermediate frequency magnetic field on differentiation of embryonic stem cell.

PubMed

Yoshie, Sachiko; Ogasawara, Yuki; Ikehata, Masateru; Ishii, Kazuyuki; Suzuki, Yukihisa; Wada, Keiji; Wake, Kanako; Nakasono, Satoshi; Taki, Masao; Ohkubo, Chiyoji

2016-01-01

The embryotoxic effect of intermediate frequency (IF) magnetic field (MF) was evaluated using murine embryonic stem (ES) cells and fibroblast cells based on the embryonic stem cell test (EST). The cells were exposed to 21 kHz IF-MF up to magnetic flux density of 3.9 mT during the cell proliferation process (7 days) or the cell differentiation process (10 days) during which an embryonic body differentiated into myocardial cells. As a result, there was no significant difference in the cell proliferation between sham- and IF-MF-exposed cells for both ES and fibroblast cells. Similarly, the ratio of the number of ES-derived cell aggregates differentiated to myocardial cells to total number of cell aggregates was not changed by IF-MF exposure. In addition, the expressions of a cardiomyocytes-specific gene, Myl2 , and an early developmental gene, Hba-x , in the exposed cell aggregate were not altered. Since the magnetic flux density adopted in this study is much higher than that generated by an inverter of the electrical railway, an induction heating (IH) cooktop, etc . in our daily lives, these results suggested that IF-MF in which the public is exposed to in general living environment would not have embryotoxic effect.

Temporal competition between differentiation programs determines cell fate choice

NASA Astrophysics Data System (ADS)

Kuchina, Anna; Espinar, Lorena; Cagatay, Tolga; Balbin, Alejandro; Alvarado, Alma; Garcia-Ojalvo, Jordi; Suel, Gurol

2011-03-01

During pluripotent differentiation, cells adopt one of several distinct fates. The dynamics of this decision-making process are poorly understood, since cell fate choice may be governed by interactions between differentiation programs that are active at the same time. We studied the dynamics of decision-making in the model organism Bacillus subtilis by simultaneously measuring the activities of competing differentiation programs (sporulation and competence) in single cells. We discovered a precise switch-like point of cell fate choice previously hidden by cell-cell variability. Engineered artificial crosslinks between competence and sporulation circuits revealed that the precision of this choice is generated by temporal competition between the key players of two differentiation programs. Modeling suggests that variable progression towards a switch-like decision might represent a general strategy to maximize adaptability and robustness of cellular decision-making.

Glycoproteomic Analysis of Glioblastoma Stem Cell Differentiation

PubMed Central

He, Jintang; Liu, Yashu; Zhu, Thant S.; Xie, Xiaolei; Costello, Mark A.; Talsma, Caroline E.; Flack, Callie G.; Crowley, Jessica G.; DiMeco, Francesco; Vescovi, Angelo L.; Fan, Xing; Lubman, David M.

2010-01-01

Cancer stem cells are responsible for tumor formation through self-renewal and differentiation into multiple cell types, and thus represent a new therapeutic target for tumors. Glycoproteins play a critical role in determining the fates of stem cells such as self-renewal, proliferation and differentiation. Here we applied a multi-lectin affinity chromatography and quantitative glycoproteomics approach to analyze alterations of glycoproteins relevant to the differentiation of a glioblastoma-derived stem cell line HSR-GBM1. Three lectins including concanavalin A (Con A), wheat germ agglutinin (WGA) and peanut agglutinin (PNA) were used to capture glycoproteins, followed by LC-MS/MS analysis. A total of 73 and 79 high-confidence (FDR < 0.01) glycoproteins were identified from the undifferentiated and differentiated cells, respectively. Label-free quantitation resulted in the discovery of 18 differentially expressed glycoproteins, wherein 9 proteins are localized in the lysosome. All of these lysosomal glycoproteins were up-regulated after differentiation, where their principal function was hydrolysis of glycosyl residues. Protein-protein interaction and functional analyses revealed the active involvement of lysosomes during the process of glioblastoma stem cell differentiation. This work provides glycoprotein markers to characterize differentiation status of glioblastoma stem cells which may be useful in stemcell therapy of glioblastoma. PMID:21110520

Retinoic acid-induced differentiation increases the rate of oxygen consumption and enhances the spare respiratory capacity of mitochondria in SH-SY5Y cells.

PubMed

Xun, Zhiyin; Lee, Do-Yup; Lim, James; Canaria, Christie A; Barnebey, Adam; Yanonne, Steven M; McMurray, Cynthia T

2012-04-01

Retinoic acid (RA) is used in differentiation therapy to treat a variety of cancers including neuroblastoma. The contributing factors for its therapeutic efficacy are poorly understood. However, mitochondria (MT) have been implicated as key effectors in RA-mediated differentiation process. Here we utilize the SH-SY5Y human neuroblastoma cell line as a model to examine how RA influences MT during the differentiation process. We find that RA confers an approximately sixfold increase in the oxygen consumption rate while the rate of glycolysis modestly increases. RA treatment does not increase the number of MT or cause measurable changes in the composition of the electron transport chain. Rather, RA treatment significantly increases the mitochondrial spare respiratory capacity. We propose a competition model for the therapeutic effects of RA. Specifically, the high metabolic rate in differentiated cells limits the availability of metabolic nutrients for use by the undifferentiated cells and suppresses their growth. Thus, RA treatment provides a selective advantage for the differentiated state. Published by Elsevier Ireland Ltd.

Retinoic acid-induced differentiation increases the rate of oxygen consumption and enhances the spare respiratory capacity of mitochondria in SH-SY5Y cells

PubMed Central

Xun, Zhiyin; Lee, Do-Yup; Lim, James; Canaria, Christie A.; Barnebey, Adam; Yanonne, Steven M.; McMurray, Cynthia T.

2012-01-01

Retinoic acid (RA) is used in differentiation therapy to treat a variety of cancers including neuroblastoma. The contributing factors for its therapeutic efficacy are poorly understood. However, mitochondria (MT) have been implicated as key effectors in RA-mediated differentiation process. Here we utilize the SH-SY5Y human neuroblastoma cell line as a model to examine how RA influences MT during the differentiation process. We find that RA confers an approximately 6-fold increase in the oxygen consumption rate while the rate of glycolysis modestly increases. RA treatment does not increase the number of MT or cause measurable changes in the composition of the electron transport chain. Rather, RA treatment significantly increases the mitochondrial spare respiratory capacity. We propose a competition model for the therapeutic effects of RA. Specifically, the high metabolic rate in differentiated cells limits the availability of metabolic nutrients for use by the undifferentiated cells and suppresses their growth. Thus, RA treatment provides a selective advantage for the differentiated state. PMID:22336883

Chromatin in embryonic stem cell neuronal differentiation.

PubMed

Meshorer, E

2007-03-01

Chromatin, the basic regulatory unit of the eukaryotic genetic material, is controlled by epigenetic mechanisms including histone modifications, histone variants, DNA methylation and chromatin remodeling. Cellular differentiation involves large changes in gene expression concomitant with alterations in genome organization and chromatin structure. Such changes are particularly evident in self-renewing pluripotent embryonic stem cells, which begin, in terms of cell fate, as a tabula rasa, and through the process of differentiation, acquire distinct identities. Here I describe the changes in chromatin that accompany neuronal differentiation, particularly of embryonic stem cells, and discuss how chromatin serves as the master regulator of cellular destiny.

Epigenetic control of CD8+ T cell differentiation.

PubMed

Henning, Amanda N; Roychoudhuri, Rahul; Restifo, Nicholas P

2018-05-01

Upon stimulation, small numbers of naive CD8 + T cells proliferate and differentiate into a variety of memory and effector cell types. CD8 + T cells can persist for years and kill tumour cells and virally infected cells. The functional and phenotypic changes that occur during CD8 + T cell differentiation are well characterized, but the epigenetic states that underlie these changes are incompletely understood. Here, we review the epigenetic processes that direct CD8 + T cell differentiation and function. We focus on epigenetic modification of DNA and associated histones at genes and their regulatory elements. We also describe structural changes in chromatin organization that affect gene expression. Finally, we examine the translational potential of epigenetic interventions to improve CD8 + T cell function in individuals with chronic infections and cancer.

Proliferation and osteo/odontogenic differentiation of stem cells from apical papilla regulated by Zinc fingers and homeoboxes 2: An in vitro study

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

Wan, Fang; VIP Center, Shandong Provincial Key Laboratory of Oral Biomedicine, School and Hospital of Stomatology, Shandong University, 44 Wenhua Xi Road, Jinan, Shandong 250012; Gao, Lifen

In the process of tooth root development, stem cells from the apical papilla (SCAPs) can differentiate into odontoblasts and form root dentin, however, molecules regulating SCAPs differentiation have not been elucidated. Zinc fingers and homeoboxes 2 (ZHX2) is a novel transcriptional inhibitor. It is reported to modulate the development of nerve cells, liver cells, B cells, red blood cells, and so on. However, the role of ZHX2 in tooth root development remains unclear. In this study, we explored the potential role of ZHX2 in the process of SCAPs differentiation. The results showed that overexpression of ZHX2 upregulated the expression ofmore » osteo/odontogenic related genes and ALP activity, inhibited the proliferation of SCAPs. Consistently, ZHX2 knockdown reduced SCAPs mineralization and promoted SCAPs proliferation. These results indicated that ZHX2 plays a critical role in the proliferation and osteo/odontogenic differentiation of SCAPs. - Highlights: • Zinc fingers and homeoboxes 2 (ZHX2) is a novel transcriptional inhibitor. • we found another new biological function of ZHX2 for the first time. • ZHX2 inhibit SCAPs proliferation. • ZHX2 promote the osteo/odontogenic differentiation of SCAPs.« less

Phosphorylated DegU Manipulates Cell Fate Differentiation in the Bacillus subtilis Biofilm

PubMed Central

Marlow, Victoria L.; Porter, Michael; Hobley, Laura; Kiley, Taryn B.; Swedlow, Jason R.; Davidson, Fordyce A.

2014-01-01

Cell differentiation is ubiquitous and facilitates division of labor and development. Bacteria are capable of multicellular behaviors that benefit the bacterial community as a whole. A striking example of bacterial differentiation occurs throughout the formation of a biofilm. During Bacillus subtilis biofilm formation, a subpopulation of cells differentiates into a specialized population that synthesizes the exopolysaccharide and the TasA amyloid components of the extracellular matrix. The differentiation process is indirectly controlled by the transcription factor Spo0A that facilitates transcription of the eps and tapA (tasA) operons. DegU is a transcription factor involved in regulating biofilm formation. Here, using a combination of genetics and live single-cell cytological techniques, we define the mechanism of biofilm inhibition at high levels of phosphorylated DegU (DegU∼P) by showing that transcription from the eps and tapA promoter regions is inhibited. Data demonstrating that this is not a direct regulatory event are presented. We demonstrate that DegU∼P controls the frequency with which cells activate transcription from the operons needed for matrix biosynthesis in favor of an off state. Subsequent experimental analysis led us to conclude that DegU∼P functions to increase the level of Spo0A∼P, driving cell fate differentiation toward the terminal developmental process of sporulation. PMID:24123822

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 differentiation directions of the bone marrow stromal cells under modeling microgravity

NASA Astrophysics Data System (ADS)

Nesterenko, Olga; Rodionova, Natalia; Katkova, Olena

Within experiments on rats simulating microgravity by base load remove from back limbs (duration of the experiment 1,5 months) on marrow stromal cells cultures (ex vivo, in vitro) comprising osteogenic cells-predecessors, extracted from femurs, studied their peculiarities of the colony formation ablity, the cell structure, some cytological and ultra-structural characteristics and differentiation direction. It was found that that under microgravity conditions there is a decline of the stromal cells colony formation intensity, decrease of the colonies size and cells mitotic activity that indicates decrease of their growth potential. Both in control and in experiment the colonies were presented by population of low-differentiated cells, differentiated cells and mature cells. The comparative cytological and morphometric analysis have shown that the studied stromal cells in colonies have the smaller sizes, more elongated shape, and higher nucleocytoplasmic ratio. Cells composition in the experiment colonies is reliably different by the ratio of the low-differentiating to being differentiated cells; a ratio of low-differentiated to already differentiated cells; ratio of differentiated cells to total number of all cells. In comparison with control group, amount of the cells passed trough a differentiation stage and mature cells in colonies is decreased by 3 to 4 times. Among the differentiated stromal cells in colonies increasing amount of adipocytes was revealed. The analysis of electron microscope microphotographs showed that in osteogenic cells differentiated under microgravity conditions, there is a reduction of the specific volume of a granular endoplasmic reticulum, Golgi's complex and quantity of nuclei reduction that indicates depression of the specific biosyntheses process intensity in cells. The increase of lysosomes and myelinic structures quantity is linked to organelles partial reduction. Consolidation of mitochondrias is an evidence of the cells’ energy metabolism disorder. In differentiated cells, disorganization and a cytoskeleton destruction was observed. Results showed that under microgravity conditions proliferative and differentiation (including osteogenic) potentialities of low-differentiated marrow stromal cells decreased, induction of their adipocytic differentiation was observes as well. Obtained results make a new contribution into gravitation sensitivity mechanisms understanding for stromal cells of the bone marrow which contain osteogenic cells- predecessors, features of the osteoporosis development.

Cyclin D1 negatively regulates the expression of differentiation genes in HT-29 M6 mucus-secreting colon cancer cells.

PubMed

Mayo, Clara; Mayol, Xavier

2009-08-28

HT-29 M6 colon cancer cells differentiate to a mucus-secreting phenotype in culture. We found that the pattern of cyclin D1 expression in HT-29 M6 cells did not correlate with instances of cell proliferation but was specifically induced during a dedifferentiation process following disaggregation of epithelial cell layers, even under conditions that did not allow cell cycle reentrance. Interestingly, ectopic expression of cyclin D1 in differentiated cells led to the inhibition of the transcriptional activity of differentiation gene promoters, such as the mucin MUC1. We thus propose that the overexpression of cyclin D1 found in colon cancer favours tumour dedifferentiation as one mechanism of tumour progression.

Cell differentiation modeled via a coupled two-switch regulatory network

NASA Astrophysics Data System (ADS)

Schittler, D.; Hasenauer, J.; Allgöwer, F.; Waldherr, S.

2010-12-01

Mesenchymal stem cells can give rise to bone and other tissue cells, but their differentiation still escapes full control. In this paper we address this issue by mathematical modeling. We present a model for a genetic switch determining the cell fate of progenitor cells which can differentiate into osteoblasts (bone cells) or chondrocytes (cartilage cells). The model consists of two switch mechanisms and reproduces the experimentally observed three stable equilibrium states: a progenitor, an osteogenic, and a chondrogenic state. Conventionally, the loss of an intermediate (progenitor) state and the entailed attraction to one of two opposite (differentiated) states is modeled as a result of changing parameters. In our model in contrast, we achieve this by distributing the differentiation process to two functional switch parts acting in concert: one triggering differentiation and the other determining cell fate. Via stability and bifurcation analysis, we investigate the effects of biochemical stimuli associated with different system inputs. We employ our model to generate differentiation scenarios on the single cell as well as on the cell population level. The single cell scenarios allow to reconstruct the switching upon extrinsic signals, whereas the cell population scenarios provide a framework to identify the impact of intrinsic properties and the limiting factors for successful differentiation.

Xiphoid Process-Derived Chondrocytes: A Novel Cell Source for Elastic Cartilage Regeneration

PubMed Central

Nam, Seungwoo; Cho, Wheemoon; Cho, Hyunji; Lee, Jungsun

2014-01-01

Reconstruction of elastic cartilage requires a source of chondrocytes that display a reliable differentiation tendency. Predetermined tissue progenitor cells are ideal candidates for meeting this need; however, it is difficult to obtain donor elastic cartilage tissue because most elastic cartilage serves important functions or forms external structures, making these tissues indispensable. We found vestigial cartilage tissue in xiphoid processes and characterized it as hyaline cartilage in the proximal region and elastic cartilage in the distal region. Xiphoid process-derived chondrocytes (XCs) showed superb in vitro expansion ability based on colony-forming unit fibroblast assays, cell yield, and cumulative cell growth. On induction of differentiation into mesenchymal lineages, XCs showed a strong tendency toward chondrogenic differentiation. An examination of the tissue-specific regeneration capacity of XCs in a subcutaneous-transplantation model and autologous chondrocyte implantation model confirmed reliable regeneration of elastic cartilage regardless of the implantation environment. On the basis of these observations, we conclude that xiphoid process cartilage, the only elastic cartilage tissue source that can be obtained without destroying external shape or function, is a source of elastic chondrocytes that show superb in vitro expansion and reliable differentiation capacity. These findings indicate that XCs could be a valuable cell source for reconstruction of elastic cartilage. PMID:25205841

Cell Expansion During Directed Differentiation of Stem Cells Toward the Hepatic Lineage.

PubMed

Raju, Ravali; Chau, David; Cho, Dong Seong; Park, Yonsil; Verfaillie, Catherine M; Hu, Wei-Shou

2017-02-15

The differentiation of human pluripotent stem cells toward the hepatocyte lineage can potentially provide an unlimited source of functional hepatocytes for transplantation and extracorporeal bioartificial liver applications. It is anticipated that the quantities of cells needed for these applications will be in the order of 10 9 -10 10 cells, because of the size of the liver. An ideal differentiation protocol would be to enable directed differentiation to the hepatocyte lineage with simultaneous cell expansion. We introduced a cell expansion stage after the commitment of human embryonic stem cells to the endodermal lineage, to allow for at least an eightfold increase in cell number, with continuation of cell maturation toward the hepatocyte lineage. The progressive changes in the transcriptome were measured by expression array, and the expression dynamics of certain lineage markers was measured by mass cytometry during the differentiation and expansion process. The findings revealed that while cells were expanding they were also capable of progressing in their differentiation toward the hepatocyte lineage. In addition, our transcriptome, protein and functional studies, including albumin secretion, drug-induced CYP450 expression and urea production, all indicated that the hepatocyte-like cells obtained with or without cell expansion are very similar. This method of simultaneous cell expansion and hepatocyte differentiation should facilitate obtaining large quantities of cells for liver cell applications.

Mitochondrial biogenesis and energy production in differentiating murine stem cells: a functional metabolic study.

PubMed

Han, Sungwon; Auger, Christopher; Thomas, Sean C; Beites, Crestina L; Appanna, Vasu D

2014-02-01

The significance of metabolic networks in guiding the fate of the stem cell differentiation is only beginning to emerge. Oxidative metabolism has been suggested to play a major role during this process. Therefore, it is critical to understand the underlying mechanisms of metabolic alterations occurring in stem cells to manipulate the ultimate outcome of these pluripotent cells. Here, using P19 murine embryonal carcinoma cells as a model system, the role of mitochondrial biogenesis and the modulation of metabolic networks during dimethyl sulfoxide (DMSO)-induced differentiation are revealed. Blue native polyacrylamide gel electrophoresis (BN-PAGE) technology aided in profiling key enzymes, such as hexokinase (HK) [EC 2.7.1.1], glucose-6-phosphate isomerase (GPI) [EC 5.3.1.9], pyruvate kinase (PK) [EC 2.7.1.40], Complex I [EC 1.6.5.3], and Complex IV [EC 1.9.3.1], that are involved in the energy budget of the differentiated cells. Mitochondrial adenosine triphosphate (ATP) production was shown to be increased in DMSO-treated cells upon exposure to the tricarboxylic acid (TCA) cycle substrates, such as succinate and malate. The increased mitochondrial activity and biogenesis were further confirmed by immunofluorescence microscopy. Collectively, the results indicate that oxidative energy metabolism and mitochondrial biogenesis were sharply upregulated in DMSO-differentiated P19 cells. This functional metabolic and proteomic study provides further evidence that modulation of mitochondrial energy metabolism is a pivotal component of the cellular differentiation process and may dictate the final destiny of stem cells.

Wnt signaling-mediated redox regulation maintains the germ line stem cell differentiation niche

PubMed Central

Wang, Su; Gao, Yuan; Song, Xiaoqing; Ma, Xing; Zhu, Xiujuan; Mao, Ying; Yang, Zhihao; Ni, Jianquan; Li, Hua; Malanowski, Kathryn E; Anoja, Perera; Park, Jungeun; Haug, Jeff; Xie, Ting

2015-01-01

Adult stem cells continuously undergo self-renewal and generate differentiated cells. In the Drosophila ovary, two separate niches control germ line stem cell (GSC) self-renewal and differentiation processes. Compared to the self-renewing niche, relatively little is known about the maintenance and function of the differentiation niche. In this study, we show that the cellular redox state regulated by Wnt signaling is critical for the maintenance and function of the differentiation niche to promote GSC progeny differentiation. Defective Wnt signaling causes the loss of the differentiation niche and the upregulated BMP signaling in differentiated GSC progeny, thereby disrupting germ cell differentiation. Mechanistically, Wnt signaling controls the expression of multiple glutathione-S-transferase family genes and the cellular redox state. Finally, Wnt2 and Wnt4 function redundantly to maintain active Wnt signaling in the differentiation niche. Therefore, this study has revealed a novel strategy for Wnt signaling in regulating the cellular redox state and maintaining the differentiation niche. DOI: http://dx.doi.org/10.7554/eLife.08174.001 PMID:26452202

A comparative study of metabolic state of stem cells during osteogenic and adipogenic differentiations via fluorescence lifetime imaging microscopy

NASA Astrophysics Data System (ADS)

Chakraborty, Sandeep; Ou, Meng-Hsin; Kuo, Jean-Cheng; Chiou, Arthur

2016-10-01

Cellular metabolic state can serve as a biomarker to indicate the differentiation potential of stem cells into other specialized cell lineages. In this study, two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was applied to determine the fluorescence lifetime and the amounts of the auto-fluorescent metabolic co-factor reduced nicotinamide adenine dinucleotide (NADH) to elucidate the cellular metabolism of human mesenchymal stem cells (hMSCs) in osteogenic and adipogenic differentiation processes. 2P-FLIM provides the free to protein-bound NADH ratio which can serve as the indicator of cellular metabolic state. We measured NADH fluorescence lifetime at 0, 7, and 14 days after hMSCs were induced for either osteogenesis or adipogenesis. In both cases, the average fluorescence lifetime increased significantly at day 14 (P < 0.001), while the ratio of free to protein-bound NADH ratio decreased significantly in 7- days (P < 0.001) and 14-days (P < 0.001). Thus, our results indicated a higher metabolic rate in both osteogenic and adipogenic differentiation processes when compared with undifferentiated hMSCs. This approach may be further utilized to study proliferation efficiency and differentiation potential of stem cells into other specialized cell lineages.

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.

Intracellular Ca2+ concentration in the N1E-115 neuronal cell line and its use for peripheric nerve regeneration.

PubMed

Rodrigues, J M; Luís, A L; Lobato, J V; Pinto, M V; Faustino, A; Hussain, N Sooraj; Lopes, M A; Veloso, A P; Freitas, M; Geuna, S; Santos, J D; Maurício, A C

2005-01-01

Entubulation repair of peripheral nerve injuries has a lengthy history. Several experimental and clinical studies have explored the effectiveness of many biodegradable and non-degradable tubes with or without addition of molecules and cells. The main objective of the present study was to develop an economical and also an easy way for culturing a neural cell line which is capable of growing, differentiating and producing locally nerve growth factors, that are otherwise extremely expensive, inside 90 PLA/10 PLG nerve guides. For this purpose the authors have chosen the N1E-115 cell line, a clone of cells derived from mouse neuroblastoma C-1300 with the perspective of using this differentiated cellular system to cover the inside of 90 PLA/10 PLG nerve guides placed to bridge a nerve gap of 10 mm in the rat sciatic nerve experimental model. The N1E-115 cells proliferate in normal culture medium but undergo neuronal differentiation in response to DMSO. Upon induction of differentiation, proliferation of N1E-115 cells ceases, extensive neurite outgrowth is observed and the membranes become highly excitable. While it is known that Ca2+ serves as an important intracellular signal for cellular various processes, such as growth and differentiation, be toxic to cells and be involved in the triggering of events leading to excitotoxic cell death in neurons. The [Ca2+]i in non-differentiated N1E-115 cells and after distinct periods of differentiation, have been determined by the epifluorescence technique using the Fura-2-AM probe. The results of this quantitative assessment, revealed that N1E-115 cells which undergo neuronal differentiation for 48 hours in the presence of 1.5% DMSO are best qualified to be used to cover the interior of the nerve guides since the [Ca2+]i was not found to be elevated indicating thus that the onset the cell death processes was not occurred.

Cell Models and Their Application for Studying Adipogenic Differentiation in Relation to Obesity: A Review

PubMed Central

Ruiz-Ojeda, Francisco Javier; Rupérez, Azahara Iris; Gomez-Llorente, Carolina; Gil, Angel; Aguilera, Concepción María

2016-01-01

Over the last several years, the increasing prevalence of obesity has favored an intense study of adipose tissue biology and the precise mechanisms involved in adipocyte differentiation and adipogenesis. Adipocyte commitment and differentiation are complex processes, which can be investigated thanks to the development of diverse in vitro cell models and molecular biology techniques that allow for a better understanding of adipogenesis and adipocyte dysfunction associated with obesity. The aim of the present work was to update the different animal and human cell culture models available for studying the in vitro adipogenic differentiation process related to obesity and its co-morbidities. The main characteristics, new protocols, and applications of the cell models used to study the adipogenesis in the last five years have been extensively revised. Moreover, we depict co-cultures and three-dimensional cultures, given their utility to understand the connections between adipocytes and their surrounding cells in adipose tissue. PMID:27376273

In Vitro Germ Cell Differentiation from Cynomolgus Monkey Embryonic Stem Cells

PubMed Central

Yamauchi, Kaori; Hasegawa, Kouichi; Chuma, Shinichiro; Nakatsuji, Norio; Suemori, Hirofumi

2009-01-01

Background Mouse embryonic stem (ES) cells can differentiate into female and male germ cells in vitro. Primate ES cells can also differentiate into immature germ cells in vitro. However, little is known about the differentiation markers and culture conditions for in vitro germ cell differentiation from ES cells in primates. Monkey ES cells are thus considered to be a useful model to study primate gametogenesis in vitro. Therefore, in order to obtain further information on germ cell differentiation from primate ES cells, this study examined the ability of cynomolgus monkey ES cells to differentiate into germ cells in vitro. Methods and Findings To explore the differentiation markers for detecting germ cells differentiated from ES cells, the expression of various germ cell marker genes was examined in tissues and ES cells of the cynomolgus monkey (Macaca fascicularis). VASA is a valuable gene for the detection of germ cells differentiated from ES cells. An increase of VASA expression was observed when differentiation was induced in ES cells via embryoid body (EB) formation. In addition, the expression of other germ cell markers, such as NANOS and PIWIL1 genes, was also up-regulated as the EB differentiation progressed. Immunocytochemistry identified the cells expressing stage-specific embryonic antigen (SSEA) 1, OCT-4, and VASA proteins in the EBs. These cells were detected in the peripheral region of the EBs as specific cell populations, such as SSEA1-positive, OCT-4-positive cells, OCT-4-positive, VASA-positive cells, and OCT-4-negative, VASA-positive cells. Thereafter, the effect of mouse gonadal cell-conditioned medium and growth factors on germ cell differentiation from monkey ES cells was examined, and this revealed that the addition of BMP4 to differentiating ES cells increased the expression of SCP1, a meiotic marker gene. Conclusion VASA is a valuable gene for the detection of germ cells differentiated from ES cells in monkeys, and the identification and characterization of germ cells derived from ES cells are possible by using reported germ cell markers in vivo, including SSEA1, OCT-4, and VASA, in vitro as well as in vivo. These findings are thus considered to help elucidate the germ cell developmental process in primates. PMID:19399191

Pleiotrophin antagonizes Brd2 during neuronal differentiation

PubMed Central

Garcia-Gutierrez, Pablo; Juarez-Vicente, Francisco; Wolgemuth, Debra J.; Garcia-Dominguez, Mario

2014-01-01

ABSTRACT Bromodomain-containing protein 2 (Brd2) is a BET family chromatin adaptor required for expression of cell-cycle-associated genes and therefore involved in cell cycle progression. Brd2 is expressed in proliferating neuronal progenitors, displays cell-cycle-stimulating activity and, when overexpressed, impairs neuronal differentiation. Paradoxically, Brd2 is also detected in differentiating neurons. To shed light on the role of Brd2 in the transition from cell proliferation to differentiation, we had previously looked for proteins that interacted with Brd2 upon induction of neuronal differentiation. Surprisingly, we identified the growth factor pleiotrophin (Ptn). Here, we show that Ptn antagonized the cell-cycle-stimulating activity associated with Brd2, thus enhancing induced neuronal differentiation. Moreover, Ptn knockdown reduced neuronal differentiation. We analyzed Ptn-mediated antagonism of Brd2 in a cell differentiation model and in two embryonic processes associated with the neural tube: spinal cord neurogenesis and neural crest migration. Finally, we investigated the mechanisms of Ptn-mediated antagonism and determined that Ptn destabilizes the association of Brd2 with chromatin. Thus, Ptn-mediated Brd2 antagonism emerges as a modulation system accounting for the balance between cell proliferation and differentiation in the vertebrate nervous system. PMID:24695857

The Methods and Mechanisms to Differentiate Endothelial-Like Cells and Smooth Muscle Cells from Mesenchymal Stem Cells for Vascularization in Vaginal Reconstruction.

PubMed

Zhang, Hua; Zhang, Jingkun; Huang, Xianghua; Li, Yanan

2018-06-01

Endothelial cells and smooth muscle cells (SMCs) are important aspects of vascularization in vaginal reconstruction. Research has confirmed that mesenchymal stem cells could differentiate into endothelial-like cells and SMCs. But the methods were more complicated and the mechanism was unknown. In the current study, we induced the bone mesenchymal stem cells (BMSCs) to differentiate into endothelial-like cells and SMCs in vitro by differentiation medium and investigated the effect of Wnt/β-catenin signaling on the differentiation process of BMSCs. Results showed that the hypoxic environment combined with VEGF and bFGF could induce increased expression of endothelial-like cells markers VEGFR1, VEGFR2, and vWF. The SMCs derived from BMSCs induced by TGF-β1 and PDGF-AB significantly expressed SMC markers SMMHC11 and α-SMA. The data also showed that activation of Wnt/β-catenin signaling could promote the differentiation of BMSCs into endothelial-like cells and SMCs. Thus, we established endothelial-like cells and SMCs in vitro by more simple methods, presented the important role of hypoxic environment on the differentiation of BMSCs into endothelial-like cells, and confirmed that the Wnt/β-catenin signaling pathway has a positive impact on the differentiation of BMSCs into endothelial-like cells and SMCs. This is important for vascular reconstruction.

Cannabidiol stimulates Aml-1a-dependent glial differentiation and inhibits glioma stem-like cells proliferation by inducing autophagy in a TRPV2-dependent manner.

PubMed

Nabissi, Massimo; Morelli, Maria Beatrice; Amantini, Consuelo; Liberati, Sonia; Santoni, Matteo; Ricci-Vitiani, Lucia; Pallini, Roberto; Santoni, Giorgio

2015-10-15

Glioma stem-like cells (GSCs) correspond to a tumor cell subpopulation, involved in glioblastoma multiforme (GBM) tumor initiation and acquired chemoresistance. Currently, drug-induced differentiation is considered as a promising approach to eradicate this tumor-driving cell population. Recently, the effect of cannabinoids (CBs) in promoting glial differentiation and inhibiting gliomagenesis has been evidenced. Herein, we demonstrated that cannabidiol (CBD) by activating transient receptor potential vanilloid-2 (TRPV2) triggers GSCs differentiation activating the autophagic process and inhibits GSCs proliferation and clonogenic capability. Above all, CBD and carmustine (BCNU) in combination overcome the high resistance of GSCs to BCNU treatment, by inducing apoptotic cell death. Acute myeloid leukemia (Aml-1) transcription factors play a pivotal role in GBM proliferation and differentiation and it is known that Aml-1 control the expression of several nociceptive receptors. So, we evaluated the expression levels of Aml-1 spliced variants (Aml-1a, b and c) in GSCs and during their differentiation. We found that Aml-1a is upregulated during GSCs differentiation, and its downregulation restores a stem cell phenotype in differentiated GSCs. Since it was demonstrated that CBD induces also TRPV2 expression and that TRPV2 is involved in GSCs differentiation, we evaluated if Aml-1a interacted directly with TRPV2 promoters. Herein, we found that Aml-1a binds TRPV2 promoters and that Aml-1a expression is upregulated by CBD treatment, in a TRPV2 and PI3K/AKT dependent manner. Altogether, these results support a novel mechanism by which CBD inducing TRPV2-dependent autophagic process stimulates Aml-1a-dependent GSCs differentiation, abrogating the BCNU chemoresistance in GSCs. © 2015 UICC.

Evidence for a terminal differentiation process in the rat liver.

PubMed

Sigal, S H; Gupta, S; Gebhard, D F; Holst, P; Neufeld, D; Reid, L M

1995-07-01

In rapidly renewing epithelia, such as skin and gut, as well as hemopoietic cells and stromal fibroblasts, the process of progenitor cell maturation, terminal differentiation and senescence from cells of a fetal phenotype is strikingly similar. To examine hepatocellular maturation, we studied embryonic, suckling and young adult rat liver cells with multiparametric fluorescence activated cell sorting (FACS), after exclusion of hemopoietic, endothelial, Kupffer, and nonviable cells. With maturation, cell granularity and autofluorescence exponentially increased from fetal liver to suckling and adult liver as the proportion of S phase cells progressively declined from 33.8% +/- 1.3% to 4.9% +/- 2.8% and 1.1% +/- 0.6% (P < 0.05), respectively. In liver from fetal and suckling rats, all hepatocytes were mononuclear and contained diploid DNA whereas 21.2% +/- 5.9% hepatocytes in adult liver were binucleated. Analysis of nuclear DNA content in adult hepatocytes demonstrated that 53.3% +/- 3.9% of the nuclei were diploid, 43.6% +/- 3.5% tetraploid and 0.5 +/- 0.6% octaploid. However, in the adult liver, small, mononuclear cells were also present with granularity and autofluorescence comparable to fetal hepatoblasts, as well as glucose-6-phosphatase activity, diploid DNA in 89.0% +/- 2.1% of the nuclei, and with increased granularity in culture. Since general features of terminal cellularity differentiation and senescence include cessation of mitotic activity, polyploidy and accumulation of autofluorescent secondary lysosomes, our data suggest that liver cells too undergo a process of terminal differentiation.

Differentiation of Mesenchymal Stem Cells Derived from Pancreatic Islets and Bone Marrow into Islet-Like Cell Phenotype

PubMed Central

Zanini, Cristina; Bruno, Stefania; Mandili, Giorgia; Baci, Denisa; Cerutti, Francesco; Cenacchi, Giovanna; Izzi, Leo; Camussi, Giovanni; Forni, Marco

2011-01-01

Background Regarding regenerative medicine for diabetes, accessible sources of Mesenchymal Stem Cells (MSCs) for induction of insular beta cell differentiation may be as important as mastering the differentiation process itself. Methodology/Principal Findings In the present work, stem cells from pancreatic islets (human islet-mesenchymal stem cells, HI-MSCs) and from human bone marrow (bone marrow mesenchymal stem cells, BM-MSCs) were cultured in custom-made serum-free medium, using suitable conditions in order to induce differentiation into Islet-like Cells (ILCs). HI-MSCs and BM-MSCs were positive for the MSC markers CD105, CD73, CD90, CD29. Following this induction, HI-MSC and BM-MSC formed evident islet-like structures in the culture flasks. To investigate functional modifications after induction to ILCs, ultrastructural analysis and immunofluorescence were performed. PDX1 (pancreatic duodenal homeobox gene-1), insulin, C peptide and Glut-2 were detected in HI-ILCs whereas BM-ILCs only expressed Glut-2 and insulin. Insulin was also detected in the culture medium following glucose stimulation, confirming an initial differentiation that resulted in glucose-sensitive endocrine secretion. In order to identify proteins that were modified following differentiation from basal MSC (HI-MSCs and BM-MSCs) to their HI-ILCs and BM-ILCs counterparts, proteomic analysis was performed. Three new proteins (APOA1, ATL2 and SODM) were present in both ILC types, while other detected proteins were verified to be unique to the single individual differentiated cells lines. Hierarchical analysis underscored the limited similarities between HI-MSCs and BM-MSCs after induction of differentiation, and the persistence of relevant differences related to cells of different origin. Conclusions/Significance Proteomic analysis highlighted differences in the MSCs according to site of origin, reflecting spontaneous differentiation and commitment. A more detailed understanding of protein assets may provide insights required to master the differentiation process of HI-MSCs to functional beta cells based only upon culture conditioning. These findings may open new strategies for the clinical use of BM-MSCs in diabetes. PMID:22194812

Plant GSK3 proteins regulate xylem cell differentiation downstream of TDIF-TDR signalling

NASA Astrophysics Data System (ADS)

Kondo, Yuki; Ito, Tasuku; Nakagami, Hirofumi; Hirakawa, Yuki; Saito, Masato; Tamaki, Takayuki; Shirasu, Ken; Fukuda, Hiroo

2014-03-01

During plant radial growth typically seen in trees, procambial and cambial cells act as meristematic cells in the vascular system to self-proliferate and differentiate into xylem cells. These two processes are regulated by a signalling pathway composed of a peptide ligand and its receptor; tracheary element differentiation inhibitory factor (TDIF) and TDIF RECEPTOR (TDR). Here we show that glycogen synthase kinase 3 proteins (GSK3s) are crucial downstream components of the TDIF signalling pathway suppressing xylem differentiation from procambial cells. TDR interacts with GSK3s at the plasma membrane and activates GSK3s in a TDIF-dependent fashion. Consistently, a specific inhibitor of plant GSK3s strongly induces xylem cell differentiation through BRI1-EMS SUPPRESSOR 1 (BES1), a well-known target transcription factor of GSK3s. Our findings provide insight into the regulation of cell fate determination in meristem maintenance.

Evidence that PC2 is the endogenous pro-neurotensin convertase in rMTC 6-23 cells and that PC1- and PC2-transfected PC12 cells differentially process pro-neurotensin.

PubMed

Rovère, C; Barbero, P; Kitabgi, P

1996-05-10

The neuropeptide precursor proneurotensin/neuromedin N (pro-NT/NN) is mainly expressed and differentially processed in the brain and in the small intestine. We showed previously that rMTC 6-23 cells process pro-NT/NN with a pattern similar to brain tissue and increase pro-NT/NN expression in response to dexamethasone, and that PC12 cells also produce pro-NT/NN but are virtually unable to process it. In addition, PC12 cells were reported to be devoid of the prohormone convertases PC1 and PC2. The present study was designed to identify the proprotein convertase(s) (PC) involved in pro-NT/NN processing in rMTC 6-23 cells and to compare PC1- and PC2-transfected PC12 cells for their ability to process pro-NT/NN. rMTC 6-23 cells were devoid of PC1, PC4, and PC5 but expressed furin and PC2. Stable expression of antisense PC2 RNA in rMTC 6-23 cells led to a 90% decrease in PC2 protein levels that correlated with a > 80% reduction of pro-NT/NN processing. PC2 expression was stimulated by dexamethasone in a time- and concentration-dependent manner. Stable PC12/PC2 transfectants processed pro-NT/NN with a pattern similar to that observed in the brain and in rMTC 6-23 cells. In contrast, stable PC12/PC1 transfectants reproduced the pro-NT/NN processing pattern seen in the gut. We conclude that (i) PC2 is the major pro-NT/NN convertase in rMTC 6-23 cells; (ii) its expression is coregulated with that of pro-NT/NN in this cell line; and (iii) PC2 and PC1 differentially process pro-NT/NN with brain and intestinal phenotype, respectively.

Progressive Chromatin Condensation and H3K9 Methylation Regulate the Differentiation of Embryonic and Hematopoietic Stem Cells

DOE PAGES

Ugarte, Fernando; Sousae, Rebekah; Cinquin, Bertrand; ...

2015-10-17

Epigenetic regulation serves as the basis for stem cell differentiation into distinct cell types, but it is unclear how global epigenetic changes are regulated during this process. Here, we tested the hypothesis that global chromatin organization affects the lineage potential of stem cells and that manipulation of chromatin dynamics influences stem cell function. Using nuclease sensitivity assays, we found a progressive decrease in chromatin digestion among pluripotent embryonic stem cells (ESCs), multipotent hematopoietic stem cells (HSCs), and mature hematopoietic cells. Quantitative high-resolution microscopy revealed that ESCs contain significantly more euchromatin than HSCs, with a further reduction in mature cells. Increasedmore » cellular maturation also led to heterochromatin localization to the nuclear periphery. Functionally, prevention of heterochromatin formation by inhibition of the histone methyltransferase G9A resulted in delayed HSC differentiation. Lastly, our results demonstrate global chromatin rearrangements during stem cell differentiation and that heterochromatin formation by H3K9 methylation regulates HSC differentiation.« less

Progressive Chromatin Condensation and H3K9 Methylation Regulate the Differentiation of Embryonic and Hematopoietic Stem Cells

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

Ugarte, Fernando; Sousae, Rebekah; Cinquin, Bertrand

Epigenetic regulation serves as the basis for stem cell differentiation into distinct cell types, but it is unclear how global epigenetic changes are regulated during this process. Here, we tested the hypothesis that global chromatin organization affects the lineage potential of stem cells and that manipulation of chromatin dynamics influences stem cell function. Using nuclease sensitivity assays, we found a progressive decrease in chromatin digestion among pluripotent embryonic stem cells (ESCs), multipotent hematopoietic stem cells (HSCs), and mature hematopoietic cells. Quantitative high-resolution microscopy revealed that ESCs contain significantly more euchromatin than HSCs, with a further reduction in mature cells. Increasedmore » cellular maturation also led to heterochromatin localization to the nuclear periphery. Functionally, prevention of heterochromatin formation by inhibition of the histone methyltransferase G9A resulted in delayed HSC differentiation. Lastly, our results demonstrate global chromatin rearrangements during stem cell differentiation and that heterochromatin formation by H3K9 methylation regulates HSC differentiation.« less

Differential effects of Notch ligands Delta-1 and Jagged-1 in human lymphoid differentiation.

PubMed

Jaleco, A C; Neves, H; Hooijberg, E; Gameiro, P; Clode, N; Haury, M; Henrique, D; Parreira, L

2001-10-01

Notch signaling is known to differentially affect the development of lymphoid B and T cell lineages, but it remains unclear whether such effects are specifically dependent on distinct Notch ligands. Using a cell coculture assay we observed that the Notch ligand Delta-1 completely inhibits the differentiation of human hematopoietic progenitors into the B cell lineage while promoting the emergence of cells with a phenotype of T cell/natural killer (NK) precursors. In contrast, Jagged-1 did not disturb either B or T cell/NK development. Furthermore, cells cultured in the presence of either Delta-1 or Jagged-1 can acquire a phenotype of NK cells, and Delta-1, but not Jagged-1, permits the emergence of a de novo cell population coexpressing CD4 and CD8. Our results thus indicate that distinct Notch ligands can mediate differential effects of Notch signaling and provide a useful system to further address cell-fate decision processes in lymphopoiesis.

Differential Effects of Notch Ligands Delta-1 and Jagged-1 in Human Lymphoid Differentiation

PubMed Central

Jaleco, Ana C.; Neves, Hélia; Hooijberg, Erik; Gameiro, Paula; Clode, Nuno; Haury, Matthias; Henrique, Domingos; Parreira, Leonor

2001-01-01

Notch signaling is known to differentially affect the development of lymphoid B and T cell lineages, but it remains unclear whether such effects are specifically dependent on distinct Notch ligands. Using a cell coculture assay we observed that the Notch ligand Delta-1 completely inhibits the differentiation of human hematopoietic progenitors into the B cell lineage while promoting the emergence of cells with a phenotype of T cell/natural killer (NK) precursors. In contrast, Jagged-1 did not disturb either B or T cell/NK development. Furthermore, cells cultured in the presence of either Delta-1 or Jagged-1 can acquire a phenotype of NK cells, and Delta-1, but not Jagged-1, permits the emergence of a de novo cell population coexpressing CD4 and CD8. Our results thus indicate that distinct Notch ligands can mediate differential effects of Notch signaling and provide a useful system to further address cell-fate decision processes in lymphopoiesis. PMID:11581320

Hacking cell differentiation: transcriptional rerouting in reprogramming, lineage infidelity and metaplasia.

PubMed

Regalo, Gonçalo; Leutz, Achim

2013-08-01

Initiating neoplastic cell transformation events are of paramount importance for the comprehension of regeneration and vanguard oncogenic processes but are difficult to characterize and frequently clinically overlooked. In epithelia, pre-neoplastic transformation stages are often distinguished by the appearance of phenotypic features of another differentiated tissue, termed metaplasia. In haemato/lymphopoietic malignancies, cell lineage ambiguity is increasingly recorded. Both, metaplasia and biphenotypic leukaemia/lymphoma represent examples of dysregulated cell differentiation that reflect a history of trans-differentiation and/or epigenetic reprogramming. Here we compare the similarity between molecular events of experimental cell trans-differentiation as an emerging therapeutic concept, with lineage confusion, as in metaplasia and dysplasia forecasting tumour development. © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.

Labeling of neuronal differentiation and neuron cells with biocompatible fluorescent nanodiamonds

PubMed Central

Hsu, Tzu-Chia; Liu, Kuang-Kai; Chang, Huan-Cheng; Hwang, Eric; Chao, Jui-I

2014-01-01

Nanodiamond is a promising carbon nanomaterial developed for biomedical applications. Here, we show fluorescent nanodiamond (FND) with the biocompatible properties that can be used for the labeling and tracking of neuronal differentiation and neuron cells derived from embryonal carcinoma stem (ECS) cells. The fluorescence intensities of FNDs were increased by treatment with FNDs in both the mouse P19 and human NT2/D1 ECS cells. FNDs were taken into ECS cells; however, FNDs did not alter the cellular morphology and growth ability. Moreover, FNDs did not change the protein expression of stem cell marker SSEA-1 of ECS cells. The neuronal differentiation of ECS cells could be induced by retinoic acid (RA). Interestingly, FNDs did not affect on the morphological alteration, cytotoxicity and apoptosis during the neuronal differentiation. Besides, FNDs did not alter the cell viability and the expression of neuron-specific marker β-III-tubulin in these differentiated neuron cells. The existence of FNDs in the neuron cells can be identified by confocal microscopy and flow cytometry. Together, FND is a biocompatible and readily detectable nanomaterial for the labeling and tracking of neuronal differentiation process and neuron cells from stem cells. PMID:24830447

Labeling of neuronal differentiation and neuron cells with biocompatible fluorescent nanodiamonds.

PubMed

Hsu, Tzu-Chia; Liu, Kuang-Kai; Chang, Huan-Cheng; Hwang, Eric; Chao, Jui-I

2014-05-16

Nanodiamond is a promising carbon nanomaterial developed for biomedical applications. Here, we show fluorescent nanodiamond (FND) with the biocompatible properties that can be used for the labeling and tracking of neuronal differentiation and neuron cells derived from embryonal carcinoma stem (ECS) cells. The fluorescence intensities of FNDs were increased by treatment with FNDs in both the mouse P19 and human NT2/D1 ECS cells. FNDs were taken into ECS cells; however, FNDs did not alter the cellular morphology and growth ability. Moreover, FNDs did not change the protein expression of stem cell marker SSEA-1 of ECS cells. The neuronal differentiation of ECS cells could be induced by retinoic acid (RA). Interestingly, FNDs did not affect on the morphological alteration, cytotoxicity and apoptosis during the neuronal differentiation. Besides, FNDs did not alter the cell viability and the expression of neuron-specific marker β-III-tubulin in these differentiated neuron cells. The existence of FNDs in the neuron cells can be identified by confocal microscopy and flow cytometry. Together, FND is a biocompatible and readily detectable nanomaterial for the labeling and tracking of neuronal differentiation process and neuron cells from stem cells.

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)

Coordinated Proliferation and Differentiation of Human-Induced Pluripotent Stem Cell-Derived Cardiac Progenitor Cells Depend on Bone Morphogenetic Protein Signaling Regulation by GREMLIN 2

PubMed Central

Bylund, Jeffery B.; Trinh, Linh T.; Awgulewitsch, Cassandra P.; Paik, David T.; Jetter, Christopher; Jha, Rajneesh; Zhang, Jianhua; Nolan, Kristof; Xu, Chunhui; Thompson, Thomas B.; Kamp, Timothy J.

2017-01-01

Heart development depends on coordinated proliferation and differentiation of cardiac progenitor cells (CPCs), but how the two processes are synchronized is not well understood. Here, we show that the secreted Bone Morphogenetic Protein (BMP) antagonist GREMLIN 2 (GREM2) is induced in CPCs shortly after cardiac mesoderm specification during differentiation of human pluripotent stem cells. GREM2 expression follows cardiac lineage differentiation independently of the differentiation method used, or the origin of the pluripotent stem cells, suggesting that GREM2 is linked to cardiogenesis. Addition of GREM2 protein strongly increases cardiomyocyte output compared to established procardiogenic differentiation methods. Our data show that inhibition of canonical BMP signaling by GREM2 is necessary to promote proliferation of CPCs. However, canonical BMP signaling inhibition alone is not sufficient to induce cardiac differentiation, which depends on subsequent JNK pathway activation specifically by GREM2. These findings may have broader implications in the design of approaches to orchestrate growth and differentiation of pluripotent stem cell-derived lineages that depend on precise regulation of BMP signaling. PMID:28125926

Coordinated Proliferation and Differentiation of Human-Induced Pluripotent Stem Cell-Derived Cardiac Progenitor Cells Depend on Bone Morphogenetic Protein Signaling Regulation by GREMLIN 2.

PubMed

Bylund, Jeffery B; Trinh, Linh T; Awgulewitsch, Cassandra P; Paik, David T; Jetter, Christopher; Jha, Rajneesh; Zhang, Jianhua; Nolan, Kristof; Xu, Chunhui; Thompson, Thomas B; Kamp, Timothy J; Hatzopoulos, Antonis K

2017-05-01

Heart development depends on coordinated proliferation and differentiation of cardiac progenitor cells (CPCs), but how the two processes are synchronized is not well understood. Here, we show that the secreted Bone Morphogenetic Protein (BMP) antagonist GREMLIN 2 (GREM2) is induced in CPCs shortly after cardiac mesoderm specification during differentiation of human pluripotent stem cells. GREM2 expression follows cardiac lineage differentiation independently of the differentiation method used, or the origin of the pluripotent stem cells, suggesting that GREM2 is linked to cardiogenesis. Addition of GREM2 protein strongly increases cardiomyocyte output compared to established procardiogenic differentiation methods. Our data show that inhibition of canonical BMP signaling by GREM2 is necessary to promote proliferation of CPCs. However, canonical BMP signaling inhibition alone is not sufficient to induce cardiac differentiation, which depends on subsequent JNK pathway activation specifically by GREM2. These findings may have broader implications in the design of approaches to orchestrate growth and differentiation of pluripotent stem cell-derived lineages that depend on precise regulation of BMP signaling.

The Maize MID-COMPLEMENTING ACTIVITY homolog CELL NUMBER REGULATOR13/NARROW ODD DWARF, coordinates organ growth and tissue patterning

USDA-ARS?s Scientific Manuscript database

Organogenesis occurs from cell division, expansion and differentiation. How these cellular processes are coordinated remains elusive. The maize leaf provides an excellent system to study cellular differentiation because it has several different tissues and cell types. The narrow odd dwarf (nod) mut...

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

Evidence for heterogeneity of astrocyte de-differentiation in vitro: astrocytes transform into intermediate precursor cells following induction of ACM from scratch-insulted astrocytes.

PubMed

Yang, Hao; Qian, Xin-Hong; Cong, Rui; Li, Jing-wen; Yao, Qin; Jiao, Xi-Ying; Ju, Gong; You, Si-Wei

2010-04-01

Our previous study definitely demonstrated that the mature astrocytes could undergo a de-differentiation process and further transform into pluripotential neural stem cells (NSCs), which might well arise from the effect of diffusible factors released from scratch-insulted astrocytes. However, these neurospheres passaged from one neurosphere-derived from de-differentiated astrocytes possessed a completely distinct characteristic in the differentiation behavior, namely heterogeneity of differentiation. The heterogeneity in cell differentiation has become a crucial but elusive issue. In this study, we show that purified astrocytes could de-differentiate into intermediate precursor cells (IPCs) with addition of scratch-insulted astrocyte-conditioned medium (ACM) to the culture, which can express NG2 and A2B5, the IPCs markers. Apart from the number of NG2(+) and A2B5(+) cells, the percentage of proliferative cells as labeled with BrdU progressively increased with prolonged culture period ranging from 1 to 10 days. Meanwhile, the protein level of A2B5 in cells also increased significantly. These results revealed that not all astrocytes could de-differentiate fully into NSCs directly when induced by ACM, rather they generated intermediate or more restricted precursor cells that might undergo progressive de-differentiation to generate NSCs.

Raman and Autofluorescence Spectrum Dynamics along the HRG-Induced Differentiation Pathway of MCF-7 Cells

PubMed Central

Morita, Shin-ichi; Takanezawa, Sota; Hiroshima, Michio; Mitsui, Toshiyuki; Ozaki, Yukihiro; Sako, Yasushi

2014-01-01

Cellular differentiation proceeds along complicated pathways, even when it is induced by extracellular signaling molecules. One of the major reasons for this complexity is the highly multidimensional internal dynamics of cells, which sometimes causes apparently stochastic responses in individual cells to extracellular stimuli. Therefore, to understand cell differentiation, it is necessary to monitor the internal dynamics of cells at single-cell resolution. Here, we used a Raman and autofluorescence spectrum analysis of single cells to detect dynamic changes in intracellular molecular components. MCF-7 cells are a human cancer-derived cell line that can be induced to differentiate into mammary-gland-like cells with the addition of heregulin (HRG) to the culture medium. We measured the spectra in the cytoplasm of MCF-7 cells during 12 days of HRG stimulation. The Raman scattering spectrum, which was the major component of the signal, changed with time. A multicomponent analysis of the Raman spectrum revealed that the dynamics of the major components of the intracellular molecules, including proteins and lipids, changed cyclically along the differentiation pathway. The background autofluorescence signals of Raman scattering also provided information about the differentiation process. Using the total information from the Raman and autofluorescence spectra, we were able to visualize the pathway of cell differentiation in the multicomponent phase space. PMID:25418290

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.

SOX2 and SOX2-MYC Reprogramming Process of Fibroblasts to the Neural Stem Cells Compromised by Senescence

PubMed Central

Winiecka-Klimek, Marta; Smolarz, Maciej; Walczak, Maciej P.; Zieba, Jolanta; Hulas-Bigoszewska, Krystyna; Kmieciak, Blazej; Piaskowski, Sylwester; Rieske, Piotr; Grzela, Dawid P.; Stoczynska-Fidelus, Ewelina

2015-01-01

Tumorigenic potential of induced pluripotent stem cells (iPSCs) infiltrating population of induced neural stem cells (iNSCs) generated from iPSCs may limit their medical applications. To overcome such a difficulty, direct reprogramming of adult somatic cells into iNSCs was proposed. The aim of this study was the systematic comparison of induced neural cells (iNc) obtained with different methods—direct reprogramming of human adult fibroblasts with either SOX2 (SiNSc-like) or SOX2 and c-MYC (SMiNSc-like) and induced pluripotent stem cells differentiation to ebiNSc—in terms of gene expression profile, differentiation potential as well as proliferation properties. Immunocytochemistry and real-time PCR analyses were used to evaluate gene expression profile and differentiation potential of various iNc types. Bromodeoxyuridine (BrdU) incorporation and senescence-associated beta-galactosidase (SA-β-gal) assays were used to estimate proliferation potential. All three types of iNc were capable of neuronal differentiation; however, astrocytic differentiation was possible only in case of ebiNSc. Contrary to ebiNSc generation, the direct reprogramming was rarely a propitious process, despite 100% transduction efficiency. The potency of direct iNSCs-like cells generation was lower as compared to iNSCs obtained by iPSCs differentiation, and only slightly improved when c-MYC was added. Directly reprogrammed iNSCs-like cells were lacking the ability to differentiate into astrocytic cells and characterized by poor efficiency of neuronal cells formation. Such features indicated that these cells could not be fully reprogrammed, as confirmed mainly with senescence detection. Importantly, SiNSc-like and SMiNSc-like cells were unable to achieve the long-term survival and became senescent, which limits their possible therapeutic applicability. Our results suggest that iNSCs-like cells, generated in the direct reprogramming attempts, were either not fully reprogrammed or reprogrammed only into neuronal progenitors, mainly because of the inaccuracies of currently available protocols. PMID:26535892

IDH1R132H in Neural Stem Cells: Differentiation Impaired by Increased Apoptosis

PubMed Central

Rosiak, Kamila; Smolarz, Maciej; Stec, Wojciech J.; Peciak, Joanna; Grzela, Dawid; Winiecka-Klimek, Marta; Stoczynska-Fidelus, Ewelina; Krynska, Barbara; Piaskowski, Sylwester; Rieske, Piotr

2016-01-01

Background The high frequency of mutations in the isocitrate dehydrogenase 1 (IDH1) gene in diffuse gliomas indicates its importance in the process of gliomagenesis. These mutations result in loss of the normal function and acquisition of the neomorphic activity converting α-ketoglutarate to 2-hydroxyglutarate. This potential oncometabolite may induce the epigenetic changes, resulting in the deregulated expression of numerous genes, including those related to the differentiation process or cell survivability. Methods Neural stem cells were derived from human induced pluripotent stem cells following embryoid body formation. Neural stem cells transduced with mutant IDH1R132H, empty vector, non-transduced and overexpressing IDH1WT controls were differentiated into astrocytes and neurons in culture. The neuronal and astrocytic differentiation was determined by morphology and expression of lineage specific markers (MAP2, Synapsin I and GFAP) as determined by real-time PCR and immunocytochemical staining. Apoptosis was evaluated by real-time observation of Caspase-3 activation and measurement of PARP cleavage by Western Blot. Results Compared with control groups, cells expressing IDH1R132H retained an undifferentiated state and lacked morphological changes following stimulated differentiation. The significant inhibitory effect of IDH1R132H on neuronal and astrocytic differentiation was confirmed by immunocytochemical staining for markers of neural stem cells. Additionally, real-time PCR indicated suppressed expression of lineage markers. High percentage of apoptotic cells was detected within IDH1R132H-positive neural stem cells population and their derivatives, if compared to normal neural stem cells and their derivatives. The analysis of PARP and Caspase-3 activity confirmed apoptosis sensitivity in mutant protein-expressing neural cells. Conclusions Our study demonstrates that expression of IDH1R132H increases apoptosis susceptibility of neural stem cells and their derivatives. Robust apoptosis causes differentiation deficiency of IDH1R132H-expressing cells. PMID:27145078

Adipogenic placenta-derived mesenchymal stem cells are not lineage restricted by withdrawing extrinsic factors: developing a novel visual angle in stem cell biology.

PubMed

Hu, C; Cao, H; Pan, X; Li, J; He, J; Pan, Q; Xin, J; Yu, X; Li, J; Wang, Y; Zhu, D; Li, L

2016-03-17

Current evidence implies that differentiated bone marrow mesenchymal stem cells (BMMSCs) can act as progenitor cells and transdifferentiate across lineage boundaries. However, whether this unrestricted lineage has specificities depending on the stem cell type is unknown. Placental-derived mesenchymal stem cells (PDMSCs), an easily accessible and less invasive source, are extremely useful materials in current stem cell therapies. No studies have comprehensively analyzed the transition in morphology, surface antigens, metabolism and multilineage potency of differentiated PDMSCs after their dedifferentiation. In this study, we showed that after withdrawing extrinsic factors, adipogenic PDMSCs reverted to a primitive cell population and retained stem cell characteristics. The mitochondrial network during differentiation and dedifferentiation may serve as a marker of absent or acquired pluripotency in various stem cell models. The new population proliferated faster than unmanipulated PDMSCs and could be differentiated into adipocytes, osteocytes and hepatocytes. The cell adhesion molecules (CAMs) signaling pathway and extracellular matrix (ECM) components modulate cell behavior and enable the cells to proliferate or differentiate during the differentiation, dedifferentiation and redifferentiation processes in our study. These observations indicate that the dedifferentiated PDMSCs are distinguishable from the original PDMSCs and may serve as a novel source in stem cell biology and cell-based therapeutic strategies. Furthermore, whether PDMSCs differentiated into other lineages can be dedifferentiated to a primitive cell population needs to be investigated.

Cell Expansion During Directed Differentiation of Stem Cells Toward the Hepatic Lineage

PubMed Central

Raju, Ravali; Chau, David; Cho, Dong Seong; Park, Yonsil; Verfaillie, Catherine M.

2017-01-01

The differentiation of human pluripotent stem cells toward the hepatocyte lineage can potentially provide an unlimited source of functional hepatocytes for transplantation and extracorporeal bioartificial liver applications. It is anticipated that the quantities of cells needed for these applications will be in the order of 109–1010 cells, because of the size of the liver. An ideal differentiation protocol would be to enable directed differentiation to the hepatocyte lineage with simultaneous cell expansion. We introduced a cell expansion stage after the commitment of human embryonic stem cells to the endodermal lineage, to allow for at least an eightfold increase in cell number, with continuation of cell maturation toward the hepatocyte lineage. The progressive changes in the transcriptome were measured by expression array, and the expression dynamics of certain lineage markers was measured by mass cytometry during the differentiation and expansion process. The findings revealed that while cells were expanding they were also capable of progressing in their differentiation toward the hepatocyte lineage. In addition, our transcriptome, protein and functional studies, including albumin secretion, drug-induced CYP450 expression and urea production, all indicated that the hepatocyte-like cells obtained with or without cell expansion are very similar. This method of simultaneous cell expansion and hepatocyte differentiation should facilitate obtaining large quantities of cells for liver cell applications. PMID:27806669

Identification of Phosphoproteins as Possible Differentiation Markers in All-Trans-Retinoic Acid-Treated Neuroblastoma Cells

PubMed Central

Mandili, Giorgia; Marini, Cristina; Carta, Franco; Zanini, Cristina; Prato, Mauro; Khadjavi, Amina

2011-01-01

Background Neuroblastic tumors account for 9–10% of pediatric tumors and neuroblastoma (NB) is the first cause of death in pre-school age children. NB is classified in four stages, depending on the extent of spreading. A fifth type of NB, so-called stage 4S (S for special), includes patients with metastatic tumors but with an overall survival that approximates 75% at five years. In most of these cases, the tumor regresses spontaneously and regression is probably associated with delayed neuroblast cell differentiation. Methodology/Principal Findings In order to identify new early markers to follow and predict this process for diagnostic and therapeutics intents, we mimicked the differentiation process treating NB cell line SJ-NK-P with all-trans-retinoic acid (ATRA) at different times; therefore the cell proteomic pattern by mass spectrometry and the phosphoproteomic pattern by a 2-DE approach coupled with anti-phosphoserine and anti-phosphotyrosine western blotting were studied. Conclusions/Significance Proteomic analysis identified only two proteins whose expression was significantly different in treated cells versus control cells: nucleoside diphosphate kinase A (NDKA) and reticulocalbin-1 (RCN1), which were both downregulated after 9 days of ATRA treatment. However, phosphoproteomic analysis identified 8 proteins that were differentially serine-phosphorylated and 3 that were differentially tyrosine-phosphorylated after ATRA treatment. All proteins were significantly regulated (at least 0.5-fold down-regulated). Our results suggest that differentially phosphorylated proteins could be considered as more promising markers of differentiation for NB than differentially expressed proteins. PMID:21573212

Effector Regulatory T Cell Differentiation and Immune Homeostasis Depend on the Transcription Factor Myb.

PubMed

Dias, Sheila; D'Amico, Angela; Cretney, Erika; Liao, Yang; Tellier, Julie; Bruggeman, Christine; Almeida, Francisca F; Leahy, Jamie; Belz, Gabrielle T; Smyth, Gordon K; Shi, Wei; Nutt, Stephen L

2017-01-17

FoxP3-expressing regulatory T (Treg) cells are essential for maintaining immune homeostasis. Activated Treg cells undergo further differentiation into an effector state that highly expresses genes critical for Treg cell function, although how this process is coordinated on a transcriptional level is poorly understood. Here, we demonstrate that mice lacking the transcription factor Myb in Treg cells succumbed to a multi-organ inflammatory disease. Myb was specifically expressed in, and required for the differentiation of, thymus-derived effector Treg cells. The combination of transcriptome and genomic footprint analyses revealed that Myb directly regulated a large proportion of the gene expression specific to effector Treg cells, identifying Myb as a critical component of the gene regulatory network controlling effector Treg cell differentiation and function. Copyright © 2017 Elsevier Inc. All rights reserved.

Simple agarose micro-confinement array and machine-learning-based classification for analyzing the patterned differentiation of mesenchymal stem cells

PubMed Central

Sato, Asako; Vogel, Viola; Tanaka, Yo

2017-01-01

The geometrical confinement of small cell colonies gives differential cues to cells sitting at the periphery versus the core. To utilize this effect, for example to create spatially graded differentiation patterns of human mesenchymal stem cells (hMSCs) in vitro or to investigate underpinning mechanisms, the confinement needs to be robust for extended time periods. To create highly repeatable micro-fabricated structures for cellular patterning and high-throughput data mining, we employed here a simple casting method to fabricate more than 800 adhesive patches confined by agarose micro-walls. In addition, a machine learning based image processing software was developed (open code) to detect the differentiation patterns of the population of hMSCs automatically. Utilizing the agarose walls, the circular patterns of hMSCs were successfully maintained throughout 15 days of cell culture. After staining lipid droplets and alkaline phosphatase as the markers of adipogenic and osteogenic differentiation, respectively, the mega-pixels of RGB color images of hMSCs were processed by the software on a laptop PC within several minutes. The image analysis successfully showed that hMSCs sitting on the more central versus peripheral sections of the adhesive circles showed adipogenic versus osteogenic differentiation as reported previously, indicating the compatibility of patterned agarose walls to conventional microcontact printing. In addition, we found a considerable fraction of undifferentiated cells which are preferentially located at the peripheral part of the adhesive circles, even in differentiation-inducing culture media. In this study, we thus successfully demonstrated a simple framework for analyzing the patterned differentiation of hMSCs in confined microenvironments, which has a range of applications in biology, including stem cell biology. PMID:28380036

Liver-enriched transcription factors are critical for the expression of hepatocyte marker genes in mES-derived hepatocyte-lineage cells.

PubMed

Kheolamai, Pakpoom; Dickson, Alan J

2009-04-23

Induction of stem cell differentiation toward functional hepatocytes is hampered by lack of knowledge of the hepatocyte differentiation processes. The overall objective of this project is to characterize key stages in the hepatocyte differentiation process. We established a mouse embryonic stem (mES) cell culture system which exhibited changes in gene expression profiles similar to those observed in the development of endodermal and hepatocyte-lineage cells previously described in the normal mouse embryo. Transgenic mES cells were established that permitted isolation of enriched hepatocyte-lineage populations. This approach has isolated mES-derived hepatocyte-lineage cells that express several markers of mature hepatocytes including albumin, glucose-6-phosphatase, tyrosine aminotransferase, cytochrome P450-3a, phosphoenolpyruvate carboxykinase and tryptophan 2,3-dioxygenase. In addition, our results show that the up-regulation of the expression levels of hepatocyte nuclear factor-3alpha, -4alpha, -6, and CCAAT-enhancer binding protein-beta might be critical for passage into late-stage differentiation towards functional hepatocytes. These data present important steps for definition of regulatory phenomena that direct specific cell fate determination. The mES cell culture system generated in this study provides a model for studying transition between stages of the hepatocyte development and has significant potential value for studying the molecular basis of hepatocyte differentiation in vitro.

Differentiation of mesenchymal stem cells into neuronal cells on fetal bovine acellular dermal matrix as a tissue engineered nerve scaffold

PubMed Central

Feng, Yuping; Wang, Jiao; Ling, Shixin; Li, Zhuo; Li, Mingsheng; Li, Qiongyi; Ma, Zongren; Yu, Sijiu

2014-01-01

The purpose of this study was to assess fetal bovine acellular dermal matrix as a scaffold for supporting the differentiation of bone marrow mesenchymal stem cells into neural cells following induction with neural differentiation medium. We performed long-term, continuous observation of cell morphology, growth, differentiation, and neuronal development using several microscopy techniques in conjunction with immunohistochemistry. We examined specific neuronal proteins and Nissl bodies involved in the differentiation process in order to determine the neuronal differentiation of bone marrow mesenchymal stem cells. The results show that bone marrow mesenchymal stem cells that differentiate on fetal bovine acellular dermal matrix display neuronal morphology with unipolar and bi/multipolar neurite elongations that express neuronal-specific proteins, including βIII tubulin. The bone marrow mesenchymal stem cells grown on fetal bovine acellular dermal matrix and induced for long periods of time with neural differentiation medium differentiated into a multilayered neural network-like structure with long nerve fibers that was composed of several parallel microfibers and neuronal cells, forming a complete neural circuit with dendrite-dendrite to axon-dendrite to dendrite-axon synapses. In addition, growth cones with filopodia were observed using scanning electron microscopy. Paraffin sectioning showed differentiated bone marrow mesenchymal stem cells with the typical features of neuronal phenotype, such as a large, round nucleus and a cytoplasm full of Nissl bodies. The data suggest that the biological scaffold fetal bovine acellular dermal matrix is capable of supporting human bone marrow mesenchymal stem cell differentiation into functional neurons and the subsequent formation of tissue engineered nerve. PMID:25598779

Stem cell technology for drug discovery and development.

PubMed

Hook, Lilian A

2012-04-01

Stem cells have enormous potential to revolutionise the drug discovery process at all stages, from target identification through to toxicology studies. Their ability to generate physiologically relevant cells in limitless supply makes them an attractive alternative to currently used recombinant cell lines or primary cells. However, realisation of the full potential of stem cells is currently hampered by the difficulty in routinely directing stem cell differentiation to reproducibly and cost effectively generate pure populations of specific cell types. In this article we discuss how stem cells have already been used in the drug discovery process and how novel technologies, particularly in relation to stem cell differentiation, can be applied to attain widespread adoption of stem cell technology by the pharmaceutical industry. Copyright © 2011 Elsevier Ltd. All rights reserved.

Biomimetics in thin film design: Niche-like wrinkles designed for i-cell progenitor cell differentiation.

PubMed

Major, Roman; Lackner, Juergen M; Sanak, Marek; Major, Boguslaw

2017-11-01

The future and development of science are in interdisciplinary areas, such as biomedical engineering. Self-assembled structures, similar to stem cell niches, inhibit rapid cellular division processes and enable the capture of stem cells from blood flow. By modifying the surface topography and stiffness properties, progenitor cells were differentiated towards the formation of endothelial cell monolayers to effectively inhibit the coagulation cascade. Wrinkled material layers in the form of thin polymeric coatings were prepared. An optimized surface topography led to proper cell differentiation and influenced the appropriate formation of endothelial cell monolayers. Blood activation was decelerated by the formed endothelium. Copyright © 2017 Elsevier B.V. All rights reserved.

Cells differentiated from mouse embryonic stem cells via embryoid bodies express renal marker molecules.

PubMed

Kramer, Jan; Steinhoff, Jürgen; Klinger, Matthias; Fricke, Lutz; Rohwedel, Jürgen

2006-03-01

Differentiation of mouse embryonic stem (ES) cells via embryoid bodies (EB) is established as a suitable model to study cellular processes of development in vitro. ES cells are known to be pluripotent because of their capability to differentiate into cell types of all three germ layers including germ cells. Here, we show that ES cells differentiate into renal cell types in vitro. We found that genes were expressed during EB cultivation, which have been previously described to be involved in renal development. Marker molecules characteristic for terminally differentiated renal cell types were found to be expressed predominantly during late stages of EB cultivation, while marker molecules involved in the initiation of nephrogenesis were already expressed during early steps of EB development. On the cellular level--using immunostaining--we detected cells expressing podocin, nephrin and wt-1, characteristic for differentiated podocytes and other cells, which expressed Tamm-Horsfall protein, a marker for distal tubule epithelial cells of kidney tissue. Furthermore, the proximal tubule marker molecules renal-specific oxido reductase, kidney androgen-related protein and 25-hydroxyvitamin D3alpha-hydroxylase were found to be expressed in EBs. In particular, we could demonstrate that cells expressing podocyte marker molecules assemble to distinct ring-like structures within the EBs. Because the differentiation efficiency into these cell types is still relatively low, application of fibroblast growth factor (FGF)-2 in combination with leukaemia inhibitory factor was tested for induction, but did not enhance ES cell-derived renal differentiation in vitro.

A model of the regulatory network involved in the control of the cell cycle and cell differentiation in the Caenorhabditis elegans vulva.

PubMed

Weinstein, Nathan; Ortiz-Gutiérrez, Elizabeth; Muñoz, Stalin; Rosenblueth, David A; Álvarez-Buylla, Elena R; Mendoza, Luis

2015-03-13

There are recent experimental reports on the cross-regulation between molecules involved in the control of the cell cycle and the differentiation of the vulval precursor cells (VPCs) of Caenorhabditis elegans. Such discoveries provide novel clues on how the molecular mechanisms involved in the cell cycle and cell differentiation processes are coordinated during vulval development. Dynamic computational models are helpful to understand the integrated regulatory mechanisms affecting these cellular processes. Here we propose a simplified model of the regulatory network that includes sufficient molecules involved in the control of both the cell cycle and cell differentiation in the C. elegans vulva to recover their dynamic behavior. We first infer both the topology and the update rules of the cell cycle module from an expected time series. Next, we use a symbolic algorithmic approach to find which interactions must be included in the regulatory network. Finally, we use a continuous-time version of the update rules for the cell cycle module to validate the cyclic behavior of the network, as well as to rule out the presence of potential artifacts due to the synchronous updating of the discrete model. We analyze the dynamical behavior of the model for the wild type and several mutants, finding that most of the results are consistent with published experimental results. Our model shows that the regulation of Notch signaling by the cell cycle preserves the potential of the VPCs and the three vulval fates to differentiate and de-differentiate, allowing them to remain completely responsive to the concentration of LIN-3 and lateral signal in the extracellular microenvironment.

Expression and regulation of long noncoding RNAs during the osteogenic differentiation of periodontal ligament stem cells in the inflammatory microenvironment.

PubMed

Zhang, Qingbin; Chen, Li; Cui, Shiman; Li, Yan; Zhao, Qi; Cao, Wei; Lai, Shixiang; Yin, Sanjun; Zuo, Zhixiang; Ren, Jian

2017-10-25

Although long noncoding RNAs (lncRNAs) have been emerging as critical regulators in various tissues and biological processes, little is known about their expression and regulation during the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) in inflammatory microenvironment. In this study, we have identified 63 lncRNAs that are not annotated in previous database. These novel lncRNAs were not randomly located in the genome but preferentially located near protein-coding genes related to particular functions and diseases, such as stem cell maintenance and differentiation, development disorders and inflammatory diseases. Moreover, we have identified 650 differentially expressed lncRNAs among different subsets of PDLSCs. Pathway enrichment analysis for neighboring protein-coding genes of these differentially expressed lncRNAs revealed stem cell differentiation related functions. Many of these differentially expressed lncRNAs function as competing endogenous RNAs that regulate protein-coding transcripts through competing shared miRNAs.

Temporal Profiling and Pulsed SILAC Labeling Identify Novel Secreted Proteins During Ex Vivo Osteoblast Differentiation of Human Stromal Stem Cells*

PubMed Central

Kristensen, Lars P.; Chen, Li; Nielsen, Maria Overbeck; Qanie, Diyako W.; Kratchmarova, Irina; Kassem, Moustapha; Andersen, Jens S.

2012-01-01

It is well established that bone forming cells (osteoblasts) secrete proteins with autocrine, paracrine, and endocrine function. However, the identity and functional role for the majority of these secreted and differentially expressed proteins during the osteoblast (OB) differentiation process, is not fully established. To address these questions, we quantified the temporal dynamics of the human stromal (mesenchymal, skeletal) stem cell (hMSC) secretome during ex vivo OB differentiation using stable isotope labeling by amino acids in cell culture (SILAC). In addition, we employed pulsed SILAC labeling to distinguish genuine secreted proteins from intracellular contaminants. We identified 466 potentially secreted proteins that were quantified at 5 time-points during 14-days ex vivo OB differentiation including 41 proteins known to be involved in OB functions. Among these, 315 proteins exhibited more than 2-fold up or down-regulation. The pulsed SILAC method revealed a strong correlation between the fraction of isotope labeling and the subset of proteins known to be secreted and involved in OB differentiation. We verified SILAC data using qRT-PCR analysis of 9 identified potential novel regulators of OB differentiation. Furthermore, we studied the biological effects of one of these proteins, the hormone stanniocalcin 2 (STC2) and demonstrated its autocrine effects in enhancing osteoblastic differentiation of hMSC. In conclusion, combining complete and pulsed SILAC labeling facilitated the identification of novel factors produced by hMSC with potential role in OB differentiation. Our study demonstrates that the secretome of osteoblastic cells is more complex than previously reported and supports the emerging evidence that osteoblastic cells secrete proteins with endocrine functions and regulate cellular processes beyond bone formation. PMID:22801418

Quantitative evaluation method for differentiation of C2C12 myoblasts by ultrasonic microscopy

NASA Astrophysics Data System (ADS)

Takanashi, Kyoichi; Washiya, Mamoru; Ota, Kazuki; Yoshida, Sachiko; Hozumi, Naohiro; Kobayashi, Kazuto

2017-07-01

Cell differentiation was evaluated by ultrasonic microscopy. However, there were some regions that showed a lower acoustic impedance than the culture liquid. It was considered that, in such regions, the cells were not perfectly in contact with the film substrate. Hence, a waveform analysis was performed, and compensated acoustic impedances in such regions were in a reasonable range of values. By the same analysis, the displacements of partially floated cells were also successfully calculated. The elapsed day transitions of the compensated acoustic impedances and displacements were successfully evaluated. In the process of differentiation, actin fibers comprising the cytoskeleton are supposed to loosen in order to induce cellular fusion. In addition, the progress in cell differentiation accompanied by a change into a three-dimensional structure can partially be assessed by the displacement between a cell and a cultured film. Hence, we believe that cell differentiation can be evaluated using an ultrasonic microscope.

Self-organization of neural patterns and structures in 3D culture of stem cells

NASA Astrophysics Data System (ADS)

Sasai, Yoshiki

2013-05-01

Over the last several years, much progress has been made for in vitro culture of mouse and human ES cells. Our laboratory focuses on the molecular and cellular mechanisms of neural differentiation from pluripotent cells. Pluripotent cells first become committed to the ectodermal fate and subsequently differentiate into uncommitted neuroectodermal cells. Both previous mammalian and amphibian studies on pluripotent cells have indicated that the neural fate is a sort of the basal direction of the differentiation of these cells while mesoendodermal differentiation requires extrinsic inductive signals. ES cells differentiate into neuroectodermal cells with a rostral-most character (telencephalon and hypothalamus) when they are cultured in the absence of strong patterning signals. In this talk, I first discuss this issue by referring to our recent data on the mechanism of spontaneous neural differentiation in serum-free culture of mouse ES cells. Then, I will talk about self-organization phenomena observed in 3D culture of ES cells, which lead to tissue-autonomous formation of regional structures such as layered cortical tissues. I also discuss our new attempt to monitor these in vitro morphogenetic processes by live imaging, in particular, self-organizing morphogenesis of the optic cup in three-dimensional cultures.

Carbon Monoxide Releasing Molecule-A1 (CORM-A1) Improves Neurogenesis: Increase of Neuronal Differentiation Yield by Preventing Cell Death.

PubMed

Almeida, Ana S; Soares, Nuno L; Vieira, Melissa; Gramsbergen, Jan Bert; Vieira, Helena L A

2016-01-01

Cerebral ischemia and neurodegenerative diseases lead to impairment or death of neurons in the central nervous system. Stem cell based therapies are promising strategies currently under investigation. Carbon monoxide (CO) is an endogenous product of heme degradation by heme oxygenase (HO) activity. Administration of CO at low concentrations produces several beneficial effects in distinct tissues, namely anti-apoptotic and anti-inflammatory. Herein the CO role on modulation of neuronal differentiation was assessed. Three different models with increasing complexity were used: human neuroblastoma SH-S5Y5 cell line, human teratocarcinoma NT2 cell line and organotypic hippocampal slice cultures (OHSC). Cell lines were differentiated into post-mitotic neurons by treatment with retinoic acid (RA) supplemented with CO-releasing molecule A1 (CORM-A1). CORM-A1 positively modulated neuronal differentiation, since it increased final neuronal production and enhanced the expression of specific neuronal genes: Nestin, Tuj1 and MAP2. Furthermore, during neuronal differentiation process, there was an increase in proliferative cell number (ki67 mRNA expressing cells) and a decrease in cell death (lower propidium iodide (PI) uptake, limitation of caspase-3 activation and higher Bcl-2 expressing cells). CO supplementation did not increase the expression of RA receptors. In the case of SH-S5Y5 model, small amounts of reactive oxygen species (ROS) generation emerges as important signaling molecules during CO-promoted neuronal differentiation. CO's improvement of neuronal differentiation yield was validated using OHSC as ex vivo model. CORM-A1 treatment of OHSC promoted higher levels of cells expressing the neuronal marker Tuj1. Still, CORM-A1 increased cell proliferation assessed by ki67 expression and also prevented cell death, which was followed by increased Bcl-2 expression, decreased levels of active caspase-3 and PI uptake. Likewise, ROS signaling emerged as key factors in CO's increasing number of differentiated neurons in OHSC. In conclusion, CO's increasing number of differentiated neurons is a novel biological role disclosed herein. CO improves neuronal yield due to its capacity to reduce cell death, promoting an increase in proliferative population. However, one cannot disregard a direct CO's effect on specific cellular processes of neuronal differentiation. Further studies are needed to evaluate how CO can potentially modulate cell mechanisms involved in neuronal differentiation. In summary, CO appears as a promising therapeutic molecule to stimulate endogenous neurogenesis or to improve in vitro neuronal production for cell therapy strategies.

Carbon Monoxide Releasing Molecule-A1 (CORM-A1) Improves Neurogenesis: Increase of Neuronal Differentiation Yield by Preventing Cell Death

PubMed Central

Almeida, Ana S.; Soares, Nuno L.; Vieira, Melissa; Gramsbergen, Jan Bert

2016-01-01

Cerebral ischemia and neurodegenerative diseases lead to impairment or death of neurons in the central nervous system. Stem cell based therapies are promising strategies currently under investigation. Carbon monoxide (CO) is an endogenous product of heme degradation by heme oxygenase (HO) activity. Administration of CO at low concentrations produces several beneficial effects in distinct tissues, namely anti-apoptotic and anti-inflammatory. Herein the CO role on modulation of neuronal differentiation was assessed. Three different models with increasing complexity were used: human neuroblastoma SH-S5Y5 cell line, human teratocarcinoma NT2 cell line and organotypic hippocampal slice cultures (OHSC). Cell lines were differentiated into post-mitotic neurons by treatment with retinoic acid (RA) supplemented with CO-releasing molecule A1 (CORM-A1). CORM-A1 positively modulated neuronal differentiation, since it increased final neuronal production and enhanced the expression of specific neuronal genes: Nestin, Tuj1 and MAP2. Furthermore, during neuronal differentiation process, there was an increase in proliferative cell number (ki67 mRNA expressing cells) and a decrease in cell death (lower propidium iodide (PI) uptake, limitation of caspase-3 activation and higher Bcl-2 expressing cells). CO supplementation did not increase the expression of RA receptors. In the case of SH-S5Y5 model, small amounts of reactive oxygen species (ROS) generation emerges as important signaling molecules during CO-promoted neuronal differentiation. CO’s improvement of neuronal differentiation yield was validated using OHSC as ex vivo model. CORM-A1 treatment of OHSC promoted higher levels of cells expressing the neuronal marker Tuj1. Still, CORM-A1 increased cell proliferation assessed by ki67 expression and also prevented cell death, which was followed by increased Bcl-2 expression, decreased levels of active caspase-3 and PI uptake. Likewise, ROS signaling emerged as key factors in CO’s increasing number of differentiated neurons in OHSC. In conclusion, CO’s increasing number of differentiated neurons is a novel biological role disclosed herein. CO improves neuronal yield due to its capacity to reduce cell death, promoting an increase in proliferative population. However, one cannot disregard a direct CO’s effect on specific cellular processes of neuronal differentiation. Further studies are needed to evaluate how CO can potentially modulate cell mechanisms involved in neuronal differentiation. In summary, CO appears as a promising therapeutic molecule to stimulate endogenous neurogenesis or to improve in vitro neuronal production for cell therapy strategies. PMID:27144388

Fixation times in differentiation and evolution in the presence of bottlenecks, deserts, and oases.

PubMed

Chou, Tom; Wang, Yu

2015-05-07

Cellular differentiation and evolution are stochastic processes that can involve multiple types (or states) of particles moving on a complex, high-dimensional state-space or "fitness" landscape. Cells of each specific type can thus be quantified by their population at a corresponding node within a network of states. Their dynamics across the state-space network involve genotypic or phenotypic transitions that can occur upon cell division, such as during symmetric or asymmetric cell differentiation, or upon spontaneous mutation. Here, we use a general multi-type branching processes to study first passage time statistics for a single cell to appear in a specific state. Our approach readily allows for nonexponentially distributed waiting times between transitions, reflecting, e.g., the cell cycle. For simplicity, we restrict most of our detailed analysis to exponentially distributed waiting times (Poisson processes). We present results for a sequential evolutionary process in which L successive transitions propel a population from a "wild-type" state to a given "terminally differentiated," "resistant," or "cancerous" state. Analytic and numeric results are also found for first passage times across an evolutionary chain containing a node with increased death or proliferation rate, representing a desert/bottleneck or an oasis. Processes involving cell proliferation are shown to be "nonlinear" (even though mean-field equations for the expected particle numbers are linear) resulting in first passage time statistics that depend on the position of the bottleneck or oasis. Our results highlight the sensitivity of stochastic measures to cell division fate and quantify the limitations of using certain approximations (such as the fixed-population and mean-field assumptions) in evaluating fixation times. Published by Elsevier Ltd.

Cellular processes involved in human epidermal cells exposed to extremely low frequency electric fields.

PubMed

Collard, J-F; Hinsenkamp, M

2015-05-01

We observed on different tissues and organisms a biological response after exposure to pulsed low frequency and low amplitude electric or electromagnetic fields but the precise mechanism of cell response remains unknown. The aim of this publication is to understand, using bioinformatics, the biological relevance of processes involved in the modification of gene expression. The list of genes analyzed was obtained after microarray protocol realized on cultures of human epidermal explants growing on deepidermized human skin exposed to a pulsed low frequency electric field. The directed acyclic graph on a WebGestalt Gene Ontology module shows six categories under the biological process root: "biological regulation", "cellular process", "cell proliferation", "death", "metabolic process" and "response to stimulus". Enriched derived categories are coherent with the type of in vitro culture, the stimulation protocol or with the previous results showing a decrease of cell proliferation and an increase of differentiation. The Kegg module on WebGestalt has highlighted "cell cycle" and "p53 signaling pathway" as significantly involved. The Kegg website brings out interactions between FoxO, MAPK, JNK, p53, p38, PI3K/Akt, Wnt, mTor or NF-KappaB. Some genes expressed by the stimulation are known to have an exclusive function on these pathways. Analyses performed with Pathway Studio linked cell proliferation, cell differentiation, apoptosis, cell cycle, mitosis, cell death etc. with our microarrays results. Medline citation generated by the software and the fold change variation confirms a diminution of the proliferation, activation of the differentiation and a less well-defined role of apoptosis or wound healing. Wnt and DKK functional classes, DKK1, MACF1, ATF3, MME, TXNRD1, and BMP-2 genes proposed in previous publications after a manual analysis are also highlighted with other genes after Pathway Studio automatic procedure. Finally, an analysis conducted on a list of genes characterized by an accelerated regulation after extremely low frequency pulsed stimulation also confirms their role in the processes of cell proliferation and differentiation. Bioinformatics approach allows in-depth research, without the bias of pre-selection, on cellular processes involved in a huge gene list. Copyright © 2015 Elsevier Inc. All rights reserved.

The APC/C Coordinates Retinal Differentiation with G1 Arrest through the Nek2-Dependent Modulation of Wingless Signaling.

PubMed

Martins, Torcato; Meghini, Francesco; Florio, Francesca; Kimata, Yuu

2017-01-09

The cell cycle is coordinated with differentiation during animal development. Here we report a cell-cycle-independent developmental role for a master cell-cycle regulator, the anaphase-promoting complex or cyclosome (APC/C), in the regulation of cell fate through modulation of Wingless (Wg) signaling. The APC/C controls both cell-cycle progression and postmitotic processes through ubiquitin-dependent proteolysis. Through an RNAi screen in the developing Drosophila eye, we found that partial APC/C inactivation severely inhibits retinal differentiation independently of cell-cycle defects. The differentiation inhibition coincides with hyperactivation of Wg signaling caused by the accumulation of a Wg modulator, Drosophila Nek2 (dNek2). The APC/C degrades dNek2 upon synchronous G1 arrest prior to differentiation, which allows retinal differentiation through local suppression of Wg signaling. We also provide evidence that decapentaplegic signaling may posttranslationally regulate this APC/C function. Thus, the APC/C coordinates cell-fate determination with the cell cycle through the modulation of developmental signaling pathways. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Different roles of TGF-β in the multi-lineage differentiation of stem cells

PubMed Central

Wang, Ming-Ke; Sun, Hui-Qin; Xiang, Ying-Chun; Jiang, Fan; Su, Yong-Ping; Zou, Zhong-Min

2012-01-01

Stem cells are a population of cells that has infinite or long-term self-renewal ability and can produce various kinds of descendent cells. Transforming growth factor β (TGF-β) family is a superfamily of growth factors, including TGF-β1, TGF-β2 and TGF-β3, bone morphogenetic proteins, activin/inhibin, and some other cytokines such as nodal, which plays very important roles in regulating a wide variety of biological processes, such as cell growth, differentiation, cell death. TGF-β, a pleiotropic cytokine, has been proved to be differentially involved in the regulation of multi-lineage differentiation of stem cells, through the Smad pathway, non-Smad pathways including mitogen-activated protein kinase pathways, phosphatidylinositol-3-kinase/AKT pathways and Rho-like GTPase signaling pathways, and their cross-talks. For instance, it is generally known that TGF-β promotes the differentiation of stem cells into smooth muscle cells, immature cardiomyocytes, chondrocytes, neurocytes, hepatic stellate cells, Th17 cells, and dendritic cells. However, TGF-β inhibits the differentiation of stem cells into myotubes, adipocytes, endothelial cells, and natural killer cells. Additionally, TGF-β can provide competence for early stages of osteoblastic differentiation, but at late stages TGF-β acts as an inhibitor. The three mammalian isoforms (TGF-β1, 2 and 3) have distinct but overlapping effects on hematopoiesis. Understanding the mechanisms underlying the regulatory effect of TGF-β in the stem cell multi-lineage differentiation is of importance in stem cell biology, and will facilitate both basic research and clinical applications of stem cells. In this article, we discuss the current status and progress in our understanding of different mechanisms by which TGF-β controls multi-lineage differentiation of stem cells. PMID:22993659

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.

Assessment of myoblast circular RNA dynamics and its correlation with miRNA during myogenic differentiation.

PubMed

Zhang, Pengpeng; Xu, Haixia; Li, Rui; Wu, Wei; Chao, Zhe; Li, Cencen; Xia, Wei; Wang, Lei; Yang, Jinzeng; Xu, Yongjie

2018-06-01

Myoblast differentiation is a highly complex process that is regulated by proteins as well as by non-coding RNAs. Circular RNAs have been identified as an emerging new class of non-coding RNA in the modulation of skeletal muscle development, whereas their expression profiles and functional regulation in myoblast differentiation remain unknown. In the present study, we performed deep RNA-sequencing of C2C12 myoblasts during cell differentiation and uncovered 37,751 unique circular RNAs derived from 6943 hosting genes. The ensuing qRT-PCR and RNA fluorescence in situ hybridization verification were carried out to confirm the RNA-sequencing results. An unbiased analysis demonstrated dynamic circular RNA expression changes in the process of myoblast differentiation, and the circular RNA abundances were independent from their cognate linear RNAs. Gene ontology analysis showed that many down-regulated circular RNAs were exclusive to cell division and the cell cycle, whereas up-regulated circular RNAs were related to the cell development process. Furthermore, interaction networks of circular RNA-microRNA were constructed. Several microRNAs well-known for myoblast regulation, such as miR-133, miR-24 and miR-23a, were in this network. In summary, this study showed that circular RNA expression dynamics changed during myoblast differentiation. Circular RNAs play a role in regulating the myoblast cell cycle and development by acting as microRNA binding sites to facilitate their regulation of gene expression during myoblast differentiation. These findings open a new avenue for future investigation of this emerging RNA class in skeletal muscle growth and development. Copyright © 2018 Elsevier Ltd. All rights reserved.

Reactive oxygen species activate differentiation gene transcription of acute myeloid leukemia cells via the JNK/c-JUN signaling pathway.

PubMed

Lam, Chung Fan; Yeung, Hoi Ting; Lam, Yuk Man; Ng, Ray Kit

2018-05-01

Reactive oxygen species (ROS) and altered cellular redox status are associated with many malignancies. Acute myeloid leukemia (AML) cells are maintained at immature state by differentiation blockade, which involves deregulation of transcription factors in myeloid differentiation. AML cells can be induced to differentiate by phorbol-12-myristate-13-acetate (PMA), which possesses pro-oxidative activity. However, the signaling events mediated by ROS in the activation of transcriptional program during AML differentiation has not been fully elucidated. Here, we investigated AML cell differentiation by treatment with PMA and ROS scavenger N-acetyl-l-cysteine (NAC). We observed elevation of intracellular ROS level in the PMA-treated AML cells, which correlated with differentiated cell morphology and increased CD11b + mature cell population. The effect of PMA can be abolished by NAC co-treatment, supporting the involvement of ROS in the process. Moreover, we demonstrated that short ROS elevation mediated cell cycle arrest, but failed to activate myeloid gene transcription; whereas prolonged ROS elevation activated JNK/c-JUN signaling pathway. Inhibition of JNK suppressed the expression of key myeloid transcriptional regulators c-JUN, SPI-1 and MAFB, and prevented AML cells from undergoing terminal differentiation. These findings provide new insights into the crucial role of JNK/c-Jun signaling pathway in the activation of transcriptional program during ROS-mediated AML differentiation. Copyright © 2018 Elsevier Ltd. All rights reserved.

Increased symplasmic permeability in barley root epidermal cells correlates with defects in root hair development

PubMed Central

Marzec, M; Muszynska, A; Melzer, M; Sas-Nowosielska, H; Kurczynska, E U; Wick, S

2014-01-01

It is well known that the process of plant cell differentiation depends on the symplasmic isolation of cells. Before starting the differentiation programme, the individual cell or group of cells should restrict symplasmic communication with neighbouring cells. We tested the symplasmic communication between epidermal cells in the different root zones of parental barley plants Hordeum vulgare L., cv. ‘Karat’ with normal root hair development, and two root hairless mutants (rhl1.a and rhl1.b). The results clearly show that symplasmic communication was limited during root hair differentiation in the parental variety, whereas in both root hairless mutants epidermal cells were still symplasmically connected in the corresponding root zone. This paper is the first report on the role of symplasmic isolation in barley root cell differentiation, and additionally shows that a disturbance in the restriction of symplasmic communication is present in root hairless mutants. PMID:23927737

Integument cell differentiation in dandelions (Taraxacum, Asteraceae, Lactuceae) with special attention paid to plasmodesmata.

PubMed

Płachno, Bartosz J; Kurczyńska, Ewa; Świątek, Piotr

2016-09-01

The aim of the paper is to determine what happens with plasmodesmata when mucilage is secreted into the periplasmic space in plant cells. Ultrastructural analysis of the periendothelial zone mucilage cells was performed on examples of the ovule tissues of several sexual and apomictic Taraxacum species. The cytoplasm of the periendothelial zone cells was dense, filled by numerous organelles and profiles of rough endoplasmic reticulum and active Golgi dictyosomes with vesicles that contained fibrillar material. At the beginning of the differentiation process of the periendothelial zone, the cells were connected by primary plasmodesmata. However, during the differentiation and the thickening of the cell walls (mucilage deposition), the plasmodesmata become elongated and associated with cytoplasmic bridges. The cytoplasmic bridges may connect the protoplast to the plasmodesmata through the mucilage layers in order to maintain cell-to-cell communication during the differentiation of the periendothelial zone cells.

Differential expression of neuroleukin in osseous tissues and its involvement in mineralization during osteoblast differentiation

NASA Technical Reports Server (NTRS)

Zhi, J.; Sommerfeldt, D. W.; Rubin, C. T.; Hadjiargyrou, M.

2001-01-01

Osteoblast differentiation is a multistep process that involves critical spatial and temporal regulation of cellular processes marked by the presence of a large number of differentially expressed molecules. To identify key functional molecules, we used differential messenger RNA (mRNA) display and compared RNA populations isolated from the defined transition phases (proliferation, matrix formation, and mineralization) of the MC3T3-E1 osteoblast-like cell line. Using this approach, a complementary DNA (cDNA) fragment was isolated and identified as neuroleukin (NLK), a multifunctional cytokine also known as autocrine motility factor (AMF), phosphoglucose isomerase (PGI; phosphohexose isomerase [PHI]), and maturation factor (MF). Northern analysis showed NLK temporal expression during MC3T3-E1 cell differentiation with a 3.5-fold increase during matrix formation and mineralization. Immunocytochemical studies revealed the presence of NLK in MC3T3-E1 cells as well as in the surrounding matrix, consistent with a secreted molecule. In contrast, the NLK receptor protein was detected primarily on the cell membrane. In subsequent studies, a high level of NLK expression was identified in osteoblasts and superficial articular chondrocytes in bone of 1-, 4-, and 8-month-old normal mice, as well as in fibroblasts, proliferating chondrocytes, and osteoblasts within a fracture callus. However, NLK was not evident in hypertrophic chondrocytes or osteocytes. In addition, treatment of MC3T3 cells with 6-phosphogluconic acid (6PGA; a NLK inhibitor) resulted in diminishing alkaline phosphatase (ALP) activity and mineralization in MC3T3-E1 cells, especially during the matrix formation stage of differentiating cells. Taken together, these data show specific expression of NLK in discrete populations of bone and cartilage cells and suggest a possible role for this secreted protein in bone development and regeneration.

Major transcriptome re-organisation and abrupt changes in signalling, cell cycle and chromatin regulation at neural differentiation in vivo.

PubMed

Olivera-Martinez, Isabel; Schurch, Nick; Li, Roman A; Song, Junfang; Halley, Pamela A; Das, Raman M; Burt, Dave W; Barton, Geoffrey J; Storey, Kate G

2014-08-01

Here, we exploit the spatial separation of temporal events of neural differentiation in the elongating chick body axis to provide the first analysis of transcriptome change in progressively more differentiated neural cell populations in vivo. Microarray data, validated against direct RNA sequencing, identified: (1) a gene cohort characteristic of the multi-potent stem zone epiblast, which contains neuro-mesodermal progenitors that progressively generate the spinal cord; (2) a major transcriptome re-organisation as cells then adopt a neural fate; and (3) increasing diversity as neural patterning and neuron production begin. Focussing on the transition from multi-potent to neural state cells, we capture changes in major signalling pathways, uncover novel Wnt and Notch signalling dynamics, and implicate new pathways (mevalonate pathway/steroid biogenesis and TGFβ). This analysis further predicts changes in cellular processes, cell cycle, RNA-processing and protein turnover as cells acquire neural fate. We show that these changes are conserved across species and provide biological evidence for reduced proteasome efficiency and a novel lengthening of S phase. This latter step may provide time for epigenetic events to mediate large-scale transcriptome re-organisation; consistent with this, we uncover simultaneous downregulation of major chromatin modifiers as the neural programme is established. We further demonstrate that transcription of one such gene, HDAC1, is dependent on FGF signalling, making a novel link between signals that control neural differentiation and transcription of a core regulator of chromatin organisation. Our work implicates new signalling pathways and dynamics, cellular processes and epigenetic modifiers in neural differentiation in vivo, identifying multiple new potential cellular and molecular mechanisms that direct differentiation. © 2014. Published by The Company of Biologists Ltd.

Acceleration of astrocytic differentiation in neural stem cells surviving X-irradiation.

PubMed

Ozeki, Ayumi; Suzuki, Keiji; Suzuki, Masatoshi; Ozawa, Hiroki; Yamashita, Shunichi

2012-03-28

Neural stem cells (NSCs) are highly susceptible to DNA double-strand breaks; however, little is known about the effects of radiation in cells surviving radiation. Although the nestin-positive NSCs predominantly became glial fibrillary acidic protein (GFAP)-positive in differentiation-permissive medium, little or no cells were GFAP positive in proliferation-permissive medium. We found that more than half of the cells surviving X-rays became GFAP positive in proliferation-permissive medium. Moreover, localized irradiation stimulated differentiation of cells outside the irradiated area. These results indicate for the first time that ionizing radiation is able to stimulate astrocyte-specific differentiation of surviving NSCs, whose process is mediated both by the direct activation of nuclear factor-κB and by the indirect bystander effect induced by X-irradiation.

Differential 3’ processing of specific transcripts expands regulatory and protein diversity across neuronal cell types

PubMed Central

Jereb, Saša; Hwang, Hun-Way; Van Otterloo, Eric; Govek, Eve-Ellen; Fak, John J; Yuan, Yuan; Hatten, Mary E

2018-01-01

Alternative polyadenylation (APA) regulates mRNA translation, stability, and protein localization. However, it is unclear to what extent APA regulates these processes uniquely in specific cell types. Using a new technique, cTag-PAPERCLIP, we discovered significant differences in APA between the principal types of mouse cerebellar neurons, the Purkinje and granule cells, as well as between proliferating and differentiated granule cells. Transcripts that differed in APA in these comparisons were enriched in key neuronal functions and many differed in coding sequence in addition to 3’UTR length. We characterize Memo1, a transcript that shifted from expressing a short 3’UTR isoform to a longer one during granule cell differentiation. We show that Memo1 regulates granule cell precursor proliferation and that its long 3’UTR isoform is targeted by miR-124, contributing to its downregulation during development. Our findings provide insight into roles for APA in specific cell types and establish a platform for further functional studies. PMID:29578408

Targeting the (Un)differentiated State of Cancer.

PubMed

Kemeny, Lajos V; Fisher, David E

2018-05-14

Dedifferentation in cancer is associated with intrinsic and acquired resistance to therapies. In this issue of Cancer Cell, Tsoi et al. identify four differentiation states in melanoma and provide evidence that melanoma cells develop drug resistance through a stepwise dedifferentiation process, making them vulnerable to ferroptotic cell death-inducing compounds. Copyright © 2018 Elsevier Inc. All rights reserved.

In vitro spatially organizing the differentiation in individual multicellular stem cell aggregates.

PubMed

Qi, Hao; Huang, Guoyou; Han, Yu Long; Lin, Wang; Li, Xiujun; Wang, Shuqi; Lu, Tian Jian; Xu, Feng

2016-01-01

With significant potential as a robust source to produce specific somatic cells for regenerative medicine, stem cells have attracted increasing attention from both academia and government. In vivo, stem cell differentiation is a process under complicated regulations to precisely build tissue with unique spatial structures. Since multicellular spheroidal aggregates of stem cells, commonly called as embryoid bodies (EBs), are considered to be capable of recapitulating the events in early stage of embryonic development, a variety of methods have been developed to form EBs in vitro for studying differentiation of embryonic stem cells. The regulation of stem cell differentiation is crucial in directing stem cells to build tissue with the correct spatial architecture for specific functions. However, stem cells within the three-dimensional multicellular aggregates undergo differentiation in a less unpredictable and spatially controlled manner in vitro than in vivo. Recently, various microengineering technologies have been developed to manipulate stem cells in vitro in a spatially controlled manner. Herein, we take the spotlight on these technologies and researches that bring us the new potential for manipulation of stem cells for specific purposes.

Evaluation of Bone Marrow Processing Protocol for Therapeutic Applications via Culture and Characterization of Mesenchymal Stem Cells.

PubMed

Verma, Poonam; Bansal, Himanshu; Agrawal, Anupama; Leon, Jerry; Sundell, I Birgitta; Koka, Prasad S

Human mesenchymal stem cells from bone marrow (hMSCs) have broad therapeutic potential. These cells can be are readily isolated from bone marrow by their property to adhere to tissue culture treated culture wares. However, the proliferation rates and other properties of the cells gradually change during expansion. This study aims to validate the protocol of isolation and differentiation of hMSCs from bone marrow for therapeutic applications. Sixty ml of bone marrow was extracted from 5 patients and MSCs were isolated. These were characterized by Flow Cytometry, CFU assay and were differentiated into bone, fat cells and neurocytes. The cells were having healthy morphology. These were positive for the markers CD105, CD90 and CD73 and negative for CD45, CD34 and HLA-DR. The cells could differentiate into fat, bone and neural cells. MSCs from the bone marrow were isolated and differentiated. These cells were morphologically healthy and passed CFU assay. The cells exhibited differentiation potential into bone, fat and neural tissue. These cells can be used in therapeutic applications.

Extended Self-Renewal and Accelerated Reprogramming in the Absence of Kdm5b

PubMed Central

Hu, Gangqing; Yu, Zu-Xi; Liu, Chengyu

2013-01-01

Embryonic stem (ES) cell pluripotency is thought to be regulated in part by H3K4 methylation. However, it is unclear how H3K4 demethylation contributes to ES cell function and participates in induced pluripotent stem (iPS) cell reprogramming. Here, we show that KDM5B, which demethylates H3K4, is important for ES cell differentiation and presents a barrier to the reprogramming process. Depletion of Kdm5b leads to an extension in the self-renewal of ES cells in the absence of LIF. Transcriptome analysis revealed the persistent expression of pluripotency genes and underexpression of developmental genes during differentiation in the absence of Kdm5b, suggesting that KDM5B plays a key role in cellular fate changes. We also observed accelerated reprogramming of differentiated cells in the absence of Kdm5b, demonstrating that KDM5B is a barrier to the reprogramming process. Expression analysis revealed that mesenchymal master regulators associated with the epithelial-to-mesenchymal transition (EMT) are downregulated during reprogramming in the absence of Kdm5b. Moreover, global analysis of H3K4me3/2 revealed that enhancers of fibroblast genes are rapidly deactivated in the absence of Kdm5b, and genes associated with EMT lose H3K4me3/2 during the early reprogramming process. These findings provide functional insight into the role for KDM5B in regulating ES cell differentiation and as a barrier to the reprogramming process. PMID:24100015

Intraportal injection of insulin-producing cells generated from human bone marrow mesenchymal stem cells decreases blood glucose level in diabetic rats.

PubMed

Tsai, Pei-Jiun; Wang, Hwai-Shi; Lin, Chi-Hung; Weng, Zen-Chung; Chen, Tien-Hua; Shyu, Jia-Fwu

2014-01-01

We studied the process of trans-differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) into insulin-producing cells. Streptozotocin (STZ)-induced diabetic rat model was used to study the effect of portal vein transplantation of these insulin-producing cells on blood sugar levels. The BM-MSCs were differentiated into insulin-producing cells under defined conditions. Real-time PCR, immunocytochemistry and glucose challenge were used to evaluate in vitro differentiation. Flow cytometry showed that hBM-MSCs were strongly positive for CD44, CD105 and CD73 and negative for hematopoietic markers CD34, CD38 and CD45. Differentiated cells expressed C-peptide as well as β-cells specific genes and hormones. Glucose stimulation increased C-peptide secretion in these cells. The insulin-producing, differentiated cells were transplanted into the portal vein of STZ-induced diabetic rats using a Port-A catheter. The insulin-producing cells were localized in the liver of the recipient rat and expressed human C-peptide. Blood glucose levels were reduced in diabetic rats transplanted with insulin-producing cells. We concluded that hBM-MSCs could be trans-differentiated into insulin-producing cells in vitro. Portal vein transplantation of insulin-producing cells alleviated hyperglycemia in diabetic rats.

Synergistic effects of TGFβ2, WNT9a, and FGFR4 signals attenuate satellite cell differentiation during skeletal muscle development.

PubMed

Zhang, Weiya; Xu, Yueyuan; Zhang, Lu; Wang, Sheng; Yin, Binxu; Zhao, Shuhong; Li, Xinyun

2018-06-04

Satellite cells play a key role in the aging, generation, and damage repair of skeletal muscle. The molecular mechanism of satellite cells in these processes remains largely unknown. This study systematically investigated for the first time the characteristics of mouse satellite cells at ten different ages. Results indicated that the number and differentiation capacity of satellite cells decreased with age during skeletal muscle development. Transcriptome analysis revealed that 2,907 genes were differentially expressed at six time points at postnatal stage. WGCNA and GO analysis indicated that 1,739 of the 2,907 DEGs were mainly involved in skeletal muscle development processes. Moreover, the results of WGCNA and protein interaction analysis demonstrated that Tgfβ2, Wnt9a, and Fgfr4 were the key genes responsible for the differentiation of satellite cells. Functional analysis showed that TGFβ2 and WNT9a inhibited, whereas FGFR4 promoted the differentiation of satellite cells. Furthermore, each two of them had a regulatory relationship at the protein level. In vivo study also confirmed that TGFβ2 could regulate the regeneration of skeletal muscle, as well as the expression of WNT9a and FGFR4. Therefore, we concluded that the synergistic effects of TGFβ2, WNT9a, and FGFR4 were responsible for attenuating of the differentiation of aging satellite cells during skeletal muscle development. This study provided new insights into the molecular mechanism of satellite cell development. The target genes and signaling pathways investigated in this study would be useful for improving the muscle growth of livestock or treating muscle diseases in clinical settings. © 2018 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

A High Proliferation Rate is Critical for Reproducible and Standardized Embryoid Body Formation from Laminin-521-Based Human Pluripotent Stem Cell Cultures.

PubMed

Dziedzicka, Dominika; Markouli, Christina; Barbé, Lise; Spits, Claudia; Sermon, Karen; Geens, Mieke

2016-12-01

When aiming for homogenous embryoid body (EB) differentiation, the use of equal-sized EBs is required to avoid a size-induced differentiation bias. In this study we developed an efficient and standardized EB formation protocol for human pluripotent stem cells (hPSC) cultured in a laminin-521-based xeno-free system. As the cell proliferation rate of the cells growing on laminin-521 strongly affected the efficiency of aggregate formation, we found that recently passaged cells, as well as the addition of ROCK inhibitor, were essential for reproducible EB formation from hPSC single-cell suspensions. EBs could be obtained in a variety of differentiation media, in 96-well round-bottom plates and in hanging drops. Gene expression studies on differentially sized EBs from three individual human embryonic stem cell lines demonstrated that the medium used for differentiation influenced the differentiation outcome to a much greater extent than the number of cells used for the initial EB formation. Our findings give a new insight into factors that influence the EB formation and differentiation process. This optimized method allows us to easily manipulate EB formation and provide an excellent starting point for downstream EB-based differentiation protocols.

Cell Fate and Differentiation of the Developing Ocular Lens

PubMed Central

Greiling, Teri M. S.; Aose, Masamoto

2010-01-01

Purpose. Even though zebrafish development does not include the formation of a lens vesicle, the authors' hypothesis is that the processes of cell differentiation are similar in zebrafish and mammals and determine cell fates in the lens. Methods. Two-photon live embryo imaging was used to follow individual fluorescently labeled cells in real-time from the placode stage at 16 hours postfertilization (hpf) until obvious morphologic differentiation into epithelium or fiber cells had occurred at approximately 28 hpf. Immunohistochemistry was used to label proliferating, differentiating, and apoptotic cells. Results. Similar to the mammal, cells in the teleost peripheral lens placode migrated to the anterior lens mass and differentiated into an anterior epithelium. Cells in the central lens placode migrated to the posterior lens mass and differentiated into primary fiber cells. Anterior and posterior polarization in the zebrafish lens mass was similar to mammalian lens vesicle polarization. Primary fiber cell differentiation was apparent at approximately 21 hpf, before separation of the lens from the surface ectoderm, as evidenced by cell elongation, exit from the cell cycle, and expression of Zl-1, a marker for fiber differentiation. TUNEL labeling demonstrated that apoptosis was not a primary mechanism for lens separation from the surface ectoderm. Conclusions. Despite the absence of a lens vesicle in the zebrafish embryo, lens organogenesis appears to be well conserved among vertebrates. Results using three-dimensional live embryo imaging of zebrafish development showed minimal differences and strong similarities in the fate of cells in the zebrafish and mammalian lens placode. PMID:19834024

Force spectroscopy of membrane hardness of SH-SY5Y neuroblastoma cells before and after differentiation

NASA Astrophysics Data System (ADS)

Kwon, Sangwoo; Yang, Woochul; Choi, Yun Kyong; Park, Jung Keuck

2014-05-01

Atomic force microscopy (AFM) is utilized in many studies for measuring the structure and the physical characteristics of soft and bio materials. In particular, the force spectroscopy function in the AFM system allows us to explore the mechanical properties of bio cells. In this study, we probe the variation in the membrane hardness of human neuroblastoma SH-SY5Y cells (SH-cells) before and after differentiation by using force spectroscopy. The SH-cell, which is usually differentiated by using a chemical treatment with retinoic acid (RA), is a neuronal cell line employed widely as an in-vitro model for neuroscience research. In force spectroscopy, the force-distance curves are obtained from both the original and the RA-treated cells while the AFM tip approaches and pushes on the cell membranes. The slope deduced from linear region in the force-distance curve is the spring constant and corresponds to the hardness of the cell membrane. The spring constant of the RA-treated cells (0.597 ± 0.010 nN/nm) was smaller than that of the original cells (0.794 ± 0.010 nN/nm), reflecting a hardness decrease in the cells differentiated with the RA treatments. The results clearly demonstrated that the differentiated cells are softer than the original cells. The change in the elasticity of the differentiated cells might be caused by morphological modification during differentiation process. We suggest that force spectroscopy can be employed as a novel method to determine the degree of differentiation of stem cells into various functional cells.

Three-Dimensional Coculture Of Human Small-Intestine Cells

NASA Technical Reports Server (NTRS)

Wolf, David; Spaulding, Glen; Goodwin, Thomas J.; Prewett, Tracy

1994-01-01

Complex three-dimensional masses of normal human epithelial and mesenchymal small-intestine cells cocultured in process involving specially designed bioreactors. Useful as tissued models for studies of growth, regulatory, and differentiation processes in normal intestinal tissues; diseases of small intestine; and interactions between cells of small intestine and viruses causing disease both in small intestine and elsewhere in body. Process used to produce other tissue models, leading to advances in understanding of growth and differentiation in developing organisms, of renewal of tissue, and of treatment of myriad of clinical conditions. Prior articles describing design and use of rotating-wall culture vessels include "Growing And Assembling Cells Into Tissues" (MSC-21559), "High-Aspect-Ratio Rotating Cell-Culture Vessel" (MSC-21662), and "In Vitro, Matrix-Free Formation Of Solid Tumor Spheroids" (MSC-21843).

Morphologic expression of glandular differentiation in the epidermoid nasal carcinomas induced by phenylglycidyl ether inhalation.

PubMed Central

Lee, K. P.; Schneider, P. W.; Trochimowicz, H. J.

1983-01-01

Charles River-CD Sprague-Dawley rats in 3 equal groups of 100 males and 100 females each were exposed to 12, 1, and 0 ppm of phenylglycidyl ether vapor for 24 months. Nasal tumors were first detected after 621 days' exposure at 12 ppm with an incidence of 11% in males and 4.4% in females. No nasal tumors were found at 1 ppm in rats exposed for 24 months. The nasal tumors, mostly epidermoid carcinomas, were derived from the respiratory epithelium and nasal glands, both of which revealed squamous metaplasia or dysplasia in the anterior nasal cavity. Most nasal tumors were confined to the anterior nasal cavity and occasionally invaded the dorsonasal bones and posterior nasal cavity. The undifferentiated glandular cells appear to differentiate to neoplastic squamous cells, because the ultrastructure of epidermoid carcinoma revealed traits of glandular cell differentiation in the neoplastic squamous cells. The features of glandular cell differentiation in the neoplastic squamous cells were intercellular or intracellular glandular lumens, secretory vesicles, mucus droplets, and intermediate cells showing both glandular and squamous differentiation. Squamous cells in the well-differentiated epidermoid carcinomas revealed abundant tonofibrils, desmosomes, glycogen particulates, and interdigitated cytoplasmic processes. These markers of squamous-cell differentiation were markedly reduced in the undifferentiated epidermoid carcinomas. The spindle-cell squamous carcinoma showed both squamous and fibroblastic-like differentiations. Some spindle cells had only fibroblastic-like differentiation, suggesting spindle-cell metaplasia of the squamous cells. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 PMID:6846500

Integrator complex plays an essential role in adipose differentiation

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

Otani, Yuichiro; Nakatsu, Yusuke; Sakoda, Hideyuki

2013-05-03

Highlights: •IntS6 and IntS11 are subunits of the Integrator complex. •Expression levels of IntS6 and IntS11 were very low in 3T3-L1 fibroblast. •IntS6 and IntS11 were upregulated during adipose differentiation. •Suppression of IntS6 or IntS11 expression inhibited adipose differentiation. -- Abstract: The dynamic process of adipose differentiation involves stepwise expressions of transcription factors and proteins specific to the mature fat cell phenotype. In this study, it was revealed that expression levels of IntS6 and IntS11, subunits of the Integrator complex, were increased in 3T3-L1 cells in the period when the cells reached confluence and differentiated into adipocytes, while being reducedmore » to basal levels after the completion of differentiation. Suppression of IntS6 or IntS11 expression using siRNAs in 3T3-L1 preadipocytes markedly inhibited differentiation into mature adipocytes, based on morphological findings as well as mRNA analysis of adipocyte-specific genes such as Glut4, perilipin and Fabp4. Although Pparγ2 protein expression was suppressed in IntS6 or IntS11-siRNA treated cells, adenoviral forced expression of Pparγ2 failed to restore the capacity for differentiation into mature adipocytes. Taken together, these findings demonstrate that increased expression of Integrator complex subunits is an indispensable event in adipose differentiation. Although further study is necessary to elucidate the underlying mechanism, the processing of U1, U2 small nuclear RNAs may be involved in cell differentiation steps.« less

New Horizons in Enhancing the Proliferation and Differentiation of Neural Stem Cells Using Stimulatory Effects of the Short Time Exposure to Radiofrequency Radiation

PubMed Central

Eghlidospour, M.; Mortazavi, S. M. J.; Yousefi, F.; Mortazavi, S. A. R.

2015-01-01

Mobile phone use and wireless communication technology have grown explosively over the past decades. This rapid growth has caused widespread global concern about the potential detrimental effects of this technology on human health. Stem cells generate specialized cell types of the tissue in which they reside through normal differentiation pathways. Considering the undeniable importance of stem cells in modern medicine, numerous studies have been performed on the effects of ionizing and non-ionizing radiation on cellular processes such as: proliferation, differentiation, cell cycle and DNA repair processes. We have conducted extensive studies on beneficial (stimulatory) or detrimental biological effects of exposure to different sources of electromagnetic fields such as mobile phones, mobile phone base stations, mobile phone jammers, radar systems, magnetic resonance imaging (MRI) systems and dentistry cavitrons over the past years. In this article, recent studies on the biological effects of non-ionizing electromagnetic radiation in the range of radiofrequency (RF) on some important features of stem cells such as their proliferation and differentiation are reviewed. Studies reviewed in this paper indicate that the stimulatory or inhibitory effects of RF radiation on the proliferation and differentiation of stem cells depend on various factors such as the biological systems, experiment conditions, the frequency and intensity of RF and the duration of exposure. PMID:26396965

New Horizons in Enhancing the Proliferation and Differentiation of Neural Stem Cells Using Stimulatory Effects of the Short Time Exposure to Radiofrequency Radiation.

PubMed

Eghlidospour, M; Mortazavi, S M J; Yousefi, F; Mortazavi, S A R

2015-09-01

Mobile phone use and wireless communication technology have grown explosively over the past decades. This rapid growth has caused widespread global concern about the potential detrimental effects of this technology on human health. Stem cells generate specialized cell types of the tissue in which they reside through normal differentiation pathways. Considering the undeniable importance of stem cells in modern medicine, numerous studies have been performed on the effects of ionizing and non-ionizing radiation on cellular processes such as: proliferation, differentiation, cell cycle and DNA repair processes. We have conducted extensive studies on beneficial (stimulatory) or detrimental biological effects of exposure to different sources of electromagnetic fields such as mobile phones, mobile phone base stations, mobile phone jammers, radar systems, magnetic resonance imaging (MRI) systems and dentistry cavitrons over the past years. In this article, recent studies on the biological effects of non-ionizing electromagnetic radiation in the range of radiofrequency (RF) on some important features of stem cells such as their proliferation and differentiation are reviewed. Studies reviewed in this paper indicate that the stimulatory or inhibitory effects of RF radiation on the proliferation and differentiation of stem cells depend on various factors such as the biological systems, experiment conditions, the frequency and intensity of RF and the duration of exposure.

Microarray Analysis of Gene Expression Alteration in Human Middle Ear Epithelial Cells Induced by Asian Sand Dust.

PubMed

Go, Yoon Young; Park, Moo Kyun; Kwon, Jee Young; Seo, Young Rok; Chae, Sung-Won; Song, Jae-Jun

2015-12-01

The primary aim of this study is to evaluate the gene expression profile of Asian sand dust (ASD)-treated human middle ear epithelial cell (HMEEC) using microarray analysis. The HMEEC was treated with ASD (400 µg/mL) and total RNA was extracted for microarray analysis. Molecular pathways among differentially expressed genes were further analyzed. For selected genes, the changes in gene expression were confirmed by real-time polymerase chain reaction. A total of 1,274 genes were differentially expressed by ASD. Among them, 1,138 genes were 2 folds up-regulated, whereas 136 genes were 2 folds down-regulated. Up-regulated genes were mainly involved in cellular processes, including apoptosis, cell differentiation, and cell proliferation. Down-regulated genes affected cellular processes, including apoptosis, cell cycle, cell differentiation, and cell proliferation. The 10 genes including ADM, CCL5, EDN1, EGR1, FOS, GHRL, JUN, SOCS3, TNF, and TNFSF10 were identified as main modulators in up-regulated genes. A total of 11 genes including CSF3, DKK1, FOSL1, FST, TERT, MMP13, PTHLH, SPRY2, TGFBR2, THBS1, and TIMP1 acted as main components of pathway associated with 2-fold down regulated genes. We identified the differentially expressed genes in ASD-treated HMEEC. Our work indicates that air pollutant like ASD, may play an important role in the pathogenesis of otitis media.

CD4+ T helper 2 cells – microbial triggers, differentiation requirements and effector functions

PubMed Central

Okoye, Isobel S; Wilson, Mark S

2011-01-01

Over the past 10 years we have made great strides in our understanding of T helper cell differentiation, expansion and effector functions. Within the context of T helper type 2 (Th2) cell development, novel innate-like cells with the capacity to secrete large amounts of interleukin-5 (IL-5), IL-13 and IL-9 as well as IL-4-producing and antigen-processing basophils have (re)-emerged onto the type 2 scene. To what extent these new players influence αβ+ CD4+ Th2 cell differentiation is discussed throughout this appraisal of the current literature. We highlight the unique features of Th2 cell development, highlighting the three necessary signals, T-cell receptor ligation, co-stimulation and cytokine receptor ligation. Finally, putting these into context, microbial and allergenic properties that trigger Th2 cell differentiation and how these influence Th2 effector function are discussed and questioned. PMID:22043920

Conceptual Challenges of the Systemic Approach in Understanding Cell Differentiation.

PubMed

Paldi, Andras

2018-01-01

The cells of a multicellular organism are derived from a single zygote and genetically identical. Yet, they are phenotypically very different. This difference is the result of a process commonly called cell differentiation. How the phenotypic diversity emerges during ontogenesis or regeneration is a central and intensely studied but still unresolved issue in biology. Cell biology is facing conceptual challenges that are frequently confused with methodological difficulties. How to define a cell type? What stability or change means in the context of cell differentiation and how to deal with the ubiquitous molecular variations seen in the living cells? What are the driving forces of the change? We propose to reframe the problem of cell differentiation in a systemic way by incorporating different theoretical approaches. The new conceptual framework is able to capture the insights made at different levels of cellular organization and considered previously as contradictory. It also provides a formal strategy for further experimental studies.

The two waves of B-lymphocyte differentiation.

PubMed

Nossal, G J

1984-01-01

The recombinant DNA revolution has struck cellular immunology with great force, but it has not yet illuminated B-lymphocyte physiology as much as it should have. The problems of the second wave of B-cell differentiation, the post-antigenic wave, will be solved relatively soon, as genetically engineered B-cell-active factors become available. Speculatively, I predict the existence of 3-4 separate factors, each possessing both growth and differentiation activity. The problems of the first set of differentiative processes, those leading to B-lymphocyte genesis, remain frustratingly inaccessible. Just to start an argument, I will speculate that IL-3 will not be involved here, but rather that we must search for non-T-cell-derived (non-lymphokine) factors produced by specialized cells (not macrophages) in the foetal liver or the bone marrow. I issue a challenge to the participants in this forum to reveal how they would go about devising a tissue culture system which at least approximates living bone marrow in its immense potential for B-cell production, because until this is done, the most interesting segment of the whole process will remain a black box.

3D patterned stem cell differentiation using thermo-responsive methylcellulose hydrogel molds.

PubMed

Lee, Wonjae; Park, Jon

2016-07-06

Tissue-specific patterned stem cell differentiation serves as the basis for the development, remodeling, and regeneration of the multicellular structure of the native tissues. We herein proposed a cytocompatible 3D casting process to recapitulate this patterned stem cell differentiation for reconstructing multicellular tissues in vitro. We first reconstituted the 2D culture conditions for stem cell fate control within 3D hydrogel by incorporating the sets of the diffusible signal molecules delivered through drug-releasing microparticles. Then, utilizing thermo-responsivity of methylcellulose (MC), we developed a cytocompatible casting process to mold these hydrogels into specific 3D configurations, generating the targeted spatial gradients of diffusible signal molecules. The liquid phase of the MC solution was viscous enough to adopt the shapes of 3D impression patterns, while the gelated MC served as a reliable mold for patterning the hydrogel prepolymers. When these patterned hydrogels were integrated together, the stem cells in each hydrogel distinctly differentiated toward individually defined fates, resulting in the formation of the multicellular tissue structure bearing the very structural integrity and characteristics as seen in vascularized bones and osteochondral tissues.

3D patterned stem cell differentiation using thermo-responsive methylcellulose hydrogel molds

NASA Astrophysics Data System (ADS)

Lee, Wonjae; Park, Jon

2016-07-01

Tissue-specific patterned stem cell differentiation serves as the basis for the development, remodeling, and regeneration of the multicellular structure of the native tissues. We herein proposed a cytocompatible 3D casting process to recapitulate this patterned stem cell differentiation for reconstructing multicellular tissues in vitro. We first reconstituted the 2D culture conditions for stem cell fate control within 3D hydrogel by incorporating the sets of the diffusible signal molecules delivered through drug-releasing microparticles. Then, utilizing thermo-responsivity of methylcellulose (MC), we developed a cytocompatible casting process to mold these hydrogels into specific 3D configurations, generating the targeted spatial gradients of diffusible signal molecules. The liquid phase of the MC solution was viscous enough to adopt the shapes of 3D impression patterns, while the gelated MC served as a reliable mold for patterning the hydrogel prepolymers. When these patterned hydrogels were integrated together, the stem cells in each hydrogel distinctly differentiated toward individually defined fates, resulting in the formation of the multicellular tissue structure bearing the very structural integrity and characteristics as seen in vascularized bones and osteochondral tissues.

3D patterned stem cell differentiation using thermo-responsive methylcellulose hydrogel molds

PubMed Central

Lee, Wonjae; Park, Jon

2016-01-01

Tissue-specific patterned stem cell differentiation serves as the basis for the development, remodeling, and regeneration of the multicellular structure of the native tissues. We herein proposed a cytocompatible 3D casting process to recapitulate this patterned stem cell differentiation for reconstructing multicellular tissues in vitro. We first reconstituted the 2D culture conditions for stem cell fate control within 3D hydrogel by incorporating the sets of the diffusible signal molecules delivered through drug-releasing microparticles. Then, utilizing thermo-responsivity of methylcellulose (MC), we developed a cytocompatible casting process to mold these hydrogels into specific 3D configurations, generating the targeted spatial gradients of diffusible signal molecules. The liquid phase of the MC solution was viscous enough to adopt the shapes of 3D impression patterns, while the gelated MC served as a reliable mold for patterning the hydrogel prepolymers. When these patterned hydrogels were integrated together, the stem cells in each hydrogel distinctly differentiated toward individually defined fates, resulting in the formation of the multicellular tissue structure bearing the very structural integrity and characteristics as seen in vascularized bones and osteochondral tissues. PMID:27381562

The transcriptional landscape of αβ T cell differentiation

PubMed Central

Mingueneau, Michael; Kreslavsky, Taras; Gray, Daniel; Heng, Tracy; Cruse, Richard; Ericson, Jeffrey; Bendall, Sean; Spitzer, Matt; Nolan, Garry; Kobayashi, Koichi; von Boehmer, Harald; Mathis, Diane; Benoist, Christophe

2013-01-01

αβT cell differentiation from thymic precursors is a complex process, explored here with the breadth of ImmGen expression datasets, analyzing how differentiation of thymic precursors gives rise to transcriptomes. After surprisingly gradual changes though early T commitment, transit through the CD4+CD8+ stage involves a shutdown or rare breadth, and correlating tightly with MYC. MHC-driven selection promotes a large-scale transcriptional reactivation. We identify distinct signatures that mark cells destined for positive selection versus apoptotic deletion. Differential expression of surprisingly few genes accompany CD4 or CD8 commitment, a similarity that carries through to peripheral T cells and their activation, revealed by mass cytometry phosphoproteomics. The novel transcripts identified as candidate mediators of key transitions help define the “known unknown” of thymocyte differentiation. PMID:23644507

Optimising parameters for the differentiation of SH-SY5Y cells to study cell adhesion and cell migration

PubMed Central

2013-01-01

Background 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. Results 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. Conclusions 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. PMID:24025096

Heterogeneity in the development of the vertebra.

PubMed

Monsoro-Burq, A H; Bontoux, M; Teillet, M A; Le Douarin, N M

1994-10-25

Vertebrae are derived from the sclerotomal moities of the somites. Sclerotomal cells migrate ventrally to surround the notochord, where they form the vertebral body, and dorsolaterally to form the neural arch, which is dorsally closed by the spinous process. Precursor cells of the spinous process as well as superficial ectoderm and roof plate express homeobox genes of the Msh family from embryonic day 2 (E2) to E6. The notochord has been shown to be responsible for the dorsoventral polarization of the somites and for the induction of sclerotomal cells into cartilage. Indeed, supernumerary notochord grafted laterally to the neural tube induces the conversion of the entire somite into cartilage. We report here that a mediodorsal graft of notochord prevents the sclerotomal cells migrating dorsally to the roof plate from differentiating into cartilage. Under these experimental conditions, expression of Msx genes is abolished. We thus demonstrate that cartilaginous, differentiation is differentially controlled in the dorsal part of the vertebra (spinous process) and in the neural arch and vertebral body.

Determination of the intracellular Ca2+ concentration in the N1E-115 neuronal cell line in perspective of its use for peripheric nerve regeneration.

PubMed

Rodrigues, J M; Luís, A L; Lobato, J V; Pinto, M V; Lopes, M A; Freitas, M; Geuna, S; Santos, J D; Maurício, A C

2005-01-01

Entubulation repair of peripheral nerve injuries has a lengthy history. Several experimental and clinical studies have explored the effectiveness of many biodegradable and non-degradable tubes with or without addition of molecules and cells. The main objective of the present study was to develop an economical and also an easy way for culturing a neural cell line which was capable of growing, differentiating and producing locally nerve growth factors that are otherwise extremely expensive, inside 90 PLA/10 PLG nerve guides. For this purpose the authors have chosen the N1E-115 cell line, a clone of cells derived from mouse neuroblastoma C-1300 with the perspective of using this differentiated cellular system to cover the inside of 90 PLA/10 PLG nerve guides placed to bridge a gap in the rat sciatic nerve experimental model. The N1E-115 cells proliferate in normal culture medium but undergo neuronal differentiation in response to DMSO. Upon induction of differentiation, proliferation of N1E-115 cells ceases, extensive neurite outgrowth is observed and the membranes become highly excitable. While it is known that Ca2+ serves as an important intracellular signal for various cellular processes, such as growth and differentiation. It is also known that can be toxic to cells and is involved in the triggering of events leading to excitotoxic cell death in neurons. The [Ca2+]i in non-differentiated N1E-115 cells and after distinct periods of differentiation, have been determined by the epifluorescence technique using the Fura-2-AM probe. The results of this quantitative assessment revealed that N1E-115 cells which undergo neuronal differentiation for 48 hours in the presence of 1.5% DMSO are best qualified to be used to cover the interior of the nerve guides since the [Ca2+]i was not found to be elevated indicating thus that the onset the cell death processes was not occurred.

PRMT5 is essential for the maintenance of chondrogenic progenitor cells in the limb bud

PubMed Central

Norrie, Jacqueline L.; Li, Qiang; Co, Swanie; Huang, Bau-Lin; Ding, Ding; Uy, Jann C.; Ji, Zhicheng; Mackem, Susan; Bedford, Mark T.; Galli, Antonella; Ji, Hongkai

2016-01-01

During embryonic development, undifferentiated progenitor cells balance the generation of additional progenitor cells with differentiation. Within the developing limb, cartilage cells differentiate from mesodermal progenitors in an ordered process that results in the specification of the correct number of appropriately sized skeletal elements. The internal pathways by which these cells maintain an undifferentiated state while preserving their capacity to differentiate is unknown. Here, we report that the arginine methyltransferase PRMT5 has a crucial role in maintaining progenitor cells. Mouse embryonic buds lacking PRMT5 have severely truncated bones with wispy digits lacking joints. This novel phenotype is caused by widespread cell death that includes mesodermal progenitor cells that have begun to precociously differentiate into cartilage cells. We propose that PRMT5 maintains progenitor cells through its regulation of Bmp4. Intriguingly, adult and embryonic stem cells also require PRMT5 for maintaining pluripotency, suggesting that similar mechanisms might regulate lineage-restricted progenitor cells during organogenesis. PMID:27827819

PRMT5 is essential for the maintenance of chondrogenic progenitor cells in the limb bud.

PubMed

Norrie, Jacqueline L; Li, Qiang; Co, Swanie; Huang, Bau-Lin; Ding, Ding; Uy, Jann C; Ji, Zhicheng; Mackem, Susan; Bedford, Mark T; Galli, Antonella; Ji, Hongkai; Vokes, Steven A

2016-12-15

During embryonic development, undifferentiated progenitor cells balance the generation of additional progenitor cells with differentiation. Within the developing limb, cartilage cells differentiate from mesodermal progenitors in an ordered process that results in the specification of the correct number of appropriately sized skeletal elements. The internal pathways by which these cells maintain an undifferentiated state while preserving their capacity to differentiate is unknown. Here, we report that the arginine methyltransferase PRMT5 has a crucial role in maintaining progenitor cells. Mouse embryonic buds lacking PRMT5 have severely truncated bones with wispy digits lacking joints. This novel phenotype is caused by widespread cell death that includes mesodermal progenitor cells that have begun to precociously differentiate into cartilage cells. We propose that PRMT5 maintains progenitor cells through its regulation of Bmp4 Intriguingly, adult and embryonic stem cells also require PRMT5 for maintaining pluripotency, suggesting that similar mechanisms might regulate lineage-restricted progenitor cells during organogenesis. © 2016. Published by The Company of Biologists Ltd.

Intracellular fragment of NLRR3 (NLRR3-ICD) stimulates ATRA-dependent neuroblastoma differentiation

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

Akter, Jesmin; Takatori, Atsushi, E-mail: atakatori@chiba-cc.jp; Islam, Md. Sazzadul

2014-10-10

Highlights: • NLRR3 is a membrane protein highly expressed in favorable neuroblastoma. • NLRR3-ICD was produced through proteolytic processing by secretases. • NLRR3-ICD was induced to be translocated into cell nucleus following ATRA exposure. • NLRR3-ICD plays a pivotal role in ATRA-mediated neuroblastoma differentiation. - Abstract: We have previously identified neuronal leucine-rich repeat protein-3 (NLRR3) gene which is preferentially expressed in favorable human neuroblastomas as compared with unfavorable ones. In this study, we have found for the first time that NLRR3 is proteolytically processed by secretases and its intracellular domain (NLRR3-ICD) is then released to translocate into cell nucleus duringmore » ATRA-mediated neuroblastoma differentiation. According to our present observations, NLRR3-ICD was induced to accumulate in cell nucleus of neuroblastoma SH-SY5Y cells following ATRA treatment. Since the proteolytic cleavage of NLRR3 was blocked by α- or γ-secretase inhibitor, it is likely that NLRR3-ICD is produced through the secretase-mediated processing of NLRR3. Intriguingly, forced expression of NLRR3-ICD in neuroblastoma SK-N-BE cells significantly suppressed their proliferation as examined by a live-cell imaging system and colony formation assay. Similar results were also obtained in neuroblastoma TGW cells. Furthermore, overexpression of NLRR3-ICD stimulated ATRA-dependent neurite elongation in SK-N-BE cells. Together, our present results strongly suggest that NLRR3-ICD produced by the secretase-mediated proteolytic processing of NLRR3 plays a crucial role in ATRA-mediated neuronal differentiation, and provide a clue to develop a novel therapeutic strategy against aggressive neuroblastomas.« less

Uniform Embryoid Body Production and Enhanced Mesendoderm Differentiation with Murine Embryonic Stem Cells in a Rotary Suspension Bioreactor.

PubMed

Lei, Xiaohua; Deng, Zhili; Duan, Enkui

2016-01-01

Embryonic stem cells (ESCs) are capable of differentiating into almost all cell types in vitro and hold great promise for drug screening, developmental studies and have a huge potential in many therapeutic areas. ESCs can aggregate to form embryoid body (EB) in static suspension culture by spontaneous differentiation, which resembles an intact embryo; while static suspension culture cannot prevent agglomeration of cells and offers little control over the size and shape of EBs, it results in aggregation of EBs into large, irregular masses, which prejudice the efficiency of differentiation of cells. Recently, bioreactor-based platforms have been shown to not only offer a beneficial effect on increasing diffusion of nutrients and oxygen which promotes cell viability and proliferation but also display local biomechanical properties (e.g., low fluid shear stresses and hydrodynamic force) in tissue development and organogenesis. This chapter describes a protocol for using a rotary suspension bioreactor to produce embryoid bodies and process the differentiation of mouse embryonic stem cells (mESCs), and to assess the efficiency of EB differentiation in the bioreactor by real-time PCR and immunostaining.

Transplantation of cells from eye-like structures differentiated from embryonic stem cells in vitro and in vivo regeneration of retinal ganglion-like cells.

PubMed

Aoki, Hitomi; Hara, Akira; Niwa, Masayuki; Motohashi, Tsutomu; Suzuki, Takashi; Kunisada, Takahiro

2008-02-01

An embryonic stem (ES) cell-derived eye-like structure, made up of neural retinal lineage cells, retinal pigment epithelial (RPE) cells, and lens cells was constructed in our laboratory. We have shown that cells from these eye-like structures can be integrated into the developing optic vesicle of chicks. The purpose of this study was to determine whether the cells from these eye-like structures can differentiate into retinal ganglion cells (RGCs) when transplanted into the vitreous of an injured adult mouse retina. ES cells were induced to differentiate into eye-like structures in vitro for 6 or 11 days. Recipient mouse eyes were injected with NMDA to injure the RGCs prior to the transplantation. Sham-treated eyes received the same amount of carrier vehicle. Cells were extracted from the eye-like structures and transplanted into the vitreous of damaged and control eyes. The host eyes were analyzed both qualitatively and quantitatively by immunohistochemistry 10 days or 8 weeks after transplantation. Cells from the ES cell-derived eye-like structures were integrated into the RGC layer, and differentiated into neurons when transplanted into control (non-NMDA-treated) adult eyes. However, they rarely expressed RGC markers. When they were transplanted into NMDA-treated eyes, the cells spread on the surface of the retina and covered a relatively large area of the host RGC layer that had been injured by the NMDA. The cells from the ES cell-derived eye cells frequently differentiated into cells expressing RGC-specific markers, and formed a new RGC layer. In addition, a small number of these ES cell-derived cells were observed to extend axon-like processes toward the optic disc of the host. However, visually evoked responses could not be recorded from the visual cortex. These findings suggest that ES cell-derived eye-like structures contain cells that can differentiate into RG-like cells and regenerate a new RGC layer. These cells also appeared to be integrated into the retina and extend axon-like processes toward the optic nerve head.

Trichinella spiralis: nurse cell formation with emphasis on analogy to muscle cell repair

PubMed Central

Wu, Zhiliang; Sofronic-Milosavljevic, Lj; Nagano, Isao; Takahashi, Yuzo

2008-01-01

Trichinella infection results in formation of a capsule in infected muscles. The capsule is a residence of the parasite which is composed of the nurse cell and fibrous wall. The process of nurse cell formation is complex and includes infected muscle cell response (de-differentiation, cell cycle re-entry and arrest) and satellite cell responses (activation, proliferation and differentiation). Some events that occur during the nurse cell formation are analogous to those occurring during muscle cell regeneration/repair. This article reviews capsule formation with emphasis on this analogy. PMID:18710582

Miz1, a Novel Target of ING4, Can Drive Prostate Luminal Epithelial Cell Differentiation.

PubMed

Berger, Penny L; Winn, Mary E; Miranti, Cindy K

2017-01-01

How prostate epithelial cells differentiate and how dysregulation of this process contributes to prostate tumorigenesis remain unclear. We recently identified a Myc target and chromatin reader protein, ING4, as a necessary component of human prostate luminal epithelial cell differentiation, which is often lost in primary prostate tumors. Furthermore, loss of ING4 in the context of oncogenic mutations is required for prostate tumorigenesis. Identifying the gene targets of ING4 can provide insight into how its loss disrupts differentiation and leads to prostate cancer. Using a combination of RNA-Seq, a best candidate approach, and chromatin immunoprecipitation (ChIP), we identified Miz1 as a new ING4 target. ING4 or Miz1 overexpression, shRNA knock-down, and a Myc-binding mutant were used in a human in vitro differentiation assay to assess the role of Miz1 in luminal cell differentiation. ING4 directly binds the Miz1 promoter and is required to induce Miz1 mRNA and protein expression during luminal cell differentiation. Miz1 mRNA was not induced in shING4 expressing cells or tumorigenic cells in which ING4 is not expressed. Miz1 dependency on ING4 was unique to differentiating luminal cells; Miz1 mRNA expression was not induced in basal cells. Although Miz1 is a direct target of ING4, and its overexpression can drive luminal cell differentiation, Miz1 was not required for differentiation. Miz1 is a newly identified ING4-induced target gene which can drive prostate luminal epithelial cell differentiation although it is not absolutely required. Prostate 77:49-59, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Autophagy is essential for the differentiation of porcine PSCs into insulin-producing cells.

PubMed

Ren, Lipeng; Yang, Hong; Cui, Yanhua; Xu, Shuanshuan; Sun, Fen; Tian, Na; Hua, Jinlian; Peng, Sha

2017-07-01

Porcine pancreatic stem cells (PSCs) are seed cells with potential use for diabetes treatment. Stem cell differentiation requires strict control of protein turnover and lysosomal digestion of organelles. Autophagy is a highly conserved process that controls the turnover of organelles and proteins within cells and contributes to the balance of cellular components. However, whether autophagy plays roles in PSC differentiation remains unknown. In this study, we successfully induced porcine PSCs into insulin-producing cells and found that autophagy was activated during the second induction stage. Inhibition of autophagy in the second stage resulted in reduced differentiational efficiency and impaired glucose-stimulated insulin secretion. Moreover, the expression of active β-catenin increased while autophagy was activated but was suppressed when autophagy was inhibited. Therefore, autophagy is essential to the formation of insulin-producing cells, and the effects of autophagy on differentiation may be regulated by canonical Wnt signalling pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

Arginine methylation catalyzed by PRMT1 is required for B cell activation and differentiation.

PubMed

Infantino, Simona; Light, Amanda; O'Donnell, Kristy; Bryant, Vanessa; Avery, Danielle T; Elliott, Michael; Tangye, Stuart G; Belz, Gabrielle; Mackay, Fabienne; Richard, Stephane; Tarlinton, David

2017-10-12

Arginine methylation catalyzed by protein arginine methyltransferases (PRMT) is a common post-translational modification in mammalian cells, regulating many important functions including cell signalling, proliferation and differentiation. Here we show the role of PRMT1 in B-cell activation and differentiation. PRMT1 expression and activity in human and mouse peripheral B cells increases in response to in vitro or in vivo activation. Deletion of the Prmt1 gene in mature B cells establishes that although the frequency and phenotype of peripheral B cell subsets seem unaffected, immune responses to T-cell-dependent and -independent antigens are substantially reduced. In vitro activation of Prmt1-deficient B cells with a variety of mitogens results in diminished proliferation, differentiation and survival, effects that are correlated with altered signal transduction from the B cell receptor. Thus PRMT1 activity in B cells is required for correct execution of multiple processes that in turn are necessary for humoral immunity.PRMT1 is an arginine methyltransferase involved in a variety of cell functions. Here the authors delete PRMT1 specifically in mature B cells to show the importance of arginine methylation for B cell proliferation, differentiation and survival, and thereby for humoral immunity.

BTG interacts with retinoblastoma to control cell fate in Dictyostelium.

PubMed

Conte, Daniele; MacWilliams, Harry K; Ceccarelli, Adriano

2010-03-12

In the genesis of many tissues, a phase of cell proliferation is followed by cell cycle exit and terminal differentiation. The latter two processes overlap: genes involved in the cessation of growth may also be important in triggering differentiation. Though conceptually distinct, they are often causally related and functional interactions between the cell cycle machinery and cell fate control networks are fundamental to coordinate growth and differentiation. A switch from proliferation to differentiation may also be important in the life cycle of single-celled organisms, and genes which arose as regulators of microbial differentiation may be conserved in higher organisms. Studies in microorganisms may thus contribute to understanding the molecular links between cell cycle machinery and the determination of cell fate choice networks. Here we show that in the amoebozoan D. discoideum, an ortholog of the metazoan antiproliferative gene btg controls cell fate, and that this function is dependent on the presence of a second tumor suppressor ortholog, the retinoblastoma-like gene product. Specifically, we find that btg-overexpressing cells preferentially adopt a stalk cell (and, more particularly, an Anterior-Like Cell) fate. No btg-dependent preference for ALC fate is observed in cells in which the retinoblastoma-like gene has been genetically inactivated. Dictyostelium btg is the only example of non-metazoan member of the BTG family characterized so far, suggesting that a genetic interaction between btg and Rb predated the divergence between dictyostelids and metazoa. While the requirement for retinoblastoma function for BTG antiproliferative activity in metazoans is known, an interaction of these genes in the control of cell fate has not been previously documented. Involvement of a single pathway in the control of mutually exclusive processes may have relevant implication in the evolution of multicellularity.

Production of Functional Glucagon-Secreting α-Cells From Human Embryonic Stem Cells

PubMed Central

Rezania, Alireza; Riedel, Michael J.; Wideman, Rhonda D.; Karanu, Francis; Ao, Ziliang; Warnock, Garth L.; Kieffer, Timothy J.

2011-01-01

OBJECTIVE Differentiation of human embryonic stem (hES) cells to fully developed cell types holds great therapeutic promise. Despite significant progress, the conversion of hES cells to stable, fully differentiated endocrine cells that exhibit physiologically regulated hormone secretion has not yet been achieved. Here we describe an efficient differentiation protocol for the in vitro conversion of hES cells to functional glucagon-producing α- cells. RESEARCH DESIGN AND METHODS Using a combination of small molecule screening and empirical testing, we developed a six-stage differentiation protocol for creating functional α-cells. An extensive in vitro and in vivo characterization of the differentiated cells was performed. RESULTS A high rate of synaptophysin expression (>75%) and robust expression of glucagon and the α-cell transcription factor ARX was achieved. After a transient polyhormonal state in which cells coexpress glucagon and insulin, maturation in vitro or in vivo resulted in depletion of insulin and other β-cell markers with concomitant enrichment of α-cell markers. After transplantation, these cells secreted fully processed, biologically active glucagon in response to physiologic stimuli including prolonged fasting and amino acid challenge. Moreover, glucagon release from transplanted cells was sufficient to reduce demand for pancreatic glucagon, resulting in a significant decrease in pancreatic α-cell mass. CONCLUSIONS These results indicate that fully differentiated pancreatic endocrine cells can be created via stepwise differentiation of hES cells. These cells may serve as a useful screening tool for the identification of compounds that modulate glucagon secretion as well as those that promote the transdifferentiation of α-cells to β-cells. PMID:20971966

Controlling Destiny through Chemistry: Small-Molecule Regulators of Cell Fate

PubMed Central

2009-01-01

Controlling cell fate is essential for embryonic development, tissue regeneration, and the prevention of human disease. With each cell in the human body sharing a common genome, achieving the appropriate spectrum of stem cells and their differentiated lineages requires the selective activation of developmental signaling pathways, the expression of specific target genes, and the maintenance of these cellular states through epigenetic mechanisms. Small molecules that target these regulatory processes are therefore valuable tools for probing and manipulating the molecular mechanisms by which stem cells self-renew, differentiate, and arise from somatic cell reprogramming. Pharmacological modulators of cell fate could also help remediate human diseases caused by dysregulated cell proliferation or differentiation, heralding a new era in molecular therapeutics. PMID:20000447

Controlling destiny through chemistry: small-molecule regulators of cell fate.

PubMed

Firestone, Ari J; Chen, James K

2010-01-15

Controlling cell fate is essential for embryonic development, tissue regeneration, and the prevention of human disease. With each cell in the human body sharing a common genome, achieving the appropriate spectrum of stem cells and their differentiated lineages requires the selective activation of developmental signaling pathways, the expression of specific target genes, and the maintenance of these cellular states through epigenetic mechanisms. Small molecules that target these regulatory processes are therefore valuable tools for probing and manipulating the molecular mechanisms by which stem cells self-renew, differentiate, and arise from somatic cell reprogramming. Pharmacological modulators of cell fate could also help remediate human diseases caused by dysregulated cell proliferation or differentiation, heralding a new era in molecular therapeutics.

Apoptosis in differentiating C2C12 muscle cells selectively targets Bcl-2-deficient myotubes

PubMed Central

Schoneich, Christian; Dremina, Elena; Galeva, Nadezhda; Sharov, Victor

2014-01-01

Muscle cell apoptosis accompanies normal muscle development and regeneration, as well as degenerative diseases and aging. C2C12 murine myoblast cells represent a common model to study muscle differentiation. Though it was already shown that myogenic differentiation of C2C12 cells is accompanied by enhanced apoptosis in a fraction of cells, either the cell population sensitive to apoptosis or regulatory mechanisms for the apoptotic response are unclear so far. In the current study we characterize apoptotic phenotypes of different types of C2C12 cells at all stages of differentiation, and report here that myotubes of differentiated C2C12 cells with low levels of anti-apoptotic Bcl-2 expression are particularly vulnerable to apoptosis even though they are displaying low levels of pro-apoptotic proteins Bax, Bak and Bad. In contrast, reserve cells exhibit higher levels of Bcl-2 and high resistance to apoptosis. The transfection of proliferating myoblasts with Bcl-2 prior to differentiation did not protect against spontaneous apoptosis accompanying differentiation of C2C12 cell but led to Bcl-2 overexpression in myotubes and to significant protection from apoptotic cell loss caused by exposure to hydrogen peroxide. Overall, our data advocate for a Bcl-2-dependent mechanism of apoptosis in differentiated muscle cells. However, downstream processes for spontaneous and hydrogen peroxide induced apoptosis are not completely similar. Apoptosis in differentiating myoblasts and myotubes is regulated not through interaction of Bcl-2 with pro-apoptotic Bcl-2 family proteins such as Bax, Bak, and Bad. PMID:24129924

Micro-Topographies Promote Late Chondrogenic Differentiation Markers in the ATDC5 Cell Line.

PubMed

Le, Bach Q; Vasilevich, Aliaksei; Vermeulen, Steven; Hulshof, Frits; Stamatialis, Dimitrios F; van Blitterswijk, Clemens A; de Boer, Jan

2017-05-01

Chemical and mechanical cues are well-established influencers of in vitro chondrogenic differentiation of ATDC5 cells. Here, we investigate the role of topographical cues in this differentiation process, a study not been explored before. Previously, using a library of surface micro-topographies we found some distinct patterns that induced alkaline phosphatase (ALP) production in human mesenchymal stromal cells. ALP is also a marker for hypertrophy, the end stage of chondrogenic differentiation preceding bone formation. Thus, we hypothesized that these patterns could influence end-stage chondrogenic differentiation of ATDC5 cells. In this study, we randomly selected seven topographies among the ALP influencing hits. Cells grown on these surfaces displayed varying nuclear shape and actin filament structure. When stimulated with insulin-transferrin-selenium (ITS) medium, nodule formation occurred and in some cases showed alignment to the topographical patterns. Gene expression analysis of cells growing on topographical surfaces in the presence of ITS medium revealed a downregulation of early markers and upregulation of late markers of chondrogenic differentiation compared to cells grown on a flat surface. In conclusion, we demonstrated that surface topography in addition to other cues can promote hypertrophic differentiation suitable for bone tissue engineering.

Diabetic human adipose tissue-derived mesenchymal stem cells fail to differentiate in functional adipocytes.

PubMed

Barbagallo, Ignazio; Li Volti, Giovanni; Galvano, Fabio; Tettamanti, Guido; Pluchinotta, Francesca R; Bergante, Sonia; Vanella, Luca

2017-05-01

Adipose tissue dysfunction represents a hallmark of diabetic patients and is a consequence of the altered homeostasis of this tissue. Mesenchymal stem cells (MSCs) and their differentiation into adipocytes contribute significantly in maintaining the mass and function of adult adipose tissue. The aim of this study was to evaluate the differentiation of MSCs from patients suffering type 2 diabetes (dASC) and how such process results in hyperplasia or rather a stop of adipocyte turnover resulting in hypertrophy of mature adipocytes. Our results showed that gene profile of all adipogenic markers is not expressed in diabetic cells after differentiation indicating that diabetic cells fail to differentiate into adipocytes. Interestingly, delta like 1, peroxisome proliferator-activated receptor alpha, and interleukin 1β were upregulated whereas Sirtuin 1 and insulin receptor substrate 1 gene expression were found downregulated in dASC compared to cells obtained from healthy subjects. Taken together our data indicate that dASC lose their ability to differentiate into mature and functional adipocytes. In conclusion, our in vitro study is the first to suggest that diabetic patients might develop obesity through a hypertrophy of existing mature adipocytes due to failure turnover of adipose tissue. Impact statement In the present manuscript, we evaluated the differentiative potential of mesenchymal stem cells (MSCs) in adipocytes obtained from healthy and diabetic patients. This finding could be of great potential interest for the field of obesity in order to exploit such results to further understand the pathophysiological processes underlying metabolic syndrome. In particular, inflammation in diabetic patients causes a dysfunction in MSCs differentiation and a decrease in adipocytes turnover leading to insulin resistance.

Second-harmonic generation microscopy used to evaluate the effect of the dimethyl sulfoxide in the cryopreservation process in collagen fibers of differentiated chondrocytes

NASA Astrophysics Data System (ADS)

Andreoli-Risso, M. F.; Duarte, A. S. S.; Ribeiro, T. B.; Bordeaux-Rego, P.; Luzo, A.; Baratti, M. O.; Adur, J.; de Thomaz, A. A.; Pelegati, V. B.; Carvalho, H. F.; Cesar, C. L.; Kharmadayan, P.; Costa, F. F.; Olalla-Saad, S. T.

2012-03-01

Cartilaginous lesions are a significant public health problem and the use of adult stem cells represents a promising therapy for this condition. Cryopreservation confers many advantages for practitioners engaged in cell-based therapies. However, conventional slow freezing has always been associated with damage and mortality due to intracellular ice formation, cryoprotectant toxicity, and dehydration. The aim of this work is to observe the effect of the usual Dimethyl Sulfoxide (DMSO) cryopreservation process on the architecture of the collagen fiber network of chondrogenic cells from mesenchymal stem cells by Second Harmonic Generation (SHG) microscopy. To perform this study we used Mesenchymal Stem Cells (MSC) derived from adipose tissue which presents the capacity to differentiate into other lineages such as osteogenic, adipogenic and chondrogenic lineages. Mesenchymal stem cells obtained after liposuction were isolated digested by collagenase type I and characterization was carried out by differentiation of mesodermic lineages, and flow cytometry using specific markers. The isolated MSCs were cryopreserved by the DMSO technique and the chondrogenic differentiation was carried out using the micromass technique. We then compared the cryopreserved vs non-cryopreserved collagen fibers which are naturally formed during the differentiation process. We observed that noncryopreserved MSCs presented a directional trend in the collagen fibers formed which was absent in the cryopreserved MSCs. We confirmed this trend quantitatively by the aspect ratio obtained by Fast Fourier Transform which was 0.76 for cryopreserved and 0.52 for non-cryopreserved MSCs, a statistical significant difference.

The Activity of Differentiation Factors Induces Apoptosis in Polyomavirus Large T-Expressing Myoblasts

PubMed Central

Fimia, Gian Maria; Gottifredi, Vanesa; Bellei, Barbara; Ricciardi, Maria Rosaria; Tafuri, Agostino; Amati, Paolo; Maione, Rossella

1998-01-01

It is commonly accepted that pathways that regulate proliferation/differentiation processes, if altered in their normal interplay, can lead to the induction of programmed cell death. In a previous work we reported that Polyoma virus Large Tumor antigen (PyLT) interferes with in vitro terminal differentiation of skeletal myoblasts by binding and inactivating the retinoblastoma antioncogene product. This inhibition occurs after the activation of some early steps of the myogenic program. In the present work we report that myoblasts expressing wild-type PyLT, when subjected to differentiation stimuli, undergo cell death and that this cell death can be defined as apoptosis. Apoptosis in PyLT-expressing myoblasts starts after growth factors removal, is promoted by cell confluence, and is temporally correlated with the expression of early markers of myogenic differentiation. The block of the initial events of myogenesis by transforming growth factor β or basic fibroblast growth factor prevents PyLT-induced apoptosis, while the acceleration of this process by the overexpression of the muscle-regulatory factor MyoD further increases cell death in this system. MyoD can induce PyLT-expressing myoblasts to accumulate RB, p21, and muscle- specific genes but is unable to induce G00 arrest. Several markers of different phases of the cell cycle, such as cyclin A, cdk-2, and cdc-2, fail to be down-regulated, indicating the occurrence of cell cycle progression. It has been frequently suggested that apoptosis can result from an unbalanced cell cycle progression in the presence of a contrasting signal, such as growth factor deprivation. Our data involve differentiation pathways, as a further contrasting signal, in the generation of this conflict during myoblast cell apoptosis. PMID:9614186

Fourier transform infrared spectroscopic analysis of cell differentiation

NASA Astrophysics Data System (ADS)

Ishii, Katsunori; Kimura, Akinori; Kushibiki, Toshihiro; Awazu, Kunio

2007-02-01

Stem cells and its differentiations have got a lot of attentions in regenerative medicine. The process of differentiations, the formation of tissues, has become better understood by the study using a lot of cell types progressively. These studies of cells and tissue dynamics at molecular levels are carried out through various approaches like histochemical methods, application of molecular biology and immunology. However, in case of using regenerative sources (cells, tissues and biomaterials etc.) clinically, they are measured and quality-controlled by non-invasive methods from the view point of safety. Recently, the use of Fourier Transform Infrared spectroscopy (FT-IR) has been used to monitor biochemical changes in cells, and has gained considerable importance. The objective of this study is to establish the infrared spectroscopy of cell differentiation as a quality control of cell sources for regenerative medicine. In the present study, as a basic study, we examined the adipose differentiation kinetics of preadipocyte (3T3-L1) and the osteoblast differentiation kinetics of bone marrow mesenchymal stem cells (Kusa-A1) to analyze the infrared absorption spectra. As a result, we achieved to analyze the adipose differentiation kinetics using the infrared absorption peak at 1739 cm-1 derived from ester bonds of triglyceride and osteoblast differentiation kinetics using the infrared absorption peak at 1030 cm-1 derived from phosphate groups of calcium phosphate.

Utilization of nuclear structural proteins for targeted therapy and detection of proliferative and differentiation disorders

DOEpatents

Lelievre, Sophie; Bissell, Mina

2001-01-01

The localization of nuclear apparatus proteins (NUMA) is used to identify tumor cells and different stages in the tumor progression and differentiation processes. There is a characteristic organization of NuMA in tumor cells and in phenotypically normal cells. NuMA distribution patterns are significantly less diffuse in proliferating non-malignant cells compared to malignant cells. The technique encompasses cell immunostaining using a NuMA specific antibody, and microscopic analysis of NuMA distribution within each nucleus.

Identification of Novel Pax8 Targets in FRTL-5 Thyroid Cells by Gene Silencing and Expression Microarray Analysis

PubMed Central

Di Palma, Tina; Conti, Anna; de Cristofaro, Tiziana; Scala, Serena; Nitsch, Lucio; Zannini, Mariastella

2011-01-01

Background The differentiation program of thyroid follicular cells (TFCs), by far the most abundant cell population of the thyroid gland, relies on the interplay between sequence-specific transcription factors and transcriptional coregulators with the basal transcriptional machinery of the cell. However, the molecular mechanisms leading to the fully differentiated thyrocyte are still the object of intense study. The transcription factor Pax8, a member of the Paired-box gene family, has been demonstrated to be a critical regulator required for proper development and differentiation of thyroid follicular cells. Despite being Pax8 well-characterized with respect to its role in regulating genes involved in thyroid differentiation, genomics approaches aiming at the identification of additional Pax8 targets are lacking and the biological pathways controlled by this transcription factor are largely unknown. Methodology/Principal Findings To identify unique downstream targets of Pax8, we investigated the genome-wide effect of Pax8 silencing comparing the transcriptome of silenced versus normal differentiated FRTL-5 thyroid cells. In total, 2815 genes were found modulated 72 h after Pax8 RNAi, induced or repressed. Genes previously reported to be regulated by Pax8 in FRTL-5 cells were confirmed. In addition, novel targets genes involved in functional processes such as DNA replication, anion transport, kinase activity, apoptosis and cellular processes were newly identified. Transcriptome analysis highlighted that Pax8 is a key molecule for thyroid morphogenesis and differentiation. Conclusions/Significance This is the first large-scale study aimed at the identification of new genes regulated by Pax8, a master regulator of thyroid development and differentiation. The biological pathways and target genes controlled by Pax8 will have considerable importance to understand thyroid disease progression as well as to set up novel therapeutic strategies. PMID:21966443

Fate vs choice: the immune system reloaded.

PubMed

Murphy, Kenneth M

2005-01-01

Development can occur by either instructive or stochastic processes. My colleagues and I have studied the contributions of these processes to differentiation of naïve CD4+ T-cells to either a Th1 or Th2 phenotype. Our initial discovery that pathogens in our in vitro priming system led to the development of Th1 cells through the action of interleukin-12 was important evidence of a link between innate and adaptive immunity. Subsequent studies in our laboratory revealed an important role for GATA-3 autoactivation in Th2 development. Other interesting projects that have emerged as a result of our Th cell differentiation studies include understanding the role of the inhibitory immunoreceptor B- and T-lymphocyte attenuator in the immune response, as well as the role of the transcription factor ERM in both T-cells and spermatogenesis. We currently maintain our interests in the Th differentiation field by trying to understand the role of type 1 interferons in Th1 development and the role of alternate promoters for the GATA-3 gene, among other things, but are also actively embarking on studies related to the choice between divergent cell types during embryonic stem cell differentiation.

Plasticity and Maintenance of Hematopoietic Stem Cells During Development

PubMed Central

Kanji, Suman; Pompili, Vincent J.; Das, Hiranmoy

2012-01-01

Maintenance of hematopoietic stem cells (HSCs) pool depends on fine balance between self-renewal and differentiation of HSCs. HSCs normally reside within the bone marrow niche of an adult mammal. The embryonic development of HSCs is a complex process that involves the migration of developing HSCs in multiple anatomical sites. Throughout the process, developing HSCs receive internal (transcriptional program) and external (HSC niche) signals, which direct them to maintain balance between self-renewal and differentiation, also to generate a pool of HSCs. In physiological condition HSCs differentiate into all mature cell types present in the blood. However, in pathological condition they may differentiate into non-hematological cells according to the need of the body. It was shown that HSCs can transdifferentiate into cell types that do not belong to the hematopoietic system suggests a complete paradigm shift of the hierarchical hematopoietic tree. This review describes the developmental origins and regulation of HSCs focusing on developmental signals that induce the adult hematopoietic stem cell program, as these informations are very critical for manipulating conditions for expansion of HSCs in ex vivo condition. This review also states clinical application and related patents using HSC. PMID:21517745

Cell migration under ultrasound irradiations in micrometer scale

NASA Astrophysics Data System (ADS)

Murakami, Shinya; Otsuka, Yo; Oshima, Yusuke; Hikita, Atsuhiko; Mitsui, Toshiyuki

2013-03-01

Cell movements, migration play an important role in many physiological processes including cell proliferation and differentiation. C2C12, a line of mouse myoblasts is known to differentiate into osteoblast under appropriate conditions. Therefore, C2C12 cells can be chosen for the differentiation studies. However, the movement of the C2C12's has not been fully investigated although the movements may provide a better understanding of the healing processes of bone repair, regeneration and differentiation. In addition, low intensity ultrasound has been thought and used to promote bone fracture healing although the microscopic mechanism of this healing is not well understood. As a first step, we have investigated single cell migration of C2C12 under optical microscopy with and without ultrasound irradiations. The ultrasound is irradiated from an apex of a sharp needle. The frequency is 1.5 MHz and the power intensity is near 24 mW/cm2. These values were similar to the ultrasound treatment values. In this conference, we will show the influence of the ultrasound irradiation on the cell movement by plotting the mean squared displacement and the velocity autocorrelation function as a function of time.

Midgut epithelium in molting silkworm: A fine balance among cell growth, differentiation, and survival.

PubMed

Franzetti, Eleonora; Casartelli, Morena; D'Antona, Paola; Montali, Aurora; Romanelli, Davide; Cappellozza, Silvia; Caccia, Silvia; Grimaldi, Annalisa; de Eguileor, Magda; Tettamanti, Gianluca

2016-07-01

The midgut of insects has attracted great attention as a system for studying intestinal stem cells (ISCs) as well as cell death-related processes, such as apoptosis and autophagy. Among insects, Lepidoptera represent a good model to analyze these cells and processes. In particular, larva-larva molting is an interesting developmental phase since the larva must deal with nutrient starvation and its organs are subjected to rearrangements due to proliferation and differentiation events. Several studies have analyzed ISCs in vitro and characterized key factors involved in their division and differentiation during molt. However, in vivo studies performed during larva-larva transition on these cells, and on the whole midgut epithelium, are fragmentary. In the present study, we analyzed the larval midgut epithelium of the silkworm, Bombyx mori, during larva-larva molting, focusing our attention on ISCs. Moreover, we investigated the metabolic changes that occur in the epithelium and evaluated the intervention of autophagy. Our data on ISCs proliferation and differentiation, autophagy activation, and metabolic and functional activities of the midgut cells shed light on the complexity of this organ during the molting phase. Copyright © 2016 Elsevier Ltd. All rights reserved.

Single-cell mechanical phenotype is an intrinsic marker of reprogramming and differentiation along the mouse neural lineage.

PubMed

Urbanska, Marta; Winzi, Maria; Neumann, Katrin; Abuhattum, Shada; Rosendahl, Philipp; Müller, Paul; Taubenberger, Anna; Anastassiadis, Konstantinos; Guck, Jochen

2017-12-01

Cellular reprogramming is a dedifferentiation process during which cells continuously undergo phenotypical remodeling. Although the genetic and biochemical details of this remodeling are fairly well understood, little is known about the change in cell mechanical properties during the process. In this study, we investigated changes in the mechanical phenotype of murine fetal neural progenitor cells (fNPCs) during reprogramming to induced pluripotent stem cells (iPSCs). We find that fNPCs become progressively stiffer en route to pluripotency, and that this stiffening is mirrored by iPSCs becoming more compliant during differentiation towards the neural lineage. Furthermore, we show that the mechanical phenotype of iPSCs is comparable with that of embryonic stem cells. These results suggest that mechanical properties of cells are inherent to their developmental stage. They also reveal that pluripotent cells can differentiate towards a more compliant phenotype, which challenges the view that pluripotent stem cells are less stiff than any cells more advanced developmentally. Finally, our study indicates that the cell mechanical phenotype might be utilized as an inherent biophysical marker of pluripotent stem cells. © 2017. Published by The Company of Biologists Ltd.

Generation of Cardiomyocytes from Pluripotent Stem Cells.

PubMed

Nakahama, Hiroko; Di Pasquale, Elisa

2016-01-01

The advent of pluripotent stem cells (PSCs) enabled a multitude of studies for modeling the development of diseases and testing pharmaceutical therapeutic potential in vitro. These PSCs have been differentiated to multiple cell types to demonstrate its pluripotent potential, including cardiomyocytes (CMs). However, the efficiency and efficacy of differentiation vary greatly between different cell lines and methods. Here, we describe two different methods for acquiring CMs from human pluripotent lines. One method involves the generation of embryoid bodies, which emulates the natural developmental process, while the other method chemically activates the canonical Wnt signaling pathway to induce a monolayer of cardiac differentiation.

Calcium: A novel and efficient inducer of differentiation of adipose-derived stem cells into neuron-like cells.

PubMed

Goudarzi, Farjam; Tayebinia, Heidar; Karimi, Jamshid; Habibitabar, Elahe; Khodadadi, Iraj

2018-06-05

This study comparatively investigated the effectiveness of calcium and other well-known inducers such as isobutylmethylxanthine (IBMX) and insulin in differentiating human adipose-derived stem cells (ADSCs) into neuronal-like cells. ADSCs were immunophenotyped and differentiated into neuron-like cells with different combinations of calcium, IBMX, and insulin. Calcium mobilization across the membrane was determined. Differentiated cells were characterized by cell cycle profiling, staining of Nissl bodies, detecting the gene expression level of markers such as neuronal nuclear antigen (NeuN), microtubule associated protein 2 (MAP2), neuron-specific enolase (NSE), doublecortin, synapsin I, glial fibrillary acidic protein (GFAP), and myelin basic protein (MBP) by quantitative real-time polymerase chain reaction (quantitative real-time polymerase chain reaction (qRT-PCR) and protein level by the immunofluorescence technique. Treatment with Ca + IBMX + Ins induced neuronal appearance and projection of neurite-like processes in the cells, accompanied with inhibition of proliferation and halt in the cell cycle. A significantly higher expression of MBP, GFAP, NeuN, NSE, synapsin 1, doublecortin, and MAP2 was detected in differentiated cells, confirming the advantages of Ca + IBMX + Ins to the other combinations of inducers. Here, we showed an efficient protocol for neuronal differentiation of ADSCs, and calcium fostered differentiation by augmenting the number of neuron-like cells and instantaneous increase in the expression of neuronal markers. © 2018 Wiley Periodicals, Inc.

The effect of low-frequency electromagnetic field on human bone marrow stem/progenitor cell differentiation

PubMed Central

Ross, Christina L.; Siriwardane, Mevan; Almeida-Porada, Graça; Porada, Christopher D.; Brink, Peter; Christ, George J.; Harrison, Benjamin S.

2015-01-01

Human bone marrow stromal cells (hBMSCs, also known as bone marrow-derived mesenchymal stem cells) are a population of progenitor cells that contain a subset of skeletal stem cells (hSSCs), able to recreate cartilage, bone, stroma that supports hematopoiesis and marrow adipocytes. As such, they have become an important resource in developing strategies for regenerative medicine and tissue engineering due to their self-renewal and differentiation capabilities. The differentiation of SSCs/BMSCs is dependent on exposure to biophysical and biochemical stimuli that favor early and rapid activation of the in vivo tissue repair process. Exposure to exogenous stimuli such as an electromagnetic field (EMF) can promote differentiation of SSCs/BMSCs via ion dynamics and small signaling molecules. The plasma membrane is often considered to be the main target for EMF signals and most results point to an effect on the rate of ion or ligand binding due to a receptor site acting as a modulator of signaling cascades. Ion fluxes are closely involved in differentiation control as stem cells move and grow in specific directions to form tissues and organs. EMF affects numerous biological functions such as gene expression, cell fate, and cell differentiation, but will only induce these effects within a certain range of low frequencies as well as low amplitudes. EMF has been reported to be effective in the enhancement of osteogenesis and chondrogenesis of hSSCs/BMSCs with no documented negative effects. Studies show specific EMF frequencies enhance hSSC/BMSC adherence, proliferation, differentiation, and viability, all of which play a key role in the use of hSSCs/BMSCs for tissue engineering. While many EMF studies report significant enhancement of the differentiation process, results differ depending on the experimental and environmental conditions. Here we review how specific EMF parameters (frequency, intensity, and time of exposure) significantly regulate hSSC/BMSC differentiation in vitro. We discuss optimal conditions and parameters for effective hSSC/BMSC differentiation using EMF treatment in an in vivo setting, and how these can be translated to clinical trials. PMID:26042793

Glimpse into Hox and tale regulation of cell differentiation and reprogramming.

PubMed

Cerdá-Esteban, Nuria; Spagnoli, Francesca M

2014-01-01

During embryonic development, cells become gradually restricted in their developmental potential and start elaborating lineage-specific transcriptional networks to ultimately acquire a unique differentiated state. Hox genes play a central role in specifying regional identities, thereby providing the cell with critical information on positional value along its differentiation path. The exquisite DNA-binding specificity of the Hox proteins is frequently dependent upon their interaction with members of the TALE family of homeodomain proteins. In addition to their function as Hox-cofactors, TALE homeoproteins control multiple crucial developmental processes through Hox-independent mechanisms. Here, we will review recent findings on the function of both Hox and TALE proteins in cell differentiation, referring mostly to vertebrate species. In addition, we will discuss the direct implications of this knowledge on cell plasticity and cell reprogramming. Copyright © 2013 Wiley Periodicals, Inc.

Phenotypic Characterization of Retinoic Acid Differentiated SH-SY5Y Cells by Transcriptional Profiling

PubMed Central

Korecka, Joanna A.; van Kesteren, Ronald E.; Blaas, Eva; Spitzer, Sonia O.; Kamstra, Jorke H.; Smit, August B.; Swaab, Dick F.; Verhaagen, Joost; Bossers, Koen

2013-01-01

Multiple genetic and environmental factors play a role in the development and progression of Parkinson’s disease (PD). The main neuropathological hallmark of PD is the degeneration of dopaminergic (DAergic) neurons in the substantia nigra pars compacta. To study genetic and molecular contributors to the disease process, there is a great need for readily accessible cells with prominent DAergic features that can be used for reproducible in vitro cellular screening. Here, we investigated the molecular phenotype of retinoic acid (RA) differentiated SH-SY5Y cells using genome wide transcriptional profiling combined with gene ontology, transcription factor and molecular pathway analysis. We demonstrated that RA induces a general neuronal differentiation program in SH-SY5Y cells and that these cells develop a predominantly mature DAergic-like neurotransmitter phenotype. This phenotype is characterized by increased dopamine levels together with a substantial suppression of other neurotransmitter phenotypes, such as those for noradrenaline, acetylcholine, glutamate, serotonin and histamine. In addition, we show that RA differentiated SH-SY5Y cells express the dopamine and noradrenalin neurotransmitter transporters that are responsible for uptake of MPP(+), a well known DAergic cell toxicant. MPP(+) treatment alters mitochondrial activity according to its proposed cytotoxic effect in DAergic neurons. Taken together, RA differentiated SH-SY5Y cells have a DAergic-like phenotype, and provide a good cellular screening tool to find novel genes or compounds that affect cytotoxic processes that are associated with PD. PMID:23724009

Tracking single hematopoietic stem cells in vivo using high-throughput sequencing in conjunction with viral genetic barcoding

PubMed Central

Lu, Rong; Neff, Norma F.; Quake, Stephen R.; Weissman, Irving L.

2011-01-01

Disentangling cellular heterogeneity is a challenge in many fields, particularly in the stem cell and cancer biology fields. Here, we demonstrate how to combine viral genetic barcoding with high-throughput sequencing to track single cells in a heterogeneous population. We use this technique to track the in vivo differentiation of unitary hematopoietic stem cells (HSCs). The results are consistent with single cell transplantation studies, but require two orders of magnitude fewer mice. In addition to its high throughput, the high sensitivity of the technique allows for a direct examination of the clonality of sparse cell populations such as HSCs. We show how these capabilities offer a clonal perspective of the HSC differentiation process. In particular, our data suggests that HSCs do not equally contribute to blood cells after irradiation-mediated transplantation, and that two distinct HSC differentiation patterns co-exist in the same recipient mouse post irradiation. This technique can be applied to any viral accessible cell type for both in vitro and in vivo processes. PMID:21964413

[Proliferation and osteogenic differentiation of mesenchymal stem cells in hydrogels of human blood plasma].

PubMed

Linero, Itali M; Doncel, Adriana; Chaparro, Orlando

2014-01-01

The use of mesenchymal stem cells in clinical practice has increased considerably in the last decade because they play a supporting role in the processes of tissue repair and regeneration, becoming the main tool of cell therapy for the treatment of diseases functionally affecting bone and cartilage tissue . To evaluate in vitro the proliferative and osteogenic differentiation ability of mesenchymal stem cells derived from human adipose tissue in a blood plasma hydrogel. Mesenchymal stem cells were obtained from human adipose tissue explants and characterized by flow cytometry. Their multipotentiality was demonstrated by their ability to differentiate to adipogenic and osteogenic lineages. Cell proliferation and osteogenic differentiation ability of the cells cultured in blood plasma hydrogels were also evaluated. Mesenchymal stem cells derived from human adipose tissue growing in human blood plasma hydrogels showed a pattern of proliferation similar to that of the cells cultured in monolayer and also maintained their ability to differentiate to osteogenic lineage. Human blood plasma hydrogels are a suitable support for proliferation and osteogenic differentiation of mesenchymal stem cells derived from human adipose tissue and provides a substrate that is autologous, biocompatible, reabsorbable, easy to use, potentially injectable and economic, which could be used as a successful strategy for the management and clinical application of cell therapy in regenerative medicine.

A kinetic investigation of interacting, stimulated T cells identifies conditions for rapid functional enhancement, minimal phenotype differentiation, and improved adoptive cell transfer tumor eradication.

PubMed

Zhou, Jing; Bethune, Michael T; Malkova, Natalia; Sutherland, Alexander M; Comin-Anduix, Begonya; Su, Yapeng; Baltimore, David; Ribas, Antoni; Heath, James R

2018-01-01

For adoptive cell transfer (ACT) immunotherapy of tumor-reactive T cells, an effective therapeutic outcome depends upon cell dose, cell expansion in vivo through a minimally differentiated phenotype, long term persistence, and strong cytolytic effector function. An incomplete understanding of the biological coupling between T cell expansion, differentiation, and response to stimulation hinders the co-optimization of these factors. We report on a biophysical investigation of how the short-term kinetics of T cell functional activation, through molecular stimulation and cell-cell interactions, competes with phenotype differentiation. T cells receive molecular stimulation for a few minutes to a few hours in bulk culture. Following this priming period, the cells are then analyzed at the transcriptional level, or isolated as single cells, with continuing molecular stimulation, within microchambers for analysis via 11-plex secreted protein assays. We resolve a rapid feedback mechanism, promoted by T cell-T cell contact interactions, which strongly amplifies T cell functional performance while yielding only minimal phenotype differentiation. When tested in mouse models of ACT, optimally primed T cells lead to complete tumor eradication. A similar kinetic process is identified in CD8+ and CD4+ T cells collected from a patient with metastatic melanoma.

Effect of β-hydroxy-β-methylbutyrate on miRNA expression in differentiating equine satellite cells exposed to hydrogen peroxide.

PubMed

Chodkowska, Karolina A; Ciecierska, Anna; Majchrzak, Kinga; Ostaszewski, Piotr; Sadkowski, Tomasz

2018-01-01

Skeletal muscle injury activates satellite cells to initiate processes of proliferation, differentiation, and hypertrophy in order to regenerate muscle fibers. The number of microRNAs and their target genes are engaged in satellite cell activation. β-Hydroxy-β-methylbutyrate (HMB) is known to prevent exercise-induced muscle damage. The purpose of this study was to evaluate the effect of HMB on miRNA and relevant target gene expression in differentiating equine satellite cells exposed to H 2 O 2 . We hypothesized that HMB may regulate satellite cell activity, proliferation, and differentiation, hence attenuate the pathological processes induced during an in vitro model of H 2 O 2 -related injury by changing the expression of miRNAs. Equine satellite cells (ESC) were isolated from the samples of skeletal muscle collected from young horses. ESC were treated with HMB (24 h) and then exposed to H 2 O 2 (1 h). For the microRNA and gene expression assessment microarrays, technique was used. Identified miRNAs and genes were validated using real-time qPCR. Cell viability, oxidative stress, and cell damage were measured using colorimetric method and flow cytometry. Analysis of miRNA and gene profile in differentiating ESC pre-incubated with HMB and then exposed to H 2 O 2 revealed difference in the expression of 27 miRNAs and 4740 genes, of which 344 were potential target genes for identified miRNAs. Special attention was focused on differentially expressed miRNAs and their target genes involved in processes related to skeletal muscle injury. Western blot analysis showed protein protection in HMB-pre-treated group compared to control. The viability test confirmed that HMB enhanced cell survival after the hydrogen peroxide exposition. Our results suggest that ESC pre-incubated with HMB and exposed to H 2 O 2 could affect expression on miRNA levels responsible for skeletal muscle development, cell proliferation and differentiation, and activation of tissue repair after injury. Enrichment analyses for targeted genes revealed that a large group of genes was associated with the regulation of signaling pathways crucial for muscle tissue development, protein metabolism, muscle injury, and regeneration, as well as with oxidative stress response.

Process to evaluate hematological parameters that reflex to manual differential cell counts in a pediatric institution.

PubMed

Guarner, Jeannette; Atuan, Maria Ana; Nix, Barbara; Mishak, Christopher; Vejjajiva, Connie; Curtis, Cheri; Park, Sunita; Mullins, Richard

2010-01-01

Each institution sets specific parameters obtained by automated hematology analyzers to trigger manual counts. We designed a process to decrease the number of manual differential cell counts without impacting patient care. We selected new criteria that prompt manual counts and studied the impact these changes had in 2 days of work and in samples of patients with newly diagnosed leukemia, sickle cell disease, and presence of left shift. By using fewer parameters and expanding our ranges we decreased the number of manual counts by 20%. The parameters that prompted manual counts most frequently were the presence of blast flags and nucleated red blood cells, 2 parameters that were not changed. The parameters that accounted for a decrease in the number of manual counts were the white blood cell count and large unstained cells. Eight of 32 patients with newly diagnosed leukemia did not show blast flags; however, other parameters triggered manual counts. In 47 patients with sickle cell disease, nucleated red cells and red cell variability prompted manual review. Bands were observed in 18% of the specimens and 4% would not have been counted manually with the new criteria, for the latter the mean band count was 2.6%. The process we followed to evaluate hematological parameters that reflex to manual differential cell counts increased efficiency without compromising patient care in our hospital system.

Knocking down of heat-shock protein 27 directs differentiation of functional glutamatergic neurons from placenta-derived multipotent cells

PubMed Central

Cheng, Yu-Che; Huang, Chi-Jung; Lee, Yih-Jing; Tien, Lu-Tai; Ku, Wei-Chi; Chien, Raymond; Lee, Fa-Kung; Chien, Chih-Cheng

2016-01-01

This study presents human placenta-derived multipotent cells (PDMCs) as a source from which functional glutamatergic neurons can be derived. We found that the small heat-shock protein 27 (HSP27) was downregulated during the neuronal differentiation process. The in vivo temporal and spatial profiles of HSP27 expression were determined and showed inverted distributions with neuronal proteins during mouse embryonic development. Overexpression of HSP27 in stem cells led to the arrest of neuronal differentiation; however, the knockdown of HSP27 yielded a substantially enhanced ability of PDMCs to differentiate into neurons. These neurons formed synaptic networks and showed positive staining for multiple neuronal markers. Additionally, cellular phenomena including the absence of apoptosis and rare proliferation in HSP27-silenced PDMCs, combined with molecular events such as cleaved caspase-3 and the loss of stemness with cleaved Nanog, indicated that HSP27 is located upstream of neuronal differentiation and constrains that process. Furthermore, the induced neurons showed increasing intracellular calcium concentrations upon glutamate treatment. These differentiated cells co-expressed the N-methyl-D-aspartate receptor, vesicular glutamate transporter, and synaptosomal-associated protein 25 but did not show expression of tyrosine hydroxylase, choline acetyltransferase or glutamate decarboxylase 67. Therefore, we concluded that HSP27-silenced PDMCs differentiated into neurons possessing the characteristics of functional glutamatergic neurons. PMID:27444754

A kinetic investigation of interacting, stimulated T cells identifies conditions for rapid functional enhancement, minimal phenotype differentiation, and improved adoptive cell transfer tumor eradication

PubMed Central

Zhou, Jing; Bethune, Michael T.; Malkova, Natalia; Sutherland, Alexander M.; Comin-Anduix, Begonya; Su, Yapeng; Baltimore, David; Ribas, Antoni

2018-01-01

For adoptive cell transfer (ACT) immunotherapy of tumor-reactive T cells, an effective therapeutic outcome depends upon cell dose, cell expansion in vivo through a minimally differentiated phenotype, long term persistence, and strong cytolytic effector function. An incomplete understanding of the biological coupling between T cell expansion, differentiation, and response to stimulation hinders the co-optimization of these factors. We report on a biophysical investigation of how the short-term kinetics of T cell functional activation, through molecular stimulation and cell-cell interactions, competes with phenotype differentiation. T cells receive molecular stimulation for a few minutes to a few hours in bulk culture. Following this priming period, the cells are then analyzed at the transcriptional level, or isolated as single cells, with continuing molecular stimulation, within microchambers for analysis via 11-plex secreted protein assays. We resolve a rapid feedback mechanism, promoted by T cell—T cell contact interactions, which strongly amplifies T cell functional performance while yielding only minimal phenotype differentiation. When tested in mouse models of ACT, optimally primed T cells lead to complete tumor eradication. A similar kinetic process is identified in CD8+ and CD4+ T cells collected from a patient with metastatic melanoma. PMID:29360859

Identification of transcription coactivator OCA-B-dependent genes involved in antigen-dependent B cell differentiation by cDNA array analyses.

PubMed

Kim, Unkyu; Siegel, Rachael; Ren, Xiaodi; Gunther, Cary S; Gaasterland, Terry; Roeder, Robert G

2003-07-22

The tissue-specific transcriptional coactivator OCA-B is required for antigen-dependent B cell differentiation events, including germinal center formation. However, the identity of OCA-B target genes involved in this process is unknown. This study has used large-scale cDNA arrays to monitor changes in gene expression patterns that accompany mature B cell differentiation. B cell receptor ligation alone induces many genes involved in B cell expansion, whereas B cell receptor and helper T cell costimulation induce genes associated with B cell effector function. OCA-B expression is induced by both B cell receptor ligation alone and helper T cell costimulation, suggesting that OCA-B is involved in B cell expansion as well as B cell function. Accordingly, several genes involved in cell proliferation and signaling, such as Lck, Kcnn4, Cdc37, cyclin D3, B4galt1, and Ms4a11, have been identified as OCA-B-dependent genes. Further studies on the roles played by these genes in B cells will contribute to an understanding of B cell differentiation.

Extracellular Matrix Degradation and Remodeling in Development and Disease

PubMed Central

Lu, Pengfei; Takai, Ken; Weaver, Valerie M.; Werb, Zena

2011-01-01

The extracellular matrix (ECM) serves diverse functions and is a major component of the cellular microenvironment. The ECM is a highly dynamic structure, constantly undergoing a remodeling process where ECM components are deposited, degraded, or otherwise modified. ECM dynamics are indispensible during restructuring of tissue architecture. ECM remodeling is an important mechanism whereby cell differentiation can be regulated, including processes such as the establishment and maintenance of stem cell niches, branching morphogenesis, angiogenesis, bone remodeling, and wound repair. In contrast, abnormal ECM dynamics lead to deregulated cell proliferation and invasion, failure of cell death, and loss of cell differentiation, resulting in congenital defects and pathological processes including tissue fibrosis and cancer. Understanding the mechanisms of ECM remodeling and its regulation, therefore, is essential for developing new therapeutic interventions for diseases and novel strategies for tissue engineering and regenerative medicine. PMID:21917992

Transcriptional and post-transcriptional down-regulation of cyclin D1 contributes to C6 glioma cell differentiation induced by forskolin.

PubMed

He, Songmin; Zhu, Wenbo; Zhou, Yuxi; Huang, Yijun; Ou, Yanqiu; Li, Yan; Yan, Guangmei

2011-09-01

Malignant gliomas are the most common and lethal intracranial tumors, and differentiation therapy shows great potential to be a promising candidate for their treatment. Here, we have elaborated that a PKA activator, forskolin, represses cell growth via cell cycle arrest in the G0/G1 phase and induces cell differentiation characteristic with elongated processes and restoration of GFAP expression. In mechanisms, we verified that forskolin significantly diminishes the mRNA and protein level of a key cell cycle regulator cyclin D1, and maintenance of low cyclin D1 expression level was required for forskolin-induced proliferation inhibition and differentiation by gain and loss of function approaches. In addition, that forskolin down-regulated the cyclin D1 by proteolytic (post-transcriptional) mechanisms was dependent on GSK-3β activation at Ser9. The pro-differentiation activity of forskolin and related molecular mechanisms imply that forskolin can be developed into a candidate for the future in differentiation therapy of glioma, and cyclin D1 is a promising target for pro-differentiation strategy. Copyright © 2011 Wiley-Liss, Inc.

MiR-144-3p regulates osteogenic differentiation and proliferation of murine mesenchymal stem cells by specifically targeting Smad4.

PubMed

Huang, Cong; Geng, Junnan; Wei, Xiajie; Zhang, Ruirui; Jiang, Siwen

2016-03-01

Despite extensive research on osteoblast differentiation and proliferation in mesenchymal stem cells (MSCs), the accurate mechanism remains to be further elucidated. MicroRNAs have been reported to be key regulators of osteoblast differentiation and proliferation. Here, we found that miR-144-3p is down-regulated during osteoblast differentiation of C3H10T1/2 cells. Overexpression of miR-144-3p inhibited osteogenic differentiation, whereas inhibition of miR-144-3p reversed this process. Furthermore, miR-144-3p inhibited the proliferation of C3H10T1/2 cells by arresting cells at the G0/G1 phase. Results from bioinformatics analysis, luciferase assay and western blotting demonstrated that miR-144-3p directly targeted Smad4. Additionally, Smad4 knockdown blocks the effects of miR-144-3p inhibitor. Therefore, we conclude that miR-144-3p negatively regulates osteogenic differentiation and proliferation of C3H10T1/2 cells by targeting Smad4. © 2016 Federation of European Biochemical Societies.

Fluorescence lifetime imaging of lipids during 3T3-L1 cell differentiation

NASA Astrophysics Data System (ADS)

Song, Young Sik; Won, Young Jae; Lee, Sang-Hak; Kim, Dug Young

2014-03-01

Obesity is becoming a big health problem in these days. Since increased body weight is due to increased number and size of the triglyceride-storing adipocytes, many researchers are working on differentiation conditions and processes of adipocytes. Adipocytes also work as regulators of whole-body energy homeostasis by secreting several proteins that regulate processes as diverse as haemostasis, blood pressure, immune function, angiogenesis and energy balance. 3T3-L1 cells are widely used cell line for studying adipogenesis because it can differentiate into an adipocyte-like phenotype under appropriate conditions. In this paper, we propose an effective fluorescence lifetime imaging technique which can easily distinguish lipids in membrane and those in lipid droplets. Nile red dyes are attached to lipids in 3T3-L1 cells. Fluorescence lifetime images were taken for 2 week during differentiation procedure of 3T3-L1 cells into adipocytes. We used 488 nm pulsed laser with 5MHz repetition rate and emission wavelength is 520 nm of Nile Red fluorescent dye. Results clearly show that the lifetime of Nile red in lipid droplets are smaller than those in cell membrane. Our results suggest that fluorescence lifetime imaging can be a very powerful tool to monitor lipid droplet formation in adipocytes from 3T3-L1 cells.

Retracted article: In vitro derivation of mammalian germ cells from stem cells and their potential therapeutic application.

PubMed

Saito, Shigeo; Lin, Ying-Chu; Murayama, Yoshinobu; Nakamura, Yukio; Eckner, Richard; Niemann, Heiner; Yokoyama, Kazunari K

2015-12-01

Pluripotent stem cells (PSCs) are a unique type of cells because they exhibit the characteristics of self-renewal and pluripotency. PSCs may be induced to differentiate into any cell type, even male and female germ cells, suggesting their potential as novel cell-based therapeutic treatment for infertility problems. Spermatogenesis is an intricate biological process that starts from self-renewal of spermatogonial stem cells (SSCs) and leads to differentiated haploid spermatozoa. Errors at any stage in spermatogenesis may result in male infertility. During the past decade, much progress has been made in the derivation of male germ cells from various types of progenitor stem cells. Currently, there are two main approaches for the derivation of functional germ cells from PSCs, either the induction of in vitro differentiation to produce haploid cell products, or combination of in vitro differentiation and in vivo transplantation. The production of mature and fertile spermatozoa from stem cells might provide an unlimited source of autologous gametes for treatment of male infertility. Here, we discuss the current state of the art regarding the differentiation potential of SSCs, embryonic stem cells, and induced pluripotent stem cells to produce functional male germ cells. We also discuss the possible use of livestock-derived PSCs as a novel option for animal reproduction and infertility treatment.

Performance evaluation of the Abbott CELL-DYN Ruby and the Sysmex XT-2000i haematology analysers.

PubMed

Leers, M P G; Goertz, H; Feller, A; Hoffmann, J J M L

2011-02-01

Two mid-range haematology analysers (Abbott CELL-DYN Ruby and Sysmex XT-2000i) were evaluated to determine their analytical performance and workflow efficiency in the haematology laboratory. In total 418 samples were processed for determining equivalence of complete blood count (CBC) measurements, and 100 for reticulocyte comparison. Blood smears served for assessing the agreement of the differential counts. Inter-instrument agreement for most parameters was good although small numbers of discrepancies were observed. Systematic biases were found for mean cell volume, reticulocytes, platelets and mean platelet volume. CELL-DYN Ruby WBC differentials were obtained with all samples while the XT-2000i suppressed differentials partially or completely in 13 samples (3.1%). WBC subpopulation counts were otherwise in good agreement with no major outliers. Following first-pass CBC/differential analysis, 88 (21%) of XT-2000i samples required further analyser processing compared to 18 (4.3%) for the CELL-DYN Ruby. Smear referrals for suspected WBC/nucleated red blood cells and platelet abnormalities were indicated for 106 (25.4%) and 95 (22.7%) of the XT-2000i and CELL-DYN Ruby samples respectively. Flagging efficiencies for both analysers were found to be similar. The Sysmex XT-2000i and Abbott CELL-DYN Ruby analysers have broadly comparable analytical performance, but the CELL-DYN Ruby showed superior first-pass efficiency. © 2010 Blackwell Publishing Ltd.

Existence and stability of periodic solutions of an impulsive differential equation and application to CD8 T-cell differentiation.

PubMed

Girel, Simon; Crauste, Fabien

2018-06-01

Unequal partitioning of the molecular content at cell division has been shown to be a source of heterogeneity in a cell population. We propose to model this phenomenon with the help of a scalar, nonlinear impulsive differential equation (IDE). To study the effect of molecular partitioning at cell division on the effector/memory cell-fate decision in a CD8 T-cell lineage, we study an IDE describing the concentration of the protein Tbet in a CD8 T-cell, where impulses are associated to cell division. We discuss how the degree of asymmetry of molecular partitioning can affect the process of cell differentiation and the phenotypical heterogeneity of a cell population. We show that a moderate degree of asymmetry is necessary and sufficient to observe irreversible differentiation. We consider, in a second part, a general autonomous IDE with fixed times of impulse and a specific form of impulse function. We establish properties of the solutions of that equation, most of them obtained under the hypothesis that impulses occur periodically. In particular, we show how to investigate the existence of periodic solutions and their stability by studying the flow of an autonomous differential equation. Then we apply those properties to prove the results presented in the first part.

Pluripotency, Differentiation, and Reprogramming: A Gene Expression Dynamics Model with Epigenetic Feedback Regulation

PubMed Central

Miyamoto, Tadashi; Furusawa, Chikara; Kaneko, Kunihiko

2015-01-01

Embryonic stem cells exhibit pluripotency: they can differentiate into all types of somatic cells. Pluripotent genes such as Oct4 and Nanog are activated in the pluripotent state, and their expression decreases during cell differentiation. Inversely, expression of differentiation genes such as Gata6 and Gata4 is promoted during differentiation. The gene regulatory network controlling the expression of these genes has been described, and slower-scale epigenetic modifications have been uncovered. Although the differentiation of pluripotent stem cells is normally irreversible, reprogramming of cells can be experimentally manipulated to regain pluripotency via overexpression of certain genes. Despite these experimental advances, the dynamics and mechanisms of differentiation and reprogramming are not yet fully understood. Based on recent experimental findings, we constructed a simple gene regulatory network including pluripotent and differentiation genes, and we demonstrated the existence of pluripotent and differentiated states from the resultant dynamical-systems model. Two differentiation mechanisms, interaction-induced switching from an expression oscillatory state and noise-assisted transition between bistable stationary states, were tested in the model. The former was found to be relevant to the differentiation process. We also introduced variables representing epigenetic modifications, which controlled the threshold for gene expression. By assuming positive feedback between expression levels and the epigenetic variables, we observed differentiation in expression dynamics. Additionally, with numerical reprogramming experiments for differentiated cells, we showed that pluripotency was recovered in cells by imposing overexpression of two pluripotent genes and external factors to control expression of differentiation genes. Interestingly, these factors were consistent with the four Yamanaka factors, Oct4, Sox2, Klf4, and Myc, which were necessary for the establishment of induced pluripotent stem cells. These results, based on a gene regulatory network and expression dynamics, contribute to our wider understanding of pluripotency, differentiation, and reprogramming of cells, and they provide a fresh viewpoint on robustness and control during development. PMID:26308610

Microtubule-regulating proteins and cAMP-dependent signaling in neuroblastoma differentiation.

PubMed

Muñoz-Llancao, Pablo; de Gregorio, Cristian; Las Heras, Macarena; Meinohl, Christopher; Noorman, Kevin; Boddeke, Erik; Cheng, Xiaodong; Lezoualc'h, Frank; Schmidt, Martina; Gonzalez-Billault, Christian

2017-03-01

Neurons are highly differentiated cells responsible for the conduction and transmission of information in the nervous system. The proper function of a neuron relies on the compartmentalization of their intracellular domains. Differentiated neuroblastoma cells have been extensively used to study and understand the physiology and cell biology of neuronal cells. Here, we show that differentiation of N1E-115 neuroblastoma cells is more pronounced upon exposure of a chemical analog of cyclic AMP (cAMP), db-cAMP. We next analysed the expression of key microtubule-regulating proteins in differentiated cells and the expression and activation of key cAMP players such as EPAC, PKA and AKAP79/150. Most of the microtubule-promoting factors were up regulated during differentiation of N1E-115 cells, while microtubule-destabilizing proteins were down regulated. We observed an increase in tubulin post-translational modifications related to microtubule stability. As expected, db-cAMP increased PKA- and EPAC-dependent signalling. Consistently, pharmacological modulation of EPAC activity instructed cell differentiation, number of neurites, and neurite length in N1E-115 cells. Moreover, disruption of the PKA-AKAP interaction reduced these morphometric parameters. Interestingly, PKA and EPAC act synergistically to induce neuronal differentiation in N1E-115. Altogether these results show that the changes observed in the differentiation of N1E-115 cells proceed by regulating several microtubule-stabilizing factors, and the acquisition of a neuronal phenotype is a process involving concerted although independent functions of EPAC and PKA. © 2017 Wiley Periodicals, Inc.

Role of the Cellular Prion Protein in Oligodendrocyte Precursor Cell Proliferation and Differentiation in the Developing and Adult Mouse CNS

PubMed Central

Bribián, Ana; Gavín, Rosalina; Reina, Manuel; García-Verdugo, José Manuel; Torres, Juan María; de Castro, Fernando; del Río, José Antonio

2012-01-01

There are numerous studies describing the signaling mechanisms that mediate oligodendrocyte precursor cell (OPC) proliferation and differentiation, although the contribution of the cellular prion protein (PrPc) to this process remains unclear. PrPc is a glycosyl-phosphatidylinositol (GPI)-anchored glycoprotein involved in diverse cellular processes during the development and maturation of the mammalian central nervous system (CNS). Here we describe how PrPc influences oligodendrocyte proliferation in the developing and adult CNS. OPCs that lack PrPc proliferate more vigorously at the expense of a delay in differentiation, which correlates with changes in the expression of oligodendrocyte lineage markers. In addition, numerous NG2-positive cells were observed in cortical regions of adult PrPc knockout mice, although no significant changes in myelination can be seen, probably due to the death of surplus cells. PMID:22529900

The Effect of Spaceflight on Cartilage Cell Cycle and Differentiation

NASA Technical Reports Server (NTRS)

Doty, Stephen B.; Stiner, Dalina; Telford, William G.

2000-01-01

In vivo studies have shown that spaceflight results in loss of bone and muscle. In an effort to understand the mechanisms of these changes, cell cultures of cartilage, bone and muscle have been subjected to spaceflight to study the microgravity effects on differentiated cells. However it now seems possible that the cell differentiation process itself may be the event(s) most affected by spaceflight. For example, osteoblast-like cells have been shown to have reduced cellular activity in microgravity due to an underdifferentiated state (Carmeliet, et al, 1997). And reduced human lymphocyte growth in spaceflight was related to increased apoptosis (Lewis, et al, 1998). Which brings us to the question of whether reduced cellular activity in space is due to an effect on the differentiated cell, an effect on the cell cycle and cell proliferation, or an effect on cell death. This question has not been specifically addressed on previous flights and was the question behind die present study.

Multi-effects of Resveratrol on stem cell characteristics: Effective dose, time, cell culture conditions and cell type-specific responses of stem cells to Resveratrol.

PubMed

Safaeinejad, Zahra; Kazeminasab, Fatemeh; Kiani-Esfahani, Abbas; Ghaedi, Kamran; Nasr-Esfahani, Mohammad Hossein

2018-06-18

Stem cells which defined by dual features of self-renewal and differentiation potential provide a unique source for repairing damaged tissues to treat a wide spectrum of diseases and injuries. Several recent studies suggest that Resveratrol (RSV), a natural polyphenol component, possesses the ability to improve either culture conditions of stem cells or their target differentiation in culture. This review covers the literature that deals with the effects of RSV and its underlying mechanisms on survival, self-renewal and lineage commitment of various stem cells. Concentration of RSV and duration of treatment with this component could exert differential effects on cellular differentiation processes and cell fate. Therefore, RSV could be accounted as an effective small molecule for a variety of cell therapies which should be implemented by a special care considering, effective concentration and duration of exposure. Copyright © 2018. Published by Elsevier Masson SAS.

PKCε as a novel promoter of skeletal muscle differentiation and regeneration.

PubMed

Di Marcantonio, D; Galli, D; Carubbi, C; Gobbi, G; Queirolo, V; Martini, S; Merighi, S; Vaccarezza, M; Maffulli, N; Sykes, S M; Vitale, M; Mirandola, P

2015-11-15

Satellite cells are muscle resident stem cells and are responsible for muscle regeneration. In this study we investigate the involvement of PKCε during muscle stem cell differentiation in vitro and in vivo. Here, we describe the identification of a previously unrecognized role for the PKCε-HMGA1 signaling axis in myoblast differentiation and regeneration processes. PKCε expression was modulated in the C2C12 cell line and primary murine satellite cells in vitro, as well as in an in vivo model of muscle regeneration. Immunohistochemistry and immunofluorescence, RT-PCR and shRNA silencing techniques were used to determine the role of PKCε and HMGA1 in myogenic differentiation. PKCε expression increases and subsequently re-localizes to the nucleus during skeletal muscle cell differentiation. In the nucleus, PKCε blocks Hmga1 expression to promote Myogenin and Mrf4 accumulation and myoblast formation. Following in vivo muscle injury, PKCε accumulates in regenerating, centrally-nucleated myofibers. Pharmacological inhibition of PKCε impairs the expression of two crucial markers of muscle differentiation, namely MyoD and Myogenin, during injury induced muscle regeneration. This work identifies the PKCε-HMGA1 signaling axis as a positive regulator of skeletal muscle differentiation. Copyright © 2015 Elsevier Inc. All rights reserved.

Microarray Detection Call Methodology as a Means to Identify and Compare Transcripts Expressed within Syncytial Cells from Soybean (Glycine max) Roots Undergoing Resistant and Susceptible Reactions to the Soybean Cyst Nematode (Heterodera glycines)

PubMed Central

Klink, Vincent P.; Overall, Christopher C.; Alkharouf, Nadim W.; MacDonald, Margaret H.; Matthews, Benjamin F.

2010-01-01

Background. A comparative microarray investigation was done using detection call methodology (DCM) and differential expression analyses. The goal was to identify genes found in specific cell populations that were eliminated by differential expression analysis due to the nature of differential expression methods. Laser capture microdissection (LCM) was used to isolate nearly homogeneous populations of plant root cells. Results. The analyses identified the presence of 13,291 transcripts between the 4 different sample types. The transcripts filtered down into a total of 6,267 that were detected as being present in one or more sample types. A comparative analysis of DCM and differential expression methods showed a group of genes that were not differentially expressed, but were expressed at detectable amounts within specific cell types. Conclusion. The DCM has identified patterns of gene expression not shown by differential expression analyses. DCM has identified genes that are possibly cell-type specific and/or involved in important aspects of plant nematode interactions during the resistance response, revealing the uniqueness of a particular cell population at a particular point during its differentiation process. PMID:20508855

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

Derivation and characterization of gut-like structures from embryonic stem cells.

PubMed

Yamada, Takatsugu; Nakajima, Yoshiyuki

2006-01-01

Embryonic stem (ES) cells have a pluripotent ability to differentiate into a variety of cell lineages of all three embryonic germ layers in vitro. The hanging drop culture of ES cell suspension in the absence of leukemia inhibitory factor induces aggregation and differentiation of the cells into simple or cystic embryoid bodies (EBs). After 6 d of hanging drop culture, the resulting EBs are plated onto plastic dishes for the outgrowth culture. At d 21 after outgrowth culture, cell populations of EBs can give rise to three-dimensional gut-like structures that exhibit spontaneous contraction and highly coordinated peristalsis. The gut-like structures have large lumens surrounded by three layers: epithelium, lamina propria, and muscularis. Ganglia are scattered along the periphery, and interstitial cells of Cajal are distributed among the smooth muscle cells. The fundamental process of formation of the in vitro organized gut-like structures is similar to embryonic gastrointestinal development in vivo. The EBs at the 6-d egg-cylinder stage may have the potential to regulate developmental programs associated with cell lineage commitment and provide an appropriate microenvironment to differentiate ES cells into enteric derivatives of all three embryonic germ layers and reproduce the gut organization process in vitro.

Ectopic Activation of Wnt/β-Catenin Signaling in Lens Fiber Cells Results in Cataract Formation and Aberrant Fiber Cell Differentiation

PubMed Central

Antosova, Barbora; Smolikova, Jana; Borkovcova, Romana; Strnad, Hynek; Lachova, Jitka; Machon, Ondrej; Kozmik, Zbynek

2013-01-01

The Wnt/β-catenin signaling pathway controls many processes during development, including cell proliferation, cell differentiation and tissue homeostasis, and its aberrant regulation has been linked to various pathologies. In this study we investigated the effect of ectopic activation of Wnt/β-catenin signaling during lens fiber cell differentiation. To activate Wnt/β-catenin signaling in lens fiber cells, the transgenic mouse referred to as αA-CLEF was generated, in which the transactivation domain of β-catenin was fused to the DNA-binding protein LEF1, and expression of the transgene was controlled by αA-crystallin promoter. Constitutive activation of Wnt/β-catenin signaling in lens fiber cells of αA-CLEF mice resulted in abnormal and delayed fiber cell differentiation. Moreover, adult αA-CLEF mice developed cataract, microphthalmia and manifested downregulated levels of γ-crystallins in lenses. We provide evidence of aberrant expression of cell cycle regulators in embryonic lenses of αA-CLEF transgenic mice resulting in the delay in cell cycle exit and in the shift of fiber cell differentiation to the central fiber cell compartment. Our results indicate that precise regulation of the Wnt/β-catenin signaling activity during later stages of lens development is essential for proper lens fiber cell differentiation and lens transparency. PMID:24205179

The AP-1 transcription factor Fra1 inhibits follicular B cell differentiation into plasma cells

PubMed Central

Grötsch, Bettina; Brachs, Sebastian; Lang, Christiane; Luther, Julia; Derer, Anja; Schlötzer-Schrehardt, Ursula; Bozec, Aline; Fillatreau, Simon; Berberich, Ingolf; Hobeika, Elias; Reth, Michael; Wagner, Erwin F.; Schett, Georg

2014-01-01

The cornerstone of humoral immunity is the differentiation of B cells into antibody-secreting plasma cells. This process is tightly controlled by a regulatory gene network centered on the transcriptional repressor B lymphocyte–induced maturation protein 1 (Blimp1). Proliferation of activated B cells is required to foster Blimp1 expression but needs to be terminated to avoid overshooting immune reactions. Activator protein 1 (AP-1) transcription factors become quickly up-regulated upon B cell activation. We demonstrate that Fra1, a Fos member of AP-1, enhances activation-induced cell death upon induction in activated B cells. Moreover, mice with B cell–specific deletion of Fra1 show enhanced plasma cell differentiation and exacerbated antibody responses. In contrast, transgenic overexpression of Fra1 blocks plasma cell differentiation and immunoglobulin production, which cannot be rescued by Bcl2. On the molecular level, Fra1 represses Blimp1 expression and interferes with binding of the activating AP-1 member c-Fos to the Blimp1 promoter. Conversely, overexpression of c-Fos in Fra1 transgenic B cells releases Blimp1 repression. As Fra1 lacks transcriptional transactivation domains, we propose that Fra1 inhibits Blimp1 expression and negatively controls plasma cell differentiation through binding to the Blimp1 promoter. In summary, we demonstrate that Fra1 negatively controls plasma cell differentiation by repressing Blimp1 expression. PMID:25288397

Involvement of the histamine H{sub 4} receptor in clozapine-induced hematopoietic toxicity: Vulnerability under granulocytic differentiation of HL-60 cells

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

Goto, Aya; Mouri, Akihiro; Nagai, Tomoko

Clozapine is an effective antipsychotic for treatment-resistant schizophrenia, but can cause fatal hematopoietic toxicity as agranulocytosis. To elucidate the mechanism of hematopoietic toxicity induced by clozapine, we developed an in vitro assay system using HL-60 cells, and investigated the effect on hematopoiesis. HL-60 cells were differentiated by all-trans retinoic acid (ATRA) into three states according to the following hematopoietic process: undifferentiated HL-60 cells, those undergoing granulocytic ATRA-differentiation, and ATRA-differentiated granulocytic cells. Hematopoietic toxicity was evaluated by analyzing cell survival, cell proliferation, granulocytic differentiation, apoptosis, and necrosis. In undifferentiated HL-60 cells and ATRA-differentiated granulocytic cells, both clozapine (50 and 100 μM)more » and doxorubicin (0.2 µM) decreased the cell survival rate, but olanzapine (1–100 µM) did not. Under granulocytic differentiation for 5 days, clozapine, even at a concentration of 25 μM, decreased survival without affecting granulocytic differentiation, increased caspase activity, and caused apoptosis rather than necrosis. Histamine H{sub 4} receptor mRNA was expressed in HL-60 cells, whereas the expression decreased under granulocytic ATRA-differentiation little by little. Both thioperamide, a histamine H{sub 4} receptor antagonist, and DEVD-FMK, a caspase-3 inhibitor, exerted protection against clozapine-induced survival rate reduction, but not of live cell counts. 4-Methylhistamine, a histamine H{sub 4} receptor agonist, decreased the survival rate and live cell counts, as did clozapine. HL-60 cells under granulocytic differentiation are vulnerable under in vitro assay conditions to hematopoietic toxicity induced by clozapine. Histamine H{sub 4} receptor is involved in the development of clozapine-induced hematopoietic toxicity through apoptosis, and may be a potential target for preventing its occurrence through granulocytic differentiation. - Highlights: • HL-60 cells under granulocytic differentiation were vulnerable for clozapine. • HL-60 cells would be in vitro assay systems for hematopoietic toxicity by clozapine. • Histamine H{sub 4} receptor was involved in hematopoietic toxicity with apoptosis. • Histamine H{sub 4} receptor may be therapeutic target to prevent hematopoietic toxicity.« less

Process-Based Expansion and Neural Differentiation of Human Pluripotent Stem Cells for Transplantation and Disease Modeling

PubMed Central

Stover, Alexander E.; Brick, David J.; Nethercott, Hubert E.; Banuelos, Maria G.; Sun, Lei; O’Dowd, Diane K.; Schwartz, Philip H.

2014-01-01

Robust strategies for developing patient-specific, human, induced pluripotent stem cell (iPSC)-based therapies of the brain require an ability to derive large numbers of highly defined neural cells. Recent progress in iPSC culture techniques includes partial-to-complete elimination of feeder layers, use of defined media, and single-cell passaging. However, these techniques still require embryoid body formation or coculture for differentiation into neural stem cells (NSCs). In addition, none of the published methodologies has employed all of the advances in a single culture system. Here we describe a reliable method for long-term, single-cell passaging of PSCs using a feeder-free, defined culture system that produces confluent, adherent PSCs that can be differentiated into NSCs. To provide a basis for robust quality control, we have devised a system of cellular nomenclature that describes an accurate genotype and phenotype of the cells at specific stages in the process. We demonstrate that this protocol allows for the efficient, large-scale, cGMP-compliant production of transplantable NSCs from all lines tested. We also show that NSCs generated from iPSCs produced with the process described are capable of forming both glia defined by their expression of S100β and neurons that fire repetitive action potentials. PMID:23893392

Generation of Arbas Cashmere Goat Induced Pluripotent Stem Cells Through Fibroblast Reprogramming.

PubMed

Tai, Dapeng; Liu, Pengxia; Gao, Jing; Jin, Muzi; Xu, Teng; Zuo, Yongchun; Liang, Hao; Liu, Dongjun

2015-08-01

Various factors affect the process of obtaining stable Arbas cashmere goat embryonic stem cells (ESCs), for example, the difficulty in isolating cells at the appropriate stage of embryonic development, the in vitro culture environment, and passage methods. With the emergence of induced pluripotent stem cell (iPSC) technology, it has become possible to use specific genes to induce somatic cell differentiation in PSCs. We transferred OCT4, SOX2, c-MYC, and KLF4 into Arbas cashmere goat fetal fibroblasts, then induced and cultured them using a drug-inducible system to obtain Arbas goat iPSCs that morphologically resembled mouse iPSCs. After identification, the obtained goat iPSCs expressed ESC markers, had a normal karyotype, could differentiate into embryoid bodies in vitro, and could differentiate into three germ layer cell types and form teratomas in vivo. We used microarray gene expression profile analysis to elucidate the reprogramming process. Our results provide the experimental basis for establishing cashmere goat iPSC lines and for future in-depth studies on molecular mechanism of cashmere goat somatic cell reprogramming.

The genetic network controlling plasma cell differentiation.

PubMed

Nutt, Stephen L; Taubenheim, Nadine; Hasbold, Jhagvaral; Corcoran, Lynn M; Hodgkin, Philip D

2011-10-01

Upon activation by antigen, mature B cells undergo immunoglobulin class switch recombination and differentiate into antibody-secreting plasma cells, the endpoint of the B cell developmental lineage. Careful quantitation of these processes, which are stochastic, independent and strongly linked to the division history of the cell, has revealed that populations of B cells behave in a highly predictable manner. Considerable progress has also been made in the last few years in understanding the gene regulatory network that controls the B cell to plasma cell transition. The mutually exclusive transcriptomes of B cells and plasma cells are maintained by the antagonistic influences of two groups of transcription factors, those that maintain the B cell program, including Pax5, Bach2 and Bcl6, and those that promote and facilitate plasma cell differentiation, notably Irf4, Blimp1 and Xbp1. In this review, we discuss progress in the definition of both the transcriptional and cellular events occurring during late B cell differentiation, as integrating these two approaches is crucial to defining a regulatory network that faithfully reflects the stochastic features and complexity of the humoral immune response. 2011 Elsevier Ltd. All rights reserved.

Inhibition of TGF-β Signaling in SHED Enhances Endothelial Differentiation.

PubMed

Xu, J G; Gong, T; Wang, Y Y; Zou, T; Heng, B C; Yang, Y Q; Zhang, C F

2018-02-01

Low efficiency of deriving endothelial cells (ECs) from adult stem cells hampers their utilization in tissue engineering studies. The purpose of this study was to investigate whether suppression of transforming growth factor beta (TGF-β) signaling could enhance the differentiation efficiency of dental pulp-derived stem cells into ECs. We initially used vascular endothelial growth factor A (VEGF-A) to stimulate 2 dental pulp-derived stem cells (dental pulp stem cells and stem cells from human exfoliated deciduous teeth [SHED]) and compared their differentiation capacity into ECs. We further evaluated whether the vascular endothelial growth factor receptor I (VEGF-RI)-specific ligand placental growth factor-1 (PlGF-1) could mediate endothelial differentiation. Finally, we investigated whether the TGF-β signaling inhibitor SB-431542 could enhance the inductive effect of VEGF-A on endothelial differentiation, as well as the underlying mechanisms involved. ECs differentiated from dental pulp-derived stem cells exhibited the typical phenotypes of primary ECs, with SHED possessing a higher endothelial differentiation potential than dental pulp stem cells. VEGFR1-specific ligand-PLGF exerted a negligible effect on SHED-ECs differentiation. Compared with VEGF-A alone, the combination of VEGF-A and SB-431542 significantly enhanced the endothelial differentiation of SHED. The presence of SB-431542 inhibited the phosphorylation of Suppressor of Mothers Against Decapentaplegic 2/3 (SMAD2/3), allowing for VEGF-A-dependent phosphorylation and upregulation of VEGFR2. Our results indicate that the combination of VEGF-A and SB-431542 could enhance the differentiation of dental pulp-derived stem cells into endothelial cells, and this process is mediated through enhancement of VEGF-A-VEGFR2 signaling and concomitant inhibition of TGF-β-SMAD2/3 signaling.

Cell-geometry-dependent changes in plasma membrane order direct stem cell signalling and fate

NASA Astrophysics Data System (ADS)

von Erlach, Thomas C.; Bertazzo, Sergio; Wozniak, Michele A.; Horejs, Christine-Maria; Maynard, Stephanie A.; Attwood, Simon; Robinson, Benjamin K.; Autefage, Hélène; Kallepitis, Charalambos; del Río Hernández, Armando; Chen, Christopher S.; Goldoni, Silvia; Stevens, Molly M.

2018-03-01

Cell size and shape affect cellular processes such as cell survival, growth and differentiation1-4, thus establishing cell geometry as a fundamental regulator of cell physiology. The contributions of the cytoskeleton, specifically actomyosin tension, to these effects have been described, but the exact biophysical mechanisms that translate changes in cell geometry to changes in cell behaviour remain mostly unresolved. Using a variety of innovative materials techniques, we demonstrate that the nanostructure and lipid assembly within the cell plasma membrane are regulated by cell geometry in a ligand-independent manner. These biophysical changes trigger signalling events involving the serine/threonine kinase Akt/protein kinase B (PKB) that direct cell-geometry-dependent mesenchymal stem cell differentiation. Our study defines a central regulatory role by plasma membrane ordered lipid raft microdomains in modulating stem cell differentiation with potential translational applications.

T follicular helper cell differentiation, function, and roles in disease

PubMed Central

Crotty, Shane

2014-01-01

Summary Follicular helper T (Tfh) cells are specialized providers of T cell help to B cells, and are essential for germinal center formation, affinity maturation, and the development of most high affinity antibodies and memory B cells. Tfh cell differentiation is a multi-stage, multi-factorial process involving B cell lymphoma 6 (Bcl6) and other transcription factors. This article reviews understanding of Tfh cell biology, including their differentiation, migration, transcriptional regulation, and B cell help functions. Tfh cells are critical components of many protective immune responses against pathogens. As such, there is strong interest in harnessing Tfh cells to improve vaccination strategies. Tfh cells also have roles in a range of other diseases, particularly autoimmune diseases. Overall, there have been dramatic advances in this young field, but there is much to be learned about Tfh cell biology in the interest of applying that knowledge to biomedical needs. PMID:25367570

Glycolysis, but not Mitochondria, responsible for intracellular ATP distribution in cortical area of podocytes.

PubMed

Ozawa, Shota; Ueda, Shuko; Imamura, Hiromi; Mori, Kiyoshi; Asanuma, Katsuhiko; Yanagita, Motoko; Nakagawa, Takahiko

2015-12-18

Differentiated podocytes, a type of renal glomerular cells, require substantial levels of energy to maintain glomerular physiology. Mitochondria and glycolysis are two major producers of ATP, but the precise roles of each in podocytes remain unknown. This study evaluated the roles of mitochondria and glycolysis in differentiated and differentiating podocytes. Mitochondria in differentiated podocytes are located in the central part of cell body while blocking mitochondria had minor effects on cell shape and migratory ability. In contrast, blocking glycolysis significantly reduced the formation of lamellipodia, a cortical area of these cells, decreased the cell migratory ability and induced the apoptosis. Consistently, the local ATP production in lamellipodia was predominantly regulated by glycolysis. In turn, synaptopodin expression was ameliorated by blocking either mitochondrial respiration or glycolysis. Similar to differentiated podocytes, the differentiating podocytes utilized the glycolysis for regulating apoptosis and lamellipodia formation while synaptopodin expression was likely involved in both mitochondrial OXPHOS and glycolysis. Finally, adult mouse podocytes have most of mitochondria predominantly in the center of the cytosol whereas phosphofructokinase, a rate limiting enzyme for glycolysis, was expressed in foot processes. These data suggest that mitochondria and glycolysis play parallel but distinct roles in differentiated and differentiating podocytes.

Glycolysis, but not Mitochondria, responsible for intracellular ATP distribution in cortical area of podocytes

PubMed Central

Ozawa, Shota; Ueda, Shuko; Imamura, Hiromi; Mori, Kiyoshi; Asanuma, Katsuhiko; Yanagita, Motoko; Nakagawa, Takahiko

2015-01-01

Differentiated podocytes, a type of renal glomerular cells, require substantial levels of energy to maintain glomerular physiology. Mitochondria and glycolysis are two major producers of ATP, but the precise roles of each in podocytes remain unknown. This study evaluated the roles of mitochondria and glycolysis in differentiated and differentiating podocytes. Mitochondria in differentiated podocytes are located in the central part of cell body while blocking mitochondria had minor effects on cell shape and migratory ability. In contrast, blocking glycolysis significantly reduced the formation of lamellipodia, a cortical area of these cells, decreased the cell migratory ability and induced the apoptosis. Consistently, the local ATP production in lamellipodia was predominantly regulated by glycolysis. In turn, synaptopodin expression was ameliorated by blocking either mitochondrial respiration or glycolysis. Similar to differentiated podocytes, the differentiating podocytes utilized the glycolysis for regulating apoptosis and lamellipodia formation while synaptopodin expression was likely involved in both mitochondrial OXPHOS and glycolysis. Finally, adult mouse podocytes have most of mitochondria predominantly in the center of the cytosol whereas phosphofructokinase, a rate limiting enzyme for glycolysis, was expressed in foot processes. These data suggest that mitochondria and glycolysis play parallel but distinct roles in differentiated and differentiating podocytes. PMID:26677804

Expression of the transforming growth factor alpha protooncogene in differentiating human promyelocytic leukemia (HL-60) cells.

PubMed

Walz, T M; Malm, C; Wasteson, A

1993-01-01

The process of myeloid differentiation in human promyelocytic leukemia cells (HL-60) is accompanied by the coordinate expression of numerous protooncogenes. To investigate the expression of transforming growth factor alpha (TGF-alpha) in myeloid differentiation, HL-60 cells were induced to differentiate into granulocytes with 1.25% dimethyl sulfoxide, 0.2 microM all-trans retinoic acid, or 500 microM N6,O2-dibutyryladenosine-3'5'-cyclic monophosphate or differentiated along the monocyte/macrophage pathway with 0.1 microM phorbol-12-myristate-13-acetate. Using Northern blot analyses, TGF-alpha transcripts were detected within 24 h of treatment in cells differentiating toward granulocytes; maximal levels of gene expression were reached after 3 days or later and remained essentially constant throughout the observation period. These cells released TGF-alpha protein, as demonstrated by analysis of the incubation medium. In contrast, no TGF-alpha RNA or protein was detectable in HL-60 cell cultures when induced with phorbol-12-myristate-13-acetate. Epidermal growth factor receptor transcripts could not be detected either in undifferentiated or in differentiated HL-60 cells; therefore it appears as if an autocrine loop involving TGF-alpha in HL-60 cells is unlikely. In conclusion, the results demonstrate, for the first time, the expression of TGF-alpha in human granulocyte precursor cells. Our findings may indicate novel regulatory pathways in hematopoiesis.

Pigment Cell Differentiation in Sea Urchin Blastula-Derived Primary Cell Cultures

PubMed Central

Ageenko, Natalya V.; Kiselev, Konstantin V.; Dmitrenok, Pavel S.; Odintsova, Nelly A.

2014-01-01

The quinone pigments of sea urchins, specifically echinochrome and spinochromes, are known for their effective antioxidant, antibacterial, antifungal, and antitumor activities. We developed in vitro technology for inducing pigment differentiation in cell culture. The intensification of the pigment differentiation was accompanied by a simultaneous decrease in cell proliferation. The number of pigment cells was two-fold higher in the cells cultivated in the coelomic fluids of injured sea urchins than in those intact. The possible roles of the specific components of the coelomic fluids in the pigment differentiation process and the quantitative measurement of the production of naphthoquinone pigments during cultivation were examined by MALDI and electrospray ionization mass spectrometry. Echinochrome A and spinochrome E were produced by the cultivated cells of the sand dollar Scaphechinus mirabilis in all tested media, while only spinochromes were found in the cultivated cells of another sea urchin, Strongylocentrotus intermedius. The expression of genes associated with the induction of pigment differentiation was increased in cells cultivated in the presence of shikimic acid, a precursor of naphthoquinone pigments. Our results should contribute to the development of new techniques in marine biotechnology, including the generation of cell cultures producing complex bioactive compounds with therapeutic potential. PMID:24979272

Morphological and proteomic analysis of early stage of osteoblast differentiation in osteoblastic progenitor cells

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

Hong, Dun; Orthopedic Department, Taizhou Hospital, Wenzhou Medical College, Linhai, Zhejiang 317000; Chen, Hai-Xiao, E-mail: Hxchen-1@163.net

Bone remodeling relies on a dynamic balance between bone formation and resorption, mediated by osteoblasts and osteoclasts, respectively. Under certain stimuli, osteoprogenitor cells may differentiate into premature osteoblasts and further into mature osteoblasts. This process is marked by increased alkaline phosphatase (ALP) activity and mineralized nodule formation. In this study, we induced osteoblast differentiation in mouse osteoprogenitor MC3T3-E1 cells and divided the process into three stages. In the first stage (day 3), the MC3T3-E1 cell under osteoblast differentiation did not express ALP or deposit a mineralized nodule. In the second stage, the MC3T3-E1 cell expressed ALP but did not formmore » a mineralized nodule. In the third stage, the MC3T3-E1 cell had ALP activity and formed mineralized nodules. In the present study, we focused on morphological and proteomic changes of MC3T3-E1 cells in the early stage of osteoblast differentiation - a period when premature osteoblasts transform into mature osteoblasts. We found that mean cell area and mean stress fiber density were increased in this stage due to enhanced cell spreading and decreased cell proliferation. We further analyzed the proteins in the signaling pathway of regulation of the cytoskeleton using a proteomic approach and found upregulation of IQGAP1, gelsolin, moesin, radixin, and Cfl1. After analyzing the focal adhesion signaling pathway, we found the upregulation of FLNA, LAMA1, LAMA5, COL1A1, COL3A1, COL4A6, and COL5A2 as well as the downregulation of COL4A1, COL4A2, and COL4A4. In conclusion, the signaling pathway of regulation of the cytoskeleton and focal adhesion play critical roles in regulating cell spreading and actin skeleton formation in the early stage of osteoblast differentiation.« less

White blood cell differential count of maturation stages in bone marrow smear using dual-stage convolutional neural networks.

PubMed

Choi, Jin Woo; Ku, Yunseo; Yoo, Byeong Wook; Kim, Jung-Ah; Lee, Dong Soon; Chai, Young Jun; Kong, Hyoun-Joong; Kim, Hee Chan

2017-01-01

The white blood cell differential count of the bone marrow provides information concerning the distribution of immature and mature cells within maturation stages. The results of such examinations are important for the diagnosis of various diseases and for follow-up care after chemotherapy. However, manual, labor-intensive methods to determine the differential count lead to inter- and intra-variations among the results obtained by hematologists. Therefore, an automated system to conduct the white blood cell differential count is highly desirable, but several difficulties hinder progress. There are variations in the white blood cells of each maturation stage, small inter-class differences within each stage, and variations in images because of the different acquisition and staining processes. Moreover, a large number of classes need to be classified for bone marrow smear analysis, and the high density of touching cells in bone marrow smears renders difficult the segmentation of single cells, which is crucial to traditional image processing and machine learning. Few studies have attempted to discriminate bone marrow cells, and even these have either discriminated only a few classes or yielded insufficient performance. In this study, we propose an automated white blood cell differential counting system from bone marrow smear images using a dual-stage convolutional neural network (CNN). A total of 2,174 patch images were collected for training and testing. The dual-stage CNN classified images into 10 classes of the myeloid and erythroid maturation series, and achieved an accuracy of 97.06%, a precision of 97.13%, a recall of 97.06%, and an F-1 score of 97.1%. The proposed method not only showed high classification performance, but also successfully classified raw images without single cell segmentation and manual feature extraction by implementing CNN. Moreover, it demonstrated rotation and location invariance. These results highlight the promise of the proposed method as an automated white blood cell differential count system.

White blood cell differential count of maturation stages in bone marrow smear using dual-stage convolutional neural networks

PubMed Central

Choi, Jin Woo; Ku, Yunseo; Yoo, Byeong Wook; Kim, Jung-Ah; Lee, Dong Soon; Chai, Young Jun; Kong, Hyoun-Joong

2017-01-01

The white blood cell differential count of the bone marrow provides information concerning the distribution of immature and mature cells within maturation stages. The results of such examinations are important for the diagnosis of various diseases and for follow-up care after chemotherapy. However, manual, labor-intensive methods to determine the differential count lead to inter- and intra-variations among the results obtained by hematologists. Therefore, an automated system to conduct the white blood cell differential count is highly desirable, but several difficulties hinder progress. There are variations in the white blood cells of each maturation stage, small inter-class differences within each stage, and variations in images because of the different acquisition and staining processes. Moreover, a large number of classes need to be classified for bone marrow smear analysis, and the high density of touching cells in bone marrow smears renders difficult the segmentation of single cells, which is crucial to traditional image processing and machine learning. Few studies have attempted to discriminate bone marrow cells, and even these have either discriminated only a few classes or yielded insufficient performance. In this study, we propose an automated white blood cell differential counting system from bone marrow smear images using a dual-stage convolutional neural network (CNN). A total of 2,174 patch images were collected for training and testing. The dual-stage CNN classified images into 10 classes of the myeloid and erythroid maturation series, and achieved an accuracy of 97.06%, a precision of 97.13%, a recall of 97.06%, and an F-1 score of 97.1%. The proposed method not only showed high classification performance, but also successfully classified raw images without single cell segmentation and manual feature extraction by implementing CNN. Moreover, it demonstrated rotation and location invariance. These results highlight the promise of the proposed method as an automated white blood cell differential count system. PMID:29228051

Heme oxygenase-1 affects generation and spontaneous cardiac differentiation of induced pluripotent stem cells.

PubMed

Stepniewski, Jacek; Pacholczak, Tomasz; Skrzypczyk, Aniela; Ciesla, Maciej; Szade, Agata; Szade, Krzysztof; Bidanel, Romain; Langrzyk, Agnieszka; Grochowski, Radoslaw; Vandermeeren, Felix; Kachamakova-Trojanowska, Neli; Jez, Mateusz; Drabik, Grazyna; Nakanishi, Mahito; Jozkowicz, Alicja; Dulak, Jozef

2018-02-01

Cellular stress can influence efficiency of iPSCs generation and their differentiation. However, the role of intracellular cytoprotective factors in these processes is still not well known. Therefore, we investigated the effect of HO-1 (Hmox1) or Nrf2 (Nfe2l2), two major cytoprotective genes. Hmox1 -/- fibroblasts demonstrated decreased reprogramming efficiency in comparison to Hmox1 +/+ cells. Reversely, pharmacological enhancement of HO-1 resulted in higher number of iPSCs colonies. Importantly, elevated level of both p53 and p53-regulated miR-34a and 14-3-3σ was observed in HO-1-deficient fibroblasts whereas downregulation of p53 in these cells markedly increased their reprogramming efficiency. In human fibroblasts HO-1 silencing also induced p53 expression and affected reprogramming outcome. Hmox1 +/+ and Hmox1 -/- iPSCs similarly differentiated in vitro to cells originating from three germ layers, however, lower number of contracting cells was observed during this process in HO-1-deficient cells indicating attenuated cardiac differentiation. Importantly, silencing of Hmox1 in murine ESC using CRISPR/Cas-9 editing also impaired their spontaneous cardiac differentiation. Decreased reprogramming efficiency was also observed in Nrf2-lacking fibroblasts. Reversely, sulforaphane, a Nrf2 activator, increased the number of iPSCs colonies. However, both Nfe2l2 +/+ and Nfe2l2 -/- iPSCs showed similar pluripotency and differentiation capacity. These results indicate that regulation of HO-1 expression can further optimize generation and cardiac differentiation of iPSCs. © 2018 IUBMB Life, 70(2):129-142, 2018. © 2018 International Union of Biochemistry and Molecular Biology.

Interleukin-32 induces the differentiation of monocytes into macrophage-like cells.

PubMed

Netea, Mihai G; Lewis, Eli C; Azam, Tania; Joosten, Leo A B; Jaekal, Jun; Bae, Su-Young; Dinarello, Charles A; Kim, Soo-Hyun

2008-03-04

After emigration from the bone marrow to the peripheral blood, monocytes enter tissues and differentiate into macrophages, the prototype scavenger of the immune system. By ingesting and killing microorganisms and removing cellular debris, macrophages also process antigens as a first step in mounting a specific immune response. IL-32 is a cytokine inducing proinflammatory cytokines and chemokines via p38-MAPK and NF-kappaB. In the present study, we demonstrate that IL-32 induces differentiation of human blood monocytes as well as THP-1 leukemic cells into macrophage-like cells with functional phagocytic activity for live bacteria. Muramyl dipepide (MDP), the ligand for the intracellular nuclear oligomerization domain (NOD) 2 receptor, has no effect on differentiation alone but augments the monocyte-to-macrophage differentiation by IL-32. Unexpectedly, IL-32 reversed GM-CSF/IL-4-induced dendritic cell differentiation to macrophage-like cells. Whereas the induction of TNFalpha, IL-1beta, and IL-6 by IL-32 is mediated by p38-MAPK, IL-32-induced monocyte-to-macrophage differentiation is mediated through nonapoptotic, caspase-3-dependent mechanisms. Thus, IL-32 not only contributes to host responses through the induction of proinflammatory cytokines but also directly affects specific immunity by differentiating monocytes into macrophage-like cells.

Down-regulation of the non-coding RNA H19 and its derived miR-675 is concomitant with up-regulation of insulin-like growth factor receptor type 1 during neural-like differentiation of human bone marrow mesenchymal stem cells.

PubMed

Farzi-Molan, Asghar; Babashah, Sadegh; Bakhshinejad, Babak; Atashi, Amir; Fakhr Taha, Masoumeh

2018-03-07

The differentiation of human bone marrow mesenchymal stem cells (BMSCs) into specific lineages offers new opportunities to use the therapeutic efficiency of these pluripotent cells in regenerative medicine. Multiple lines of evidence have revealed that non-coding RNAs play major roles in the differentiation of BMSCs into neural cells. Here, we applied a cocktail of neural inducing factors (NIFs) to differentiate BMSCs into neural-like cells. Our data demonstrated that during neurogenic induction, BMSCs obtained a neuron-like morphology. Also, the results of gene expression analysis by qRT-PCR showed progressively increasing expression levels of neuron-specific enolase (NSE) as well as microtubule-associated protein 2 (MAP-2) and immunocytochemical staining detected the expression of these neuron-specific markers along differentiated BMSC bodies and cytoplasmic processes, confirming the differentiation of BMSCs into neuronal lineages. We also compared differences in the expression levels of the long non-coding RNA (lncRNA) H19 and H19-derived miR-675 between undifferentiated and neurally differentiated BMSCs and found that during neural differentiation down-regulation of the lncRNA H19/miR-675 axis is concomitant with up-regulation of insulin-like growth factor type-1 (IGF-1R), a well-established target of miR-675 involved in neurogenesis. The findings of the current study provide support for the hypothesis that miR-675 may confer functionality to H19, suggesting a key role for this miRNA in the neural differentiation of BSMCs. However, further investigation is required to gain deeper insights into the biological roles of this miRNA in the complex process of neurogenesis. © 2018 International Federation for Cell Biology.

In vitro differentiation of HT-29 M6 mucus-secreting colon cancer cells involves a trychostatin A and p27(KIP1)-inducible transcriptional program of gene expression.

PubMed

Mayo, Clara; Lloreta, Josep; Real, Francisco X; Mayol, Xavier

2007-07-01

Tumor cell dedifferentiation-such as the loss of cell-to-cell adhesion in epithelial tumors-is associated with tumor progression. To better understand the mechanisms that maintain carcinoma cells in a differentiated state, we have dissected in vitro differentiation pathways in the mucus-secretor HT-29 M6 colon cancer cell line, which spontaneously differentiates in postconfluent cultures. By lowering the extracellular calcium concentration to levels that prevent intercellular adhesion and epithelial polarization, our results reveal that differentiation is calcium-dependent and involves: (i) a process of cell cycle exit to G(0) and (ii) the induction of a transcriptional program of differentiation gene expression (i.e., mucins MUC1 and MUC5AC, and the apical membrane peptidase DPPIV). In calcium-deprived, non-differentiated postconfluent cultures, differentiation gene promoters are repressed by a trichostatin A (TSA)-sensitive mechanism, indicating that loss of gene expression by dedifferentiation is driven by histone deacetylases (HDAC). Since TSA treatment or extracellular calcium restoration allow gene promoter activation to similar levels, we suggest that induction of differentiation is one mechanism of HDAC inhibitor antitumor action. Moreover, transcriptional de-repression can also be induced in non-differentiating culture conditions by overexpressing the cyclin-dependent kinase inhibitor p27(KIP1), which is normally induced during spontaneous differentiation. Since p27(KIP1) downregulation in colon cancer is associated with poor prognosis independently of tumor cell division rates, we propose that p27 (KIP1) may prevent tumor progression by, at least in part, enhancing the expression of some differentiation genes. Therefore, the HT-29 M6 model allows the identification of some basic mechanisms of cancer cell differentiation control, so far revealing HDAC and p27(KIP1) as key regulatory factors of differentiation gene expression.

Intestinal development and differentiation

PubMed Central

Noah, Taeko K.; Donahue, Bridgitte; Shroyer, Noah F.

2011-01-01

In this review, we present an overview of intestinal development and cellular differentiation of the intestinal epithelium. The review is separated into two sections: Section one summarizes organogenesis of the small and large intestines, including endoderm and gut tube formation in early embryogenesis, villus morphogenesis, and crypt formation. Section two reviews cell fate specification and differentiation of each cell type within the intestinal epithelium. Growth factor and transcriptional networks that regulate these developmental processes are summarized. PMID:21978911

Biogenesis of protein bodies during legumin accumulation in developing olive (Olea europaea L.) seed.

PubMed

Jimenez-Lopez, Jose C; Zienkiewicz, Agnieszka; Zienkiewicz, Krzysztof; Alché, Juan D; Rodríguez-García, Maria I

2016-03-01

Much of our current knowledge about seed development and differentiation regarding reserves synthesis and accumulation come from monocot (cereals) plants. Studies in dicotyledonous seeds differentiation are limited to a few species and in oleaginous species are even scarcer despite their agronomic and economic importance. We examined the changes accompanying the differentiation of olive endosperm and cotyledon with a focus on protein bodies (PBs) biogenesis during legumin protein synthesis and accumulation, with the aim of getting insights and a better understanding of the PBs' formation process. Cotyledon and endosperm undergo differentiation during seed development, where an asynchronous time-course of protein synthesis, accumulation, and differential PB formation patterns was found in both tissues. At the end of seed maturation, a broad population of PBs, particularly in cotyledon cells, was distinguishable in terms of number per cell and morphometric and cytochemical features. Olive seed development is a tissue-dependent process characterized by differential rates of legumin accumulation and PB formation in the main tissues integrating seed. One of the main features of the impressive differentiation process is the specific formation of a broad group of PBs, particularly in cotyledon cells, which might depend on selective accumulation and packaging of proteins and specific polypeptides into PBs. The nature and availability of the major components detected in the PBs of olive seed are key parameters in order to consider the potential use of this material as a suitable source of carbon and nitrogen for animal or even human use.

Global transcriptome analysis of spore formation in Myxococcus xanthus reveals a locus necessary for cell differentiation

PubMed Central

2010-01-01

Background Myxococcus xanthus is a Gram negative bacterium that can differentiate into metabolically quiescent, environmentally resistant spores. Little is known about the mechanisms involved in differentiation in part because sporulation is normally initiated at the culmination of a complex starvation-induced developmental program and only inside multicellular fruiting bodies. To obtain a broad overview of the sporulation process and to identify novel genes necessary for differentiation, we instead performed global transcriptome analysis of an artificial chemically-induced sporulation process in which addition of glycerol to vegetatively growing liquid cultures of M. xanthus leads to rapid and synchronized differentiation of nearly all cells into myxospore-like entities. Results Our analyses identified 1 486 genes whose expression was significantly regulated at least two-fold within four hours of chemical-induced differentiation. Most of the previously identified sporulation marker genes were significantly upregulated. In contrast, most genes that are required to build starvation-induced multicellular fruiting bodies, but which are not required for sporulation per se, were not significantly regulated in our analysis. Analysis of functional gene categories significantly over-represented in the regulated genes, suggested large rearrangements in core metabolic pathways, and in genes involved in protein synthesis and fate. We used the microarray data to identify a novel operon of eight genes that, when mutated, rendered cells unable to produce viable chemical- or starvation-induced spores. Importantly, these mutants displayed no defects in building fruiting bodies, suggesting these genes are necessary for the core sporulation process. Furthermore, during the starvation-induced developmental program, these genes were expressed in fruiting bodies but not in peripheral rods, a subpopulation of developing cells which do not sporulate. Conclusions These results suggest that microarray analysis of chemical-induced spore formation is an excellent system to specifically identify genes necessary for the core sporulation process of a Gram negative model organism for differentiation. PMID:20420673

Th9 cells: differentiation and disease

PubMed Central

Kaplan, Mark H.

2014-01-01

Summary CD4+ T-helper cells regulate immunity and inflammation through the acquisition of potential to secrete specific cytokines. The acquisition of cytokine-secreting potential, in a process termed T-helper cell differentiation, is a response to multiple environmental signals including the cytokine milieu. The most recently defined subset of T-helper cells are termed Th9 and are identified by the potent production of interleukin-9 (IL-9). Given the pleiotropic functions of IL-9, Th9 cells might be involved in pathogen immunity and immune-mediated disease. In this review, I focus on recent developments in understanding the signals that promote Th9 differentiation, the transcription factors that regulate IL-9 expression, and finally the potential roles for Th9 cells in immunity in vivo. PMID:23405898

Activation of the kinase activity of ATM by retinoic acid is required for CREB-dependent differentiation of neuroblastoma cells.

PubMed

Fernandes, Norvin D; Sun, Yingli; Price, Brendan D

2007-06-01

The ATM protein kinase is mutated in ataxia telangiectasia, a genetic disease characterized by defective DNA repair, neurodegeneration, and growth factor signaling defects. The activity of ATM kinase is activated by DNA damage, and this activation is required for cells to survive genotoxic events. In addition to this well characterized role in DNA repair, we now demonstrate a novel role for ATM in the retinoic acid (RA)-induced differentiation of SH-SY5Y neuroblastoma cells into post-mitotic, neuronal-like cells. RA rapidly activates the activity of ATM kinase, leading to the ATM-dependent phosphorylation of the CREB protein, extrusion of neuritic processes, and differentiation of SH-SY5Y cells into neuronal-like cells. When ATM protein expression was suppressed by short hairpin RNA, the ATM-dependent phosphorylation of CREB was blocked. Furthermore, ATM-negative cells failed to differentiate into neuronal-like cells when exposed to retinoic acid; instead, they underwent cell death. Expression of a constitutively active CREBVP16 construct, or exposure to forskolin to induce CREB phosphorylation, rescued ATM negative cells and restored differentiation. Furthermore, when dominant negative CREB proteins with mutations in either the CREB phosphorylation site (CREBS133A) or the DNA binding domain (KCREB) were introduced into SH-SY5Y cells, retinoic acid-induced differentiation was blocked and the cells underwent cell death. The results demonstrate that ATM is required for the retinoic acid-induced differentiation of SH-SY5Y cells through the ATM dependent-phosphorylation of serine 133 of CREB. These results therefore define a novel mechanism for activation of the activity of ATM kinase by RA, and implicate ATM in the regulation of CREB function during RA-induced differentiation.

c-FLIP is involved in erythropoietin-mediated protection of erythroid-differentiated cells from TNF-alpha-induced apoptosis.

PubMed

Vittori, Daniela; Vota, Daiana; Callero, Mariana; Chamorro, María E; Nesse, Alcira

2010-05-04

The TNF-alpha (tumour necrosis factor) affects a wide range of biological activities, such as cell proliferation and apoptosis. Cell life or death responses to this cytokine might depend on cell conditions. This study focused on the modulation of factors that would affect the sensitivity of erythroid-differentiated cells to TNF-alpha. Hemin-differentiated K562 cells showed higher sensitivity to TNF-induced apoptosis than undifferentiated cells. At the same time, hemin-induced erythroid differentiation reduced c-FLIP (cellular FLICE-inhibitory protein) expression. However, this negative effect was prevented by prior treatment with Epo (erythropoietin), which allowed the cell line to maintain c-FLIP levels. On the other hand, erythroid-differentiated UT-7 cells - dependent on Epo for survival - showed resistance to TNF-alpha pro-apoptotic action. Only after the inhibition of PI3K (phosphatidylinositol-3 kinase)-mediated pathways, which was accompanied by negative c-FLIP modulation and increased erythroid differentiation, were UT-7 cells sensitive to TNF-alpha-triggered apoptosis. In summary, erythroid differentiation might deregulate the balance between growth promotion and death signals induced by TNF-alpha, depending on cell type and environmental conditions. The role of c-FLIP seemed to be critical in the protection of erythroid-differentiated cells from apoptosis or in the determination of their sensitivity to TNF-mediated programmed cell death. Epo, which for the first time was found to be involved in the prevention of c-FLIP down-regulation, proved to have an anti-apoptotic effect against the pro-inflammatory factor. The identification of signals related to cell life/death switching would have significant implications in the control of proliferative diseases and would contribute to the understanding of mechanisms underlying the anaemia associated with inflammatory processes.

Drosophila sessile hemocyte clusters are true hematopoietic tissues that regulate larval blood cell differentiation

PubMed Central

Leitão, Alexandre B; Sucena, Élio

2015-01-01

Virtually all species of coelomate animals contain blood cells that display a division of labor necessary for homeostasis. This functional partition depends upon the balance between proliferation and differentiation mostly accomplished in the hematopoietic organs. In Drosophila melanogaster, the lymph gland produces plasmatocytes and crystal cells that are not released until pupariation. Yet, throughout larval development, both hemocyte types increase in numbers. Mature plasmatocytes can proliferate but it is not known if crystal cell numbers increase by self-renewal or by de novo differentiation. We show that new crystal cells in third instar larvae originate through a Notch-dependent process of plasmatocyte transdifferentiation. This process occurs in the sessile clusters and is contingent upon the integrity of these structures. The existence of this hematopoietic tissue, relying on structure-dependent signaling events to promote blood homeostasis, creates a new paradigm for addressing outstanding questions in Drosophila hematopoiesis and establishing further parallels with vertebrate systems. DOI: http://dx.doi.org/10.7554/eLife.06166.001 PMID:25650737

Auxin minimum triggers the developmental switch from cell division to cell differentiation in the Arabidopsis root

PubMed Central

De Ruvo, Micol; Pacifici, Elena; Salvi, Elena; Sozzani, Rosangela; Benfey, Philip N.; Di Paola, Luisa; Marée, Athanasius F. M.; Costantino, Paolo; Grieneisen, Verônica A.; Sabatini, Sabrina

2017-01-01

In multicellular organisms, a stringent control of the transition between cell division and differentiation is crucial for correct tissue and organ development. In the Arabidopsis root, the boundary between dividing and differentiating cells is positioned by the antagonistic interaction of the hormones auxin and cytokinin. Cytokinin affects polar auxin transport, but how this impacts the positional information required to establish this tissue boundary, is still unknown. By combining computational modeling with molecular genetics, we show that boundary formation is dependent on cytokinin’s control on auxin polar transport and degradation. The regulation of both processes shapes the auxin profile in a well-defined auxin minimum. This auxin minimum positions the boundary between dividing and differentiating cells, acting as a trigger for this developmental transition, thus controlling meristem size. PMID:28831001

Fank1 and Jazf1 promote multiciliated cell differentiation in the mouse airway epithelium

PubMed Central

Johnson, Jo-Anne; Watson, Julie K.

2018-01-01

ABSTRACT The airways are lined by secretory and multiciliated cells which function together to remove particles and debris from the respiratory tract. The transcriptome of multiciliated cells has been extensively studied, but the function of many of the genes identified is unknown. We have established an assay to test the ability of over-expressed transcripts to promote multiciliated cell differentiation in mouse embryonic tracheal explants. Overexpression data indicated that Fibronectin type 3 and ankyrin repeat domains 1 (Fank1) and JAZF zinc finger 1 (Jazf1) promoted multiciliated cell differentiation alone, and cooperatively with the canonical multiciliated cell transcription factor Foxj1. Moreover, knock-down of Fank1 or Jazf1 in adult mouse airway epithelial cultures demonstrated that these factors are both required for ciliated cell differentiation in vitro. This analysis identifies Fank1 and Jazf1 as novel regulators of multiciliated cell differentiation. Moreover, we show that they are likely to function downstream of IL6 signalling and upstream of Foxj1 activity in the process of ciliated cell differentiation. In addition, our in vitro explant assay provides a convenient method for preliminary investigation of over-expression phenotypes in the developing mouse airways. This article has an associated First Person interview with the first author of the paper. PMID:29661797

Synchrotron FTIR microspectroscopy reveals early adipogenic differentiation of human mesenchymal stem cells at single-cell level

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

Liu, Zhixiao; University of Chinese Academy of Science, Beijing 100049; Tang, Yuzhao

Human mesenchymal stem cells (hMSCs) have been used as an ideal in vitro model to study human adipogenesis. However, little knowledge of the early stage differentiation greatly hinders our understanding on the mechanism of the adipogenesis processes. In this study, synchrotron radiation-based Fourier transform infrared (SR-FTIR) microspectroscopy was applied to track the global structural and compositional changes of lipids, proteins and nucleic acids inside individual hMSCs along the time course. The multivariate analysis of the SR-FTIR spectra distinguished the dynamic and significant changes of the lipids and nucleic acid at early differentiation stage. Importantly, changes of lipid structure during early daysmore » (Day 1–3) of differentiation might serve as a potential biomarker in identifying the state in early differentiation at single cell level. These results proved that SR-FTIR is a powerful tool to study the stem cell fate determination and early lipogenesis events. - Highlights: • Molecular events occur in the early adipogenic differentiation stage of hMSCs are studied by SR-FTIR. • SR-FTIR data suggest that lipids may play an important role in hMSCs determination. • As potential biomarkers, lipids peaks can identify the state of cell in early differentiation stage at single-cell level.« less

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

Lipid deregulation in UV irradiated skin cells: Role of 25-hydroxycholesterol in keratinocyte differentiation during photoaging.

PubMed

Olivier, Elodie; Dutot, Mélody; Regazzetti, Anne; Dargère, Delphine; Auzeil, Nicolas; Laprévote, Olivier; Rat, Patrice

2017-05-01

Skin photoaging due to UV irradiation is a degenerative process that appears more and more as a growing concern. Lipids, including oxysterols, are involved in degenerative processes; as skin cells contain various lipids, the aim of our study was to evaluate first, changes in keratinocyte lipid levels induced by UV exposure and second, cellular effects of oxysterols in cell morphology and several hallmarks of keratinocyte differentiation. Our mass spectrometry results demonstrated that UV irradiation induces changes in lipid profile of cultured keratinocytes; in particular, ceramides and oxysterols, specifically 25-hydroxycholesterol (25-OH), were increased. Using holography and confocal microscopy analyses, we highlighted cell thickening and cytoskeletal disruption after incubation of keratinocytes with 25-OH. These alterations were associated with keratinocyte differentiation patterns: autophagy stimulation and intracellular calcium increase as measured by cytofluorometry, and increased involucrin level detected by immunocytochemistry. To conclude, oxysterol deregulation could be considered as a common marker of degenerative disorders. During photoaging, 25-OH seems to play a key role inducing morphological changes and keratinocyte differentiation. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Stem cells and regenerative medicine for diabetes mellitus.

PubMed

Sumi, Shoichiro; Gu, Yuanjun; Hiura, Akihito; Inoue, Kazutomo

2004-10-01

A profound knowledge of the development and differentiation of pancreatic tissues, especially islets of Langerhans, is necessary for developing regenerative therapy for severe diabetes mellitus. A recent developmental study showed that PTF-1a is expressed in almost all parts of pancreatic tissues, in addition to PDX-1, a well-known transcription factor that is essential for pancreas development. Another study suggested that alpha cells and beta cells individually, but not sequentially, differentiated from neurogenin-3--expressing precursor cells. Under strong induction of pancreas regeneration, it is likely that pancreatic duct cells dedifferentiate to grow, express PDX-1, and re-differentiate toward other cell types including islet cells. Duct epithelium-like cells can be cultivated from crude pancreatic exocrine cells and can be induced to differentiate toward islet-like cell clusters under some culture conditions. These cell clusters made from murine pancreas have been shown to control hyperglycemia when transplanted into diabetic mice. Liver-derived oval cells and their putative precursor H-CFU-C have been shown to differentiate toward pancreatic cells. Furthermore, extrapancreatic cells contained in bone marrow and amniotic membrane are reported to become insulin-producing cells. However, their exact characterization and relationship between these cell types remain to be elucidated. Our recent study has shown that islet-like cell clusters can be differentiated from mouse embryonic stem cells. Transplantation of these clusters could ameliorate hyperglycemia of STZ-induced diabetic mice without forming teratomas. Interestingly, these cells expressed several genes specific to exocrine pancreatic tissue in addition to islet-related genes, suggesting that stable and efficient differentiation toward certain tissues can only be achieved through a process mimicking normal development of the tissue. Perhaps recent developments in these fields may rapidly lead to an established regenerative therapy for diabetes mellitus.

The expression of PTEN in the development of mouse cochlear lateral wall.

PubMed

Dong, Y; Sui, L; Yamaguchi, F; Kamitori, K; Hirata, Y; Hossain, A; Noguchi, C; Katagi, A; Nishio, M; Suzuki, A; Lou, X; Tokuda, M

2014-01-31

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a tumor suppressor gene that regulates various cell processes including proliferation, growth, synaptogenesis, neural and glioma stem/progenitor cell renewal. In addition, PTEN can regulate sensory cell proliferation and differentiation of hair bundles in the mammalian cochlea. In this study we use immunofluorescence, Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR) to reveal the expression of PTEN in the developing cochlear lateral wall, which is crucial for regulating K(+) homeostasis. Relatively high levels of PTEN are initially expressed in the marginal cells (MCs) of the lateral wall at embryonic day (E) 17.5 when they start to differentiate. Similarly high levels are subsequently expressed in differentiating root cells (RCs) at postnatal day (P) 3 and then in spiral ligament fibrocytes (SLFs) at P 10. In the mature cochlea, PTEN expression is low or undetectable in MCs and SLFs but it remains high in RCs and their processes. The expression pattern for PTEN in the developing lateral wall suggests that it plays a critical role in the differentiation of the cellular pathways that regulate K(+) homeostasis in the cochlea. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

Endocrine disrupting chemicals affect the adipogenic differentiation of mesenchymal stem cells in distinct ontogenetic windows

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

Biemann, Ronald, E-mail: ronald.biemann@medizin.uni-halle.de; Navarrete Santos, Anne; Navarrete Santos, Alexander

Highlights: Black-Right-Pointing-Pointer Endocrine disrupting chemicals affect adipogenesis in mesenchymal stem cells (MSC). Black-Right-Pointing-Pointer The adipogenic impact depends strongly on the window of exposure. Black-Right-Pointing-Pointer Bisphenol A reduces the potential of MSC to differentiate into adipocytes. Black-Right-Pointing-Pointer DEHP and TBT trigger the adipogenic differentiation of mesenchymal stem cells. Black-Right-Pointing-Pointer BPA, DEHP and TBT did not affect adipogenesis in embryonic stem cells. -- Abstract: Endocrine disrupting chemicals (EDC) like bisphenol A (BPA), bis(2-ethylhexyl)phthalate (DEHP) and tributyltin (TBT) are ubiquitously present in the environment and in human tissues. They bind to nuclear hormone receptors and affect cellular and developmental processes. In this study,more » we show that BPA, DEHP and TBT affect the adipogenic differentiation of murine mesenchymal stem cells (MSC, C3H/10T1/2) in a concentration-, stage- and compound-specific manner. C3H/10T1/2 cells and embryonic stem cells (CGR8) were exposed to BPA, DEHP or TBT at different stages of cell determination and differentiation (undifferentiated growth, adipogenic induction and terminal adipogenic differentiation). The final amount of differentiated adipocytes, cellular triglyceride content and mRNA expression of adipogenic marker genes (adiponectin, FABP4, PPAR{gamma}2, LPL) were quantified and compared with corresponding unexposed cells. BPA (10 {mu}M) decreased subsequent adipogenic differentiation of MSC, when cells were exposed during undifferentiated growth. In contrast, DEHP (100 {mu}M) during the hormonal induction period, and TBT (100 nM) in all investigated stages, enhanced adipogenesis. Importantly, exposure of undifferentiated murine embryonic stem cells did not show any effect of the investigated EDC on subsequent adipogenic differentiation.« less

Myostatin inhibits myosatellite cell proliferation and consequently activates differentiation: evidence for endocrine-regulated transcript processing.

PubMed

Garikipati, Dilip K; Rodgers, Buel D

2012-10-01

Myostatin is a potent negative regulator of muscle growth in mammals. Despite high structural conservation, functional conservation in nonmammalian species is only assumed. This is particularly true for fish due to the presence of several myostatin paralogs: two in most species and four in salmonids (MSTN-1a, -1b, -2a, and -2b). Rainbow trout are a rich source of primary myosatellite cells as hyperplastic muscle growth occurs even in adult fish. These cells were therefore used to determine myostatin's effects on proliferation whereas our earlier studies reported its effects on quiescent cells. As in mammals, recombinant myostatin suppressed proliferation with no changes in cell morphology. Expression of MSTN-1a was several fold higher than the other paralogs and was autoregulated by myostatin, which also upregulated the expression of key differentiation markers: Myf5, MyoD1, myogenin, and myosin light chain. Thus, myostatin-stimulated cellular growth inhibition activates rather than represses differentiation. IGF-1 stimulated proliferation but had minimal and delayed effects on differentiation and its actions were suppressed by myostatin. However, IGF-1 upregulated MSTN-2a expression and the processing of its transcript, which is normally unprocessed. Myostatin therefore appears to partly mediate IGF-stimulated myosatellite differentiation in rainbow trout. This also occurs in mammals, although the IGF-stimulated processing of MSTN-2a transcripts is highly unique and is indicative of subfunctionalization within the gene family. These studies also suggest that the myokine's actions, including its antagonistic relationship with IGF-1, are conserved and that the salmonid gene family is functionally diverging.

The Effect of Mechanical Stimulation on Mineralization in Differentiating Osteoblasts in Collagen-I Scaffolds

PubMed Central

Damaraju, Swathi; Matyas, John R.; Rancourt, Derrick E.

2014-01-01

Developing a viable and functional bone scaffold in vitro that is capable of surviving and bearing mechanical load in vivo requires an understanding of the cell biology of osteoprogenitor cells, particularly how they are influenced by mechanical stimulation during cell differentiation and maturation. In this study, mechanical load was applied using a modified FlexCell plate to impart confined compression to collagen-I scaffolds seeded with undifferentiated murine embryonic stem cells. The activity, presence, and expression of osteoblast-cadherin (OB-Cad) and connexin-43, as well as various pluripotent and osteogenic markers were examined at 5–30 days of differentiation as cells were stimulated to differentiate to osteoblasts with and without applied mechanical load. Fluorescence recovery after photobleaching, immunofluorescence, viability, von Kossa, and real-time polymerase chain reaction assessments revealed that mechanical prestimulation of this cell-seeded scaffold altered the expression of OB-Cad and connexin-43 and resulted in significant differences in the structure and organization of mineralization present in the collagen matrix. Specifically, cells in gels that were loaded for 40 h after 5 days of differentiation and then left to fully differentiate for 30 days produced a highly structured honeycomb-shaped mineralization in the matrix; an outcome that was previously shown to be indicative of late osteoblast/early osteocyte activity. This study highlights the potential of mechanical load to accelerate differentiation and enhance osteoblast communication and function during the differentiation process, and highlights a time point of cell differentiation within this scaffold to apply load in order to most effectively transduce a mechanical signal. PMID:24851936

Nucleosomal occupancy changes locally over key regulatory regions during cell differentiation and reprogramming.

PubMed

West, Jason A; Cook, April; Alver, Burak H; Stadtfeld, Matthias; Deaton, Aimee M; Hochedlinger, Konrad; Park, Peter J; Tolstorukov, Michael Y; Kingston, Robert E

2014-08-27

Chromatin structure determines DNA accessibility. We compare nucleosome occupancy in mouse and human embryonic stem cells (ESCs), induced-pluripotent stem cells (iPSCs) and differentiated cell types using MNase-seq. To address variability inherent in this technique, we developed a bioinformatic approach to identify regions of difference (RoD) in nucleosome occupancy between pluripotent and somatic cells. Surprisingly, most chromatin remains unchanged; a majority of rearrangements appear to affect a single nucleosome. RoDs are enriched at genes and regulatory elements, including enhancers associated with pluripotency and differentiation. RoDs co-localize with binding sites of key developmental regulators, including the reprogramming factors Klf4, Oct4/Sox2 and c-Myc. Nucleosomal landscapes in ESC enhancers are extensively altered, exhibiting lower nucleosome occupancy in pluripotent cells than in somatic cells. Most changes are reset during reprogramming. We conclude that changes in nucleosome occupancy are a hallmark of cell differentiation and reprogramming and likely identify regulatory regions essential for these processes.

On the Application of Pattern Recognition and AI Technique to the Cytoscreening of Vaginal Smears by Computer

NASA Astrophysics Data System (ADS)

Bow, Sing T.; Wang, Xia-Fang

1989-05-01

In this paper the concepts of pattern recognition, image processing and artificial intelligence are applied to the development of an intelligent cytoscreening system to differentiate the abnormal cytological objects from the normal ones in vaginal smears. To achieve this goal,work listed below are involved: 1. Enhancement of the microscopic images of the smears; 2. Elevation of the qualitative differentiation under the microscope by cytologists to a quantitative differentiation plateau on the epithelial cells, ciliated cells, vacuolated cells, foreign-body-giant cells, plasma cells, lymph cells, white blood cells, red blood cells, etc. These knowledges are to be inputted into our intelligent cyto-screening system to ameliorate machine differentiation; 3. Selection of a set of effective features to characterize the cytological objects onto various regions of the multiclustered by computer algorithms; and 4. Systematical summarization of the knowledge that a gynecologist has and the way he/she follows when dealing with a case.

New insights in osteogenic differentiation revealed by mass spectrometric assessment of phosphorylated substrates in murine skin mesenchymal cells

PubMed Central

2013-01-01

Background Bone fractures and loss represent significant costs for the public health system and often affect the patients quality of life, therefore, understanding the molecular basis for bone regeneration is essential. Cytokines, such as IL-6, IL-10 and TNFα, secreted by inflammatory cells at the lesion site, at the very beginning of the repair process, act as chemotactic factors for mesenchymal stem cells, which proliferate and differentiate into osteoblasts through the autocrine and paracrine action of bone morphogenetic proteins (BMPs), mainly BMP-2. Although it is known that BMP-2 binds to ActRI/BMPR and activates the SMAD 1/5/8 downstream effectors, little is known about the intracellular mechanisms participating in osteoblastic differentiation. We assessed differences in the phosphorylation status of different cellular proteins upon BMP-2 osteogenic induction of isolated murine skin mesenchymal stem cells using Triplex Stable Isotope Dimethyl Labeling coupled with LC/MS. Results From 150 μg of starting material, 2,264 proteins were identified and quantified at five different time points, 235 of which are differentially phosphorylated. Kinase motif analysis showed that several substrates display phosphorylation sites for Casein Kinase, p38, CDK and JNK. Gene ontology analysis showed an increase in biological processes related with signaling and differentiation at early time points after BMP2 induction. Moreover, proteins involved in cytoskeleton rearrangement, Wnt and Ras pathways were found to be differentially phosphorylated during all timepoints studied. Conclusions Taken together, these data, allow new insights on the intracellular substrates which are phosphorylated early on during differentiation to BMP2-driven osteoblastic differentiation of skin-derived mesenchymal stem cells. PMID:24148232

Differential encoding of spatial information among retinal on cone bipolar cells

PubMed Central

Purgert, Robert J.

2015-01-01

The retina is the first stage of visual processing. It encodes elemental features of visual scenes. Distinct cone bipolar cells provide the substrate for this to occur. They encode visual information, such as color and luminance, a principle known as parallel processing. Few studies have directly examined whether different forms of spatial information are processed in parallel among cone bipolar cells. To address this issue, we examined the spatial information encoded by mouse ON cone bipolar cells, the subpopulation excited by increments in illumination. Two types of spatial processing were identified. We found that ON cone bipolar cells with axons ramifying in the central inner plexiform layer were tuned to preferentially encode small stimuli. By contrast, ON cone bipolar cells with axons ramifying in the proximal inner plexiform layer, nearest the ganglion cell layer, were tuned to encode both small and large stimuli. This dichotomy in spatial tuning is attributable to amacrine cells providing stronger inhibition to central ON cone bipolar cells compared with proximal ON cone bipolar cells. Furthermore, background illumination altered this difference in spatial tuning. It became less pronounced in bright light, as amacrine cell-driven inhibition became pervasive among all ON cone bipolar cells. These results suggest that differential amacrine cell input determined the distinct spatial encoding properties among ON cone bipolar cells. These findings enhance the known parallel processing capacity of the retina. PMID:26203104

A Model of Differential Growth-Guided Apical Hook Formation in Plants

PubMed Central

Žádníková, Petra; Wabnik, Krzysztof; Abuzeineh, Anas; Prusinkiewicz, Przemysław

2016-01-01

Differential cell growth enables flexible organ bending in the presence of environmental signals such as light or gravity. A prominent example of the developmental processes based on differential cell growth is the formation of the apical hook that protects the fragile shoot apical meristem when it breaks through the soil during germination. Here, we combined in silico and in vivo approaches to identify a minimal mechanism producing auxin gradient-guided differential growth during the establishment of the apical hook in the model plant Arabidopsis thaliana. Computer simulation models based on experimental data demonstrate that asymmetric expression of the PIN-FORMED auxin efflux carrier at the concave (inner) versus convex (outer) side of the hook suffices to establish an auxin maximum in the epidermis at the concave side of the apical hook. Furthermore, we propose a mechanism that translates this maximum into differential growth, and thus curvature, of the apical hook. Through a combination of experimental and in silico computational approaches, we have identified the individual contributions of differential cell elongation and proliferation to defining the apical hook and reveal the role of auxin-ethylene crosstalk in balancing these two processes. PMID:27754878

Two domains in vertebral development: antagonistic regulation by SHH and BMP4 proteins.

PubMed

Watanabe, Y; Duprez, D; Monsoro-Burq, A H; Vincent, C; Le Douarin, N M

1998-07-01

It has previously been shown that the notochord grafted laterally to the neural tube enhances the differentiation of the vertebral cartilage at the expense of the derivatives of the dermomyotome. In contrast, the dorsomedial graft of a notochord inhibits cartilage differentiation of the dorsal part of the vertebra carrying the spinous process. Cartilage differentiation is preceded by the expression of transcription factors of the Pax family (Pax1/Pax9) in the ventrolateral domain and of the Msx family in the dorsal domain. The proliferation and differentiation of Msx-expressing cells in the dorsal precartilaginous domain of the vertebra are stimulated by BMP4, which acts upstream of Msx genes. It has previously been shown that the SHH protein arising from the notochord (and floor plate) is necessary for the survival and further development of Pax1/Pax9-expressing sclerotomal cells. We show here that SHH acts antagonistically to BMP4. SHH-producing cells grafted dorsally to the neural tube at E2 inhibit expression of Bmp4 and Msx genes and also inhibits the differentiation of the spinous process. We present a model that accounts for cartilage differentiation in the vertebra.

Mesoporous silica nanoparticle-based substrates for cell directed delivery of Notch signalling modulators to control myoblast differentiation

NASA Astrophysics Data System (ADS)

Böcking, Dominique; Wiltschka, Oliver; Niinimäki, Jenni; Shokry, Hussein; Brenner, Rolf; Lindén, Mika; Sahlgren, Cecilia

2014-01-01

Biochemical cues are critical to control stem cell function and can be utilized to develop smart biomaterials for stem cell engineering. The challenge is to deliver these cues in a restricted manner with spatial and temporal control. Here we have developed bilayer films of mesoporous silica nanoparticles for delayed cellular delivery of Notch modulators to promote muscle stem cell differentiation. We demonstrate that drug-loaded particles are internalized from the particle-covered surface, which allows for direct delivery of the drug into the cell and a delayed and confined drug release. Substrates of particles loaded with γ-secretase-inhibitors, which block the Notch signalling pathway, promoted efficient differentiation of myoblasts. The particle substrates were fully biocompatible and did not interfere with the inherent differentiation process. We further demonstrate that impregnating commercially available, biocompatible polymer scaffolds with MSNs allows for a free standing substrate for cell directed drug delivery.Biochemical cues are critical to control stem cell function and can be utilized to develop smart biomaterials for stem cell engineering. The challenge is to deliver these cues in a restricted manner with spatial and temporal control. Here we have developed bilayer films of mesoporous silica nanoparticles for delayed cellular delivery of Notch modulators to promote muscle stem cell differentiation. We demonstrate that drug-loaded particles are internalized from the particle-covered surface, which allows for direct delivery of the drug into the cell and a delayed and confined drug release. Substrates of particles loaded with γ-secretase-inhibitors, which block the Notch signalling pathway, promoted efficient differentiation of myoblasts. The particle substrates were fully biocompatible and did not interfere with the inherent differentiation process. We further demonstrate that impregnating commercially available, biocompatible polymer scaffolds with MSNs allows for a free standing substrate for cell directed drug delivery. Electronic supplementary information (ESI) available: (1) Particle characterization. (2) Immunohistochemistry and SEM analyses of C2C12 cells grown on films for 3, 6, 24 and 72 h. Light microscopy and WST1 analyses of cells grown on cover slips and films for 6, 24 and 72 h (3) Quantification of protein levels of C2C12 cells differentiating on cover slips versus MSN films. (4) Stability of MSN films in biological solution and the influence on cell viability. (5) Cell internalization of particles from MSN films and intracellular drug release at 12 and 24 h (6) Cell internalization and intracellular DiI release of MSNs from (3Dtro®) fiber scaffolds impregnated with MSNs. See DOI: 10.1039/c3nr04022d

Transcriptomic Analysis Provides Insights into Grafting Union Development in Pecan (Carya illinoinensis).

PubMed

Mo, Zhenghai; Feng, Gang; Su, Wenchuan; Liu, Zhuangzhuang; Peng, Fangren

2018-02-05

Pecan ( Carya illinoinensis ), as a popular nut tree, has been widely planted in China in recent years. Grafting is an important technique for its cultivation. For a successful grafting, graft union development generally involves the formation of callus and vascular bundles at the graft union. To explore the molecular mechanism of graft union development, we applied high throughput RNA sequencing to investigate the transcriptomic profiles of graft union at four timepoints (0 days, 8 days, 15 days, and 30 days) during the pecan grafting process. After de novo assembly, 83,693 unigenes were obtained, and 40,069 of them were annotated. A total of 12,180 differentially expressed genes were identified between by grafting. Genes involved in hormone signaling, cell proliferation, xylem differentiation, cell elongation, secondary cell wall deposition, programmed cell death, and reactive oxygen species (ROS) scavenging showed significant differential expression during the graft union developmental process. In addition, we found that the content of auxin, cytokinin, and gibberellin were accumulated at the graft unions during the grafting process. These results will aid in our understanding of successful grafting in the future.

Transcriptomic Analysis Provides Insights into Grafting Union Development in Pecan (Carya illinoinensis)

PubMed Central

Mo, Zhenghai; Feng, Gang; Su, Wenchuan; Liu, Zhuangzhuang; Peng, Fangren

2018-01-01

Pecan (Carya illinoinensis), as a popular nut tree, has been widely planted in China in recent years. Grafting is an important technique for its cultivation. For a successful grafting, graft union development generally involves the formation of callus and vascular bundles at the graft union. To explore the molecular mechanism of graft union development, we applied high throughput RNA sequencing to investigate the transcriptomic profiles of graft union at four timepoints (0 days, 8 days, 15 days, and 30 days) during the pecan grafting process. After de novo assembly, 83,693 unigenes were obtained, and 40,069 of them were annotated. A total of 12,180 differentially expressed genes were identified between by grafting. Genes involved in hormone signaling, cell proliferation, xylem differentiation, cell elongation, secondary cell wall deposition, programmed cell death, and reactive oxygen species (ROS) scavenging showed significant differential expression during the graft union developmental process. In addition, we found that the content of auxin, cytokinin, and gibberellin were accumulated at the graft unions during the grafting process. These results will aid in our understanding of successful grafting in the future. PMID:29401757

Generation of a human airway epithelium derived basal cell line with multipotent differentiation capacity

PubMed Central

2013-01-01

Background As the multipotent progenitor population of the airway epithelium, human airway basal cells (BC) replenish the specialized differentiated cell populations of the mucociliated airway epithelium during physiological turnover and repair. Cultured primary BC divide a limited number of times before entering a state of replicative senescence, preventing the establishment of long-term replicating cultures of airway BC that maintain their original phenotype. Methods To generate an immortalized human airway BC cell line, primary human airway BC obtained by brushing the airway epithelium of healthy nonsmokers were infected with a retrovirus expressing human telomerase (hTERT). The resulting immortalized cell line was then characterized under non-differentiating and differentiating air-liquid interface (ALI) culture conditions using ELISA, TaqMan quantitative PCR, Western analysis, and immunofluorescent and immunohistochemical staining analysis for cell type specific markers. In addition, the ability of the cell line to respond to environmental stimuli under differentiating ALI culture was assessed. Results We successfully generated an immortalized human airway BC cell line termed BCi-NS1 via expression of hTERT. A single cell derived clone from the parental BCi-NS1 cells, BCi-NS1.1, retains characteristics of the original primary cells for over 40 passages and demonstrates a multipotent differentiation capacity into secretory (MUC5AC, MUC5B), goblet (TFF3), Clara (CC10) and ciliated (DNAI1, FOXJ1) cells on ALI culture. The cells can respond to external stimuli such as IL-13, resulting in alteration of the normal differentiation process. Conclusion Development of immortalized human airway BC that retain multipotent differentiation capacity over long-term culture should be useful in understanding the biology of BC, the response of BC to environmental stress, and as a target for assessment of pharmacologic agents. PMID:24298994

Pituitary cell differentiation from stem cells and other cells: toward restorative therapy for hypopituitarism?

PubMed

Willems, Christophe; Vankelecom, Hugo

2014-01-01

The pituitary gland, key regulator of our endocrine system, produces multiple hormones that steer essential physiological processes. Hence, deficient pituitary function (hypopituitarism) leads to severe disorders. Hypopituitarism can be caused by defective embryonic development, or by damage through tumor growth/resection and traumatic brain injury. Lifelong hormone replacement is needed but associated with significant side effects. It would be more desirable to restore pituitary tissue and function. Recently, we showed that the adult (mouse) pituitary holds regenerative capacity in which local stem cells are involved. Repair of deficient pituitary may therefore be achieved by activating these resident stem cells. Alternatively, pituitary dysfunction may be mended by cell (replacement) therapy. The hormonal cells to be transplanted could be obtained by (trans-)differentiating various kinds of stem cells or other cells. Here, we summarize the studies on pituitary cell regeneration and on (trans-)differentiation toward hormonal cells, and speculate on restorative therapies for pituitary deficiency.

Inflammatory and immunological aspects of dental pulp repair

PubMed Central

Goldberg, Michel; Farges, Jean-Christophe; Lacerda-Pinheiro, Sally; Six, Ngampis; Jegat, Nadège; Decup, Frank; Septier, Dominique; Carrouel, Florence; Durand, Stéphanie; Chaussain-Miller, Catherine; DenBesten, Pamela; Veis, Arthur; Poliard, Anne

2010-01-01

The repair of dental pulp by direct capping with calcium hydroxide or by implantation of bioactive extracellular matrix (ECM) molecules implies a cascade of four steps: a moderate inflammation, the commitment of adult reserve stem cells, their proliferation and terminal differentiation. The link between the initial inflammation and cell commitment is not yet well established but appears as a potential key factor in the reparative process. Either the release of cytokines due to inflammatory events activates resident stem (progenitor) cells, or inflammatory cells or pulp fibroblasts undergo a phenotypic conversion into osteoblast/odontoblast-like progenitors implicated in reparative dentin formation. Activation of antigen-presenting dendritic cells by mild inflammatory processes may also promote osteoblast/odontoblast-like differentiation and expression of ECM molecules implicated in mineralization. Recognition of bacteria by specific odontoblast and fibroblast membrane receptors triggers an inflammatory and immune response within the pulp tissue that would also modulate the repair process. PMID:18602009

In vitro induction effect of 1,25(OH)2D3 on differentiation of hair follicle stem cell into keratinocyte.

PubMed

Joulai Veijouyeh, Sanaz; Mashayekhi, Farhad; Yari, Abazar; Heidari, Fatemeh; Sajedi, Nayereh; Moghani Ghoroghi, Fatemeh; Nobakht, Maliheh

2017-02-01

Stem cells are characterized by self-renewal and differentiation capabilities. The bulge hair follicle stem cells (HFSCs) are able to convert to epithelial components. The active metabolite of vitamin D, 1,25(OH) 2 D 3 , plays important roles in this differentiation process. In the present study has found that 1,25(OH) 2 D 3 induces the HFSCs differentiation into keratinocyte. HFSCs are isolated from rat whiskers and cultivated in DMEM medium. To isolate bulge stem cell population, flow cytometry and immunocytochemistry using K15, CD34 and nestin biomarkers were performed. In order to accelerate the HFSCs differentiation into eratinocyte, HFSCs were treated with 10 -12 M, 1,25(OH) 2 D 3 every 48 h for a week. Immunocytochemistry results showed that bulge stem cells are nestin and CD34 positive but K15 negative before differentiation. Subsequently flow cytometry results, showed that the expression of nestin, CD34 and K15 were 70.96%, 93.03% and 6.88% respectively. After differentiation, the immunocytochemical and flow cytometry results indicated that differentiated cells have positive reaction to K15 with 68.94% expression level. It was concluded that 10 -12 M, 1,25(OH) 2 D 3 could induce the HFSCs differentiation into keratinocytes. Copyright © 2017 Chang Gung University. Published by Elsevier B.V. All rights reserved.

Three-dimensional co-culture process

NASA Technical Reports Server (NTRS)

Wolf, David A. (Inventor); Goodwin, Thomas J. (Inventor)

1992-01-01

The present invention relates to a 3-dimensional co-culture process, more particularly to methods or co-culturing at least two types of cells in a culture environment, either in space or in unit gravity, with minimum shear stress, freedom for 3-dimensional spatial orientation of the suspended particles and localization of particles with differing or similar sedimentation properties in a similar spatial region to form 3-dimensional tissue-like structures. Several examples of multicellular 3-dimensional experiences are included. The protocol and procedure are also set forth. The process allows simultaneous culture of multiple cell types and supporting substrates in a manner which does not disrupt the 3-dimensional spatial orientation of these components. The co-cultured cells cause a mutual induction effect which mimics the natural hormonal signals and cell interactions found in the intact organism. This causes the tissues to differentiate and form higher 3-dimensional structures such as glands, junctional complexes polypoid geometries, and microvilli which represent the corresponding in-vitro structures to a greater degree than when the cell types are cultured individually or by conventional processes. This process was clearly demonstrated for the case of two epithelial derived colon cancer lines, each co-cultured with normal human fibroblasts and with microcarrier bead substrates. The results clearly demonstrate increased 3-dimensional tissue-like structure and biochemical evidence of an increased differentiation state. With the present invention a variety of cells may be co-cultured to produce tissue which has 3-dimensionality and has some of the characteristics of in-vitro tissue. The process provides enhanced 3-dimensional tissue which create a multicellular organoid differentiation model.

Comparison of differentiation potential of male mouse adipose tissue and bone marrow derived-mesenchymal stem cells into germ cells

PubMed Central

Hosseinzadeh Shirzeily, Maryam; Pasbakhsh, Parichehr; Amidi, Fardin; Mehrannia, Kobra; Sobhani, Aligholi

2013-01-01

Background: Recent publications about differentiation of stem cells to germ cells have motivated researchers to make new approaches to infertility. In vitro production of germ cells improves understanding differentiation process of male and female germ cells. Due to the problem of using embryonic stem cells (ESC), it’s necessary the mentioned cells be replaced with some adult multi-potent stem cells in laboratories. Objective: The aim of this study was to obtain germ cells from appropriate source beyond ESC and compare differential potentials of adipocytes derived stem cells (ADMSCs) with bone marrow derived stem cells (BMMSCs). Materials and Methods: To find multi-potential entity, after providing purified ADMSCs and BMMSCs, differentiation to osteoblast and adipocyte was confirmed by using appropriate culture medium. To confirm mesenchymal lineage production superficial markers (expression of CD90 and CD44 and non-expression of CD45 and CD31) were investigated by flowcytometry. Then the cells were differentiated to germ cells in inductive medium containing retinoic acid for 7days. To evaluate germ cells characteristic markers [Dazl (Deleted in azoospermia-like), Mvh (Mouse vasa homolog gene), Stra8 (Stimulated by retinoic acid) and Scp3 (Synaptonemal complex protein 3)] flowcytometry, imunoflorescence and real time PCR were used. Results: Both types of cells were able to differentiate into osteoblast and adipocyte cells and presentation of stem cell superficial markers (CD90, CD44) and absence of endothelial and blood cell markers (CD31, CD45) were confirmative The flowcytometry, imunoflorescence and real time PCR results showed remarkable expression of germ cells characteristic markers (Mvh, Dazl, Stra8, and Scp3). Conclusion: It was found that although ADMSCs were attained easier and also cultured and differentiated rapidly, germ cell markers were expressed in BMMSCs significantly more than ADMSCs. This article extracted from M.Sc. thesis. (Maryam Hosseinzadeh Shirzeily) PMID:24639722

Differential proteome analysis of the cell differentiation regulated by BCC, CRH, CXCR4, GnRH, GPCR, IL1 signaling pathways in Chinese fire-bellied newt limb regeneration.

PubMed

Geng, Xiaofang; Xu, Tiantian; Niu, Zhipeng; Zhou, Xiaochun; Zhao, Lijun; Xie, Zhaohui; Xue, Deming; Zhang, Fuchun; Xu, Cunshuan

2014-01-01

Following amputation, the newt has the remarkable ability to regenerate its limb, and this process involves dedifferentiation, proliferation and differentiation. To investigate the potential proteome during a dynamic network of Chinese fire-bellied newt limb regeneration (CNLR), two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mass spectrum (MS) were applied to examine changes in the proteome that occurred at 11 time points after amputation. Meanwhile, several proteins were selected to validate their expression levels by Western blot. The results revealed that 1476 proteins had significantly changed as compared to the control group. Gene Ontology annotation and protein network analysis by Ingenuity Pathway Analysis 9.0 (IPA) software suggested that the differentially expressed proteins were involved in 33 kinds of physiological activities including signal transduction, cell proliferation, cell differentiation, etc. Among these proteins, 407 proteins participated in cell differentiation with 212 proteins in the differentiation of skin cell, myocyte, neurocyte, chondrocyte and osteocyte, and 37 proteins participated in signaling pathways of BCC, CRH, CXCR4, GnRH, GPCR and IL1 which regulated cell differentiation and redifferentiation. On the other hand, the signal transduction activity and cell differentiation activity were analyzed by IPA based on the changes in the expression of these proteins. The results showed that BCC, CRH, CXCR4, GnRH, GPCR and IL1 signaling pathways played an important role in regulating the differentiation of skin cell, myocyte, neurocyte, chondrocyte and osteocyte during CNLR. Copyright © 2014 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Closed-channel culture system for efficient and reproducible differentiation of human pluripotent stem cells into islet cells

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

Hirano, Kunio; Konagaya, Shuhei; Turner, Alexander

Human pluripotent stem cells (hPSCs) are thought to be a promising cell-source solution for regenerative medicine due to their indefinite proliferative potential and ability to differentiate to functional somatic cells. However, issues remain with regard to achieving reproducible differentiation of cells with the required functionality for realizing human transplantation therapies and with regard to reducing the potential for bacterial or fungal contamination. To meet these needs, we have developed a closed-channel culture device and corresponding control system. Uniformly-sized spheroidal hPSCs aggregates were formed inside wells within a closed-channel and maintained continuously throughout the culture process. Functional islet-like endocrine cell aggregatesmore » were reproducibly induced following a 30-day differentiation protocol. Our system shows an easily scalable, novel method for inducing PSC differentiation with both purity and functionality. - Highlights: • A simple, closed-channel-based, semi-automatic culture system is proposed. • Uniform cell aggregate formation and culture is realized in microwell structure. • Functional islet cells are successfully induced following 30-plus-day protocol. • System requires no daily medium replacement and reduces contamination risk.« less

Prostaglandin E2 promotes intestinal repair through an adaptive cellular response of the epithelium.

PubMed

Miyoshi, Hiroyuki; VanDussen, Kelli L; Malvin, Nicole P; Ryu, Stacy H; Wang, Yi; Sonnek, Naomi M; Lai, Chin-Wen; Stappenbeck, Thaddeus S

2017-01-04

Adaptive cellular responses are often required during wound repair. Following disruption of the intestinal epithelium, wound-associated epithelial (WAE) cells form the initial barrier over the wound. Our goal was to determine the critical factor that promotes WAE cell differentiation. Using an adaptation of our in vitro primary epithelial cell culture system, we found that prostaglandin E2 (PGE 2 ) signaling through one of its receptors, Ptger4, was sufficient to drive a differentiation state morphologically and transcriptionally similar to in vivo WAE cells. WAE cell differentiation was a permanent state and dominant over enterocyte differentiation in plasticity experiments. WAE cell differentiation was triggered by nuclear β-catenin signaling independent of canonical Wnt signaling. Creation of WAE cells via the PGE 2 -Ptger4 pathway was required in vivo, as mice with loss of Ptger4 in the intestinal epithelium did not produce WAE cells and exhibited impaired wound repair. Our results demonstrate a mechanism by which WAE cells are formed by PGE 2 and suggest a process of adaptive cellular reprogramming of the intestinal epithelium that occurs to ensure proper repair to injury. © 2016 The Authors.

Neutral dynamics and cell renewal of colonic crypts in homeostatic regime

NASA Astrophysics Data System (ADS)

Fendrik, A. J.; Romanelli, L.; Rotondo, E.

2018-05-01

The self renewal process in colonic crypts is the object of several studies. We present here a new compartment model with the following characteristics: (a) we distinguish different classes of cells: stem cells, six generations of transit amplifying cells and the differentiated cells; (b) in order to take into account the monoclonal character of crypts in homeostatic regimes we include symmetric divisions of the stem cells. We first consider the dynamic differential equations that describe the evolution of the mean values of the populations, but the small observed value of the total number of cells involved plus the huge dispersion of experimental data found in the literature leads us to study the stochastic discrete process. This analysis allows us to study fluctuations, the neutral drift that leads to monoclonality, and the effects of the fixation of mutant clones.

Prohibitin 2 Regulates the Proliferation and Lineage-Specific Differentiation of Mouse Embryonic Stem Cells in Mitochondria

PubMed Central

Komazaki, Shinji; Enomoto, Kei; Seki, Yasuhiro; Wang, Ying Ying; Ishigaki, Yohei; Ninomiya, Naoto; Noguchi, Taka-aki K.; Kokubu, Yuko; Ohnishi, Keigoh; Nakajima, Yoshiro; Kato, Kaoru; Intoh, Atsushi; Takada, Hitomi; Yamakawa, Norio; Wang, Pi-Chao; Asashima, Makoto; Kurisaki, Akira

2014-01-01

Background The pluripotent state of embryonic stem (ES) cells is controlled by a network of specific transcription factors. Recent studies also suggested the significant contribution of mitochondria on the regulation of pluripotent stem cells. However, the molecules involved in these regulations are still unknown. Methodology/Principal Findings In this study, we found that prohibitin 2 (PHB2), a pleiotrophic factor mainly localized in mitochondria, is a crucial regulatory factor for the homeostasis and differentiation of ES cells. PHB2 was highly expressed in undifferentiated mouse ES cells, and the expression was decreased during the differentiation of ES cells. Knockdown of PHB2 induced significant apoptosis in pluripotent ES cells, whereas enhanced expression of PHB2 contributed to the proliferation of ES cells. However, enhanced expression of PHB2 strongly inhibited ES cell differentiation into neuronal and endodermal cells. Interestingly, only PHB2 with intact mitochondrial targeting signal showed these specific effects on ES cells. Moreover, overexpression of PHB2 enhanced the processing of a dynamin-like GTPase (OPA1) that regulates mitochondrial fusion and cristae remodeling, which could induce partial dysfunction of mitochondria. Conclusions/Significance Our results suggest that PHB2 is a crucial mitochondrial regulator for homeostasis and lineage-specific differentiation of ES cells. PMID:24709813

Physalis angulata induces in vitro differentiation of murine bone marrow cells into macrophages.

PubMed

da Silva, Bruno José Martins; Rodrigues, Ana Paula D; Farias, Luis Henrique S; Hage, Amanda Anastácia P; Do Nascimento, Jose Luiz M; Silva, Edilene O

2014-10-03

The bone marrow is a hematopoietic tissue that, in the presence of cytokines and growth factors, generates all of the circulating blood cells. These cells are important for protecting the organism against pathogens and for establishing an effective immune response. Previous studies have shown immunomodulatory effects of different products isolated from plant extracts. This study aimed to evaluate the immunomodulatory properties of aqueous Physalis angulata (AEPa) extract on the differentiation of bone marrow cells. Increased cellular area, higher spreading ability and several cytoplasmatic projections were observed in the treated cells, using optical microscopy, suggesting cell differentiation. Furthermore, AEPa did not promote the proliferation of lymphocytes and polymorphonuclear leukocytes, however promotes increased the number of macrophages in the culture. The ultrastructural analysis by Transmission Electron Microscopy of treated cells showed spreading ability, high number of cytoplasmatic projections and increase of autophagic vacuoles. Moreover, a high level of LC3b expression by treated cells was detected by flow cytometry, suggesting an autophagic process. Cell surface expression of F4/80 and CD11b also indicated that AEPa may stimulate differentiation of bone marrow cells mainly into macrophages. In addition, AEPa did not differentiate cells into dendritic cells, as assessed by CD11c analysis. Furthermore, no cytotoxic effects were observed in the cells treated with AEPa. Results demonstrate that AEPa promotes the differentiation of bone marrow cells, particularly into macrophages and may hold promise as an immunomodulating agent.

Physalis angulata induces in vitro differentiation of murine bone marrow cells into macrophages

PubMed Central

2014-01-01

Background The bone marrow is a hematopoietic tissue that, in the presence of cytokines and growth factors, generates all of the circulating blood cells. These cells are important for protecting the organism against pathogens and for establishing an effective immune response. Previous studies have shown immunomodulatory effects of different products isolated from plant extracts. This study aimed to evaluate the immunomodulatory properties of aqueous Physalis angulata (AEPa) extract on the differentiation of bone marrow cells. Results Increased cellular area, higher spreading ability and several cytoplasmatic projections were observed in the treated cells, using optical microscopy, suggesting cell differentiation. Furthermore, AEPa did not promote the proliferation of lymphocytes and polymorphonuclear leukocytes, however promotes increased the number of macrophages in the culture. The ultrastructural analysis by Transmission Electron Microscopy of treated cells showed spreading ability, high number of cytoplasmatic projections and increase of autophagic vacuoles. Moreover, a high level of LC3b expression by treated cells was detected by flow cytometry, suggesting an autophagic process. Cell surface expression of F4/80 and CD11b also indicated that AEPa may stimulate differentiation of bone marrow cells mainly into macrophages. In addition, AEPa did not differentiate cells into dendritic cells, as assessed by CD11c analysis. Furthermore, no cytotoxic effects were observed in the cells treated with AEPa. Conclusion Results demonstrate that AEPa promotes the differentiation of bone marrow cells, particularly into macrophages and may hold promise as an immunomodulating agent. PMID:25281406

AKT1 provides an essential survival signal required for differentiation and stratification of primary human keratinocytes.

PubMed

Thrash, Barry R; Menges, Craig W; Pierce, Robert H; McCance, Dennis J

2006-04-28

Keratinocyte differentiation and stratification are complex processes involving multiple signaling pathways, which convert a basal proliferative cell into an inviable rigid squame. Loss of attachment to the basement membrane triggers keratinocyte differentiation, while in other epithelial cells, detachment from the extracellular matrix leads to rapid programmed cell death or anoikis. The potential role of AKT in providing a survival signal necessary for stratification and differentiation of primary human keratinocytes was investigated. AKT activity increased during keratinocyte differentiation and was attributed to the specific activation of AKT1 and AKT2. Targeted reduction of AKT1 expression, but not AKT2, by RNA interference resulted in an abnormal epidermis in organotypic skin cultures with a thin parabasal region and a pronounced but disorganized cornified layer. This abnormal stratification was due to significant cell death in the suprabasal layers and was alleviated by caspase inhibition. Normal expression patterns of both early and late markers of keratinocyte differentiation were also disrupted, producing a poorly developed stratum corneum.

Transient Downregulation of Nanog and Oct4 Induced by DETA/NO Exposure in Mouse Embryonic Stem Cells Leads to Mesodermal/Endodermal Lineage Differentiation

PubMed Central

Mora-Castilla, Sergio; Tejedo, Juan R.; Díaz, Irene; Hitos, Ana B.; Cahuana, Gladys M.; Hmadcha, Abdelkrim; Martín, Franz; Soria, Bernat

2014-01-01

The function of pluripotency genes in differentiation is a matter of investigation. We report here that Nanog and Oct4 are reexpressed in two mouse embryonic stem cell (mESC) lines following exposure to the differentiating agent DETA/NO. Both cell lines express a battery of both endoderm and mesoderm markers following induction of differentiation with DETA/NO-based protocols. Confocal analysis of cells undergoing directed differentiation shows that the majority of cells expressing Nanog express also endoderm genes such as Gata4 and FoxA2 (75.4% and 96.2%, resp.). Simultaneously, mRNA of mesodermal markers Flk1 and Mef2c are also regulated by the treatment. Acetylated histone H3 occupancy at the promoter of Nanog is involved in the process of reexpression. Furthermore, Nanog binding to the promoter of Brachyury leads to repression of this gene, thus disrupting mesendoderm transition. PMID:25544848

The Hippo pathway regulates human megakaryocytic differentiation.

PubMed

Lorthongpanich, Chanchao; Jiamvoraphong, Nittaya; Supraditaporn, Kantpitchar; Klaihmon, Phatchanat; U-Pratya, Yaowalak; Issaragrisil, Surapol

2017-01-05

The Hippo pathway is involved in several biological processes in both flies and mammals. Recent studies have shown that the Hippo pathway regulates Drosophila's haematopoiesis; however, understanding of its role in mammalian haematopoiesis is still limited. In flies, deletion of the Hippo component gene, Warts, affects crystal cell differentiation. We explored the role of the Hippo pathway in human haematopoiesis focusing on megakaryopoiesis. To investigate the role of LATS1/2 (a mammalian homolog of Warts) in human megakaryoblastic cell differentiation and platelet formation, megakaryoblastic cell (MEG-01) line was used as a model to gain insight into mechanism of the Hippo pathway in mammalian megakaryopoiesis. Effect of LATS1/2 on megakaryoblastic cell differentiation and platelet production were determined by functional changes. We found that depletion of LATS1/2 resulted in an increase of CD41 + megakaryocytes with impaired platelet biogenesis. Our study shows that the Hippo signalling pathway plays a crucial role in human megakaryoblastic cell differentiation and thrombopoiesis.

Enhanced osteogenic differentiation of MC3T3-E1 cells on grid-topographic surface and evidence for involvement of YAP mediator.

PubMed

Zhang, Yingying; Gong, He; Sun, Yan; Huang, Yan; Fan, Yubo

2016-05-01

Numerous studies have shown that surface topography can promote cell-substrate associations and deeply influence cell fate. The intracellular mechanism or how micro- or nano-patterned extracellular signal is ultimately linked to activity of nuclear transcription factors remains unknown. It has been reported that Yes-associated protein (YAP) can respond to extracellular matrix microenvironment signals, thus regulates stem cell differentiation process. We propose that YAP may play a role in mediating the topography induced cell differentiation. To this end, we fabricated polydimethylsiloxane (PDMS) micropatterns with grid topology (GT) (3 μm pattern width, 2 μm pattern interval length, 7 μm pattern height); nonpatterned PDMS substrates were used as the planar controls. The MC3T3-E1 cells were then cultured on these surfaces, respectively, in osteogenic inducing medium. Cell differentiation in terms of osteogenesis related gene expression, protein levels, alkaline phosphatase activity and extracellular matrix mineralization was assessed. It was shown that the cells on GT surfaces had stronger osteogenesis capacity. In addition, expression level of YAP was increased when MC3T3-E1 cells grew on GT substrates, which was similar to the levels of osteogenic differentiation markers. It was also shown that YAP knockdown attenuated GT substrates-induced MC3T3-E1 differentiation, which reduced the osteogenic differentiation effect of the GT substrates. Collectively, our findings indicate that GT substrates-induced MC3T3-E1 differentiation may be associated with YAP. This paper provides new target points for transcriptional mechanism research of microenvironment induced cell differentiation and a useful approach to obtain more biofunctionalization scaffolds for tissue engineering. © 2016 Wiley Periodicals, Inc.

Notch signaling pathways in human thoracic ossification of the ligamentum flavum.

PubMed

Qu, Xiaochen; Chen, Zhongqiang; Fan, Dongwei; Sun, Chuiguo; Zeng, Yan; Hou, Xiaofei; Ning, Shanglong

2016-08-01

This study investigated the pathological process of Notch signaling in the osteogenesis of ligamentum flavum tissues and cells, and the associated regulatory mechanisms. Notch receptors, ligands, and target genes were identified by quantitative polymerase chain reaction (qPCR) in ligamentum flavum cells and immunohistochemistry in ligamentum flavum sections from ossification of the ligamentum flavum (OLF) patients and controls. The temporospatial expression patterns of JAG1/Notch2/HES1 in human ligamentum flavum cells during osteogenic differentiation were determined by qPCR. Lentiviral vectors for Notch2 overexpression and knockdown were constructed and transfected into ligamentum flavum cells before osteogenic differentiation to examine the function of Notch signaling pathways in the osteogenic differentiation of ligamentum flavum cells. Alkaline phosphatase, Runx2, Osterix, osteocalcin, and osteopontin mRNA levels, alkaline phosphatase activity, and Alizarin Red staining were used as indicators of osteogenic differentiation. JAG1/Notch2/HES1 mRNA levels were up-regulated in ligamentum flavum cells from OLF patients, which increased during osteogenic differentiation. Immunohistochemical analysis suggested positive Notch2 expression at the ossification front. Down-regulation of Notch2 expression decelerated osteogenic differentiation of ligamentum flavum cells, and Notch2 overexpression promoted osteogenic differentiation of ligamentum flavum cells. Expression of Runx2 and Osterix increased in a manner similar to that of Notch2 during osteogenic differentiation of ligamentum flavum cells, and Notch2 knockdown and overexpression influenced their expression levels. Notch signaling plays an important role in OLF, and Notch may affect the osteogenic differentiation of ligamentum flavum cells via interactions with Runx2 and Osterix.© 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1481-1491, 2016. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Gravity, an Regulation Factor in BMSCs Differentiation to osteoblasts

NASA Astrophysics Data System (ADS)

Yan, Huang; Yinghui, Li; Fen, Yang; Zhongquan, Dai

PURPOSE Most studies of regulatory mechanisms of adult stem cell differentiation are concentrated in chemical factors but few efforts are put into physical factors Recent space life science studies indicate mechanical factors participate in the differentiation of cells The aim of this study is to investigate the effects of simulated microgravity or hypergravity on the osteogenic differentiation of rat bone marrow mesenchymal stem cells BMSCs METHODOLOGY The BMSCs at day 7 were added osteogenic inducer 10nM dexamethasone 10mM beta -glycerophosphate and 50 mu M asorbic acid-2-phosphate for 7 days and cultured under simulated microgravity or hypergravity 2g for 1 day 3 days 5 days or 7 days RESULTS After treating BMSCs with osteogenic inducer and hypergravity the cells expressed more ColIA1 Cbfa1 and ALP than in single steogenic inducer treatment Reversely the cells treated with osteogenic inducer and simulated microgravity expressed less ColIA1 Cbfa1 and ALP CONCLUSIONS Our study suggests that hypergravity promotes the osteogenic differentiation of BMSCs and simulated microgravity inhibits this process Gravity is an important regulation factor in BMSCs differentiation to osteoblasts

Loss of EGF binding and cation transport response during differentiation of mouse neuroblastoma cells.

PubMed

Mummery, C L; van der Saag, P T; de Laat, S W

1983-01-01

Mouse neuroblastoma cells (clone N1E-115) differentiate in culture upon withdrawal of serum growth factors and acquire the characteristics of neurons. We have shown tht exponentially growing N1E-115 cells possess functional epidermal growth factor (EGF) receptors but that the capacity for binding EGF and for stimulation of DNA synthesis is lost as the cells differentiate. Furthermore, in exponentially growing cells, EGF induces a rapid increase in amiloride-sensitive Na+ influx, followed by stimulation of the (Na+-K+)ATPase, indicating that activation of the Na+/H+ exchange mechanism in N1E-115 cells [1] may be induced by EGF. The ionic response is also lost during differentiation, but we have shown that the stimulation of both Na+ and K+ influx is directly proportional to the number of occupied receptors in all cells whether exponentially growing or differentiating, thus only indirectly dependent on the external EGF concentration. The linearity of the relationships indicates that there is no rate-limiting step between EGF binding and the ionic response. Our data would suggest that as neuroblastoma cells differentiate and acquire neuronal properties, their ability to respond to mitogens, both biologically and in the activation of cation transport processes, progressively decreases owing to the loss of the appropriate receptors.

Hepatocyte nuclear factor-4alpha induces transdifferentiation of hematopoietic cells into hepatocytes.

PubMed

Khurana, Satish; Jaiswal, Amit K; Mukhopadhyay, Asok

2010-02-12

Hematopoietic stem cells can directly transdifferentiate into hepatocytes because of cellular plasticity, but the molecular basis of transdifferentiation is not known. Here, we show the molecular basis using lineage-depleted oncostatin M receptor beta-expressing (Lin(-)OSMRbeta(+)) mouse bone marrow cells in a hepatic differentiation culture system. Differentiation of the cells was marked by the expression of albumin. Hepatocyte nuclear factor (HNF)-4alpha was expressed and translocated into the nuclei of the differentiating cells. Suppression of its activation in OSM-neutralized culture medium inhibited cellular differentiation. Ectopic expression of full-length HNF4alpha in 32D myeloid cells resulted in decreased myeloid colony-forming potential and increased expression of hepatocyte-specific genes and proteins. Nevertheless, the neohepatocytes produced in culture expressed active P450 enzyme. The obligatory role of HNF4alpha in hepatic differentiation was confirmed by transfecting Lin(-)OSMRbeta(+) cells with dominant negative HNF4alpha in the differentiation culture because its expression inhibited the transcription of the albumin and tyrosine aminotransferase genes. The loss and gain of functional activities strongly suggested that HNF4alpha plays a central role in the transdifferentiation process. For the first time, this report demonstrates the mechanism of transdifferentiation of hematopoietic cells into hepatocytes, in which HNF4alpha serves as a molecular switch.

Kinetics of the maintenance of the epidermis

NASA Astrophysics Data System (ADS)

Zhdanov, Vladimir P.; Cho, Nam-Joon

2013-08-01

The epidermis is the outermost layer of skin. It is comprised of keratin-containing cells called keratinocytes. Functionally, the epidermis serves as a physical barrier that can prevent infection and regulate body hydration. Maintenance and repair of the epidermis are important for human health. Mechanistically, these processes occur primarily via proliferation and differentiation of stem cells located in the basal monolayer. These processes are believed to depend on cell-cell communication and spatial constraints but existing kinetic models focus mainly on proliferation and differentiation. To address this issue, we present a mean-field kinetic model that takes these additional factors into account and describes the epidermis at a biosystem level. The corresponding equations operate with the populations of stem cells and differentiated cells in the basal layer. The keratinocytes located above the basal layer are treated at a more coarse-grained level by considering the thickness of the epidermis. The model clarifies the likely role of various negative feedbacks that may control the epidermis and, accordingly, provides insight into the cellular mechanisms underlying complex biological phenomena such as wound healing.

Involvement of Rictor/mTORC2 in cardiomyocyte differentiation of mouse embryonic stem cells in vitro

PubMed Central

Zheng, Bei; Wang, Jiadan; Tang, Leilei; Tan, Chao; Zhao, Zhe; Xiao, Yi; Ge, Renshan; Zhu, Danyan

2017-01-01

Rictor is a key regulatory/structural subunit of the mammalian target of rapamycin complex 2 (mTORC2) and is required for phosphorylation of Akt at serine 473. It plays an important role in cell survival, actin cytoskeleton organization and other processes in embryogenesis. However, the role of Rictor/mTORC2 in the embryonic cardiac differentiation has been uncovered. In the present study, we examined a possible link between Rictor expression and cardiomyocyte differentiation of the mouse embryonic stem (mES) cells. Knockdown of Rictor by shRNA significantly reduced the phosphorylation of Akt at serine 473 followed by a decrease in cardiomyocyte differentiation detected by beating embryoid bodies. The protein levels of brachyury (mesoderm protein), Nkx2.5 (cardiac progenitor cell protein) and α-Actinin (cardiomyocyte biomarker) decreased in Rictor knockdown group during cardiogenesis. Furthermore, knockdown of Rictor specifically inhibited the ventricular-like cells differentiation of mES cells with reduced level of ventricular-specific protein, MLC-2v. Meanwhile, patch-clamp analysis revealed that shRNA-Rictor significantly increased the number of cardiomyocytes with abnormal electrophysiology. In addition, the expressions and distribution patterns of cell-cell junction proteins (Cx43/Desmoplakin/N-cadherin) were also affected in shRNA-Rictor cardiomyocytes. Taken together, the results demonstrated that Rictor/mTORC2 might play an important role in the cardiomyocyte differentiation of mES cells. Knockdown of Rictor resulted in inhibiting ventricular-like myocytes differentiation and induced arrhythmias symptom, which was accompanied by interfering the expression and distribution patterns of cell-cell junction proteins. Rictor/mTORC2 might become a new target for regulating cardiomyocyte differentiation and a useful reference for application of the induced pluripotent stem cells. PMID:28123351

Well-differentiated liposarcoma of the tongue.

PubMed

Nunes, F D; Loducca, S V L; de Oliveira, E M F; de Araújo, V C

2002-01-01

Intraoral liposarcomas are rare, with most reported cases being of the myxoid histological type. We present a well-differentiated liposarcoma of the tongue, in a 65-year-old man. The tumour presented lipoblasts in various stages of differentiation, lipocytes in different sizes and shapes, mesenchymal and signet-ring cells. Lipoma, spindle-cell lipoma, myxoma, hibernoma, angiolipoma, fibrolipoma, pseudosarcomatous faciitis and malignant hysticytoma were considered in the diagnosis process. The patient was treated surgically and so far is free of disease.

Three-dimensional slice cultures from murine fetal gut for investigations of the enteric nervous system.

PubMed

Metzger, Marco; Bareiss, Petra M; Nikolov, Ivan; Skutella, Thomas; Just, Lothar

2007-01-01

Three-dimensional intestinal cultures offer new possibilities for the examination of growth potential, analysis of time specific gene expression, and spatial cellular arrangement of enteric nervous system in an organotypical environment. We present an easy to produce in vitro model of the enteric nervous system for analysis and manipulation of cellular differentiation processes. Slice cultures of murine fetal colon were cultured on membrane inserts for up to 2 weeks without loss of autonomous contractility. After slice preparation, cultured tissue reorganized within the first days in vitro. Afterward, the culture possessed more than 35 cell layers, including high prismatic epithelial cells, smooth muscle cells, glial cells, and neurons analyzed by immunohistochemistry. The contraction frequency of intestinal slice culture could be modulated by the neurotransmitter serotonin and the sodium channel blocker tetrodotoxin. Coculture experiments with cultured neurospheres isolated from enhanced green fluorescent protein (eGFP) transgenic mice demonstrated that differentiating eGFP-positive neurons were integrated into the intestinal tissue culture. This slice culture model of enteric nervous system proved to be useful for studying cell-cell interactions, cellular signaling, and cell differentiation processes in a three-dimensional cell arrangement.

Secretome analysis to elucidate metalloprotease-dependent ectodomain shedding of glycoproteins during neuronal differentiation.

PubMed

Tsumagari, Kazuya; Shirakabe, Kyoko; Ogura, Mayu; Sato, Fuminori; Ishihama, Yasushi; Sehara-Fujisawa, Atsuko

2017-02-01

Many membrane proteins are subjected to limited proteolyses at their juxtamembrane regions, processes referred to as ectodomain shedding. Shedding ectodomains of membrane-bound ligands results in activation of downstream signaling pathways, whereas shedding those of cell adhesion molecules causes loss of cell-cell contacts. Secreted proteomics (secretomics) using high-resolution mass spectrometry would be strong tools for both comprehensive identification and quantitative measurement of membrane proteins that undergo ectodomain shedding. In this study, to elucidate the ectodomain shedding events that occur during neuronal differentiation, we establish a strategy for quantitative secretomics of glycoproteins released from differentiating neuroblastoma cells into culture medium with or without GM6001, a broad-spectrum metalloprotease inhibitor. Considering that most of transmembrane and secreted proteins are N-glycosylated, we include a process of N-glycosylated peptides enrichment as well as isotope tagging in our secretomics workflow. Our results show that differentiating N1E-115 neurons secrete numerous glycosylated polypeptides in metalloprotease-dependent manners. They are derived from cell adhesion molecules such as NCAM1, CADM1, L1CAM, various transporters and receptor proteins. These results show the landscape of ectodomain shedding and other secretory events in differentiating neurons and/or during axon elongation, which should help elucidate the mechanism of neurogenesis and the pathogenesis of neurological disorders. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Placenta-an alternative source of stem cells

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

Matikainen, Tiina; Laine, Jarmo

2005-09-01

The two most promising practical applications of human stem cells are cellular replacement therapies in human disease and toxicological screening of candidate drug molecules. Both require a source of human stem cells that can be isolated, purified, expanded in number and differentiated into the cell type of choice in a controlled manner. Currently, uses of both embryonic and adult stem cells are investigated. While embryonic stem cells are pluripotent and can differentiate into any specialised cell type, their use requires establishment of embryonic stem cell lines using the inner cell mass of an early pre-implantation embryo. As the blastocyst ismore » destroyed during the process, ethical issues need to be carefully considered. The use of embryonic stem cells is also limited by the difficulties in growing large numbers of the cells without inducing spontaneous differentiation, and the problems in controlling directed differentiation of the cells. The use of adult stem cells, typically derived from bone marrow, but also from other tissues, is ethically non-controversial but their differentiation potential is more limited than that of the embryonic stem cells. Since human cord blood, umbilical cord, placenta and amnion are normally discarded at birth, they provide an easily accessible alternative source of stem cells. We review the potential and current status of the use of adult stem cells derived from the placenta or umbilical cord in therapeutic and toxicological applications.« less

T cell exit from quiescence and differentiation into Th2 cells depend on Raptor-mTORC1-mediated metabolic programming

PubMed Central

Yang, Kai; Shrestha, Sharad; Zeng, Hu; Karmaus, Peer W.F.; Neale, Geoffrey; Vogel, Peter; Guertin, David A.; Lamb, Richard F.; Chi, Hongbo

2014-01-01

SUMMARY Naïve T cells respond to antigen stimulation by exiting from quiescence and initiating clonal expansion and functional differentiation, but the control mechanism is elusive. Here we describe that Raptor-mTORC1-dependent metabolic programming is a central determinant of this transitional process. Loss of Raptor abrogated T cell priming and Th2 cell differentiation, although Raptor function is less important for continuous proliferation of actively cycling cells. mTORC1 coordinated multiple metabolic programs in T cells including glycolysis, lipid synthesis and oxidative phosphorylation to mediate antigen-triggered exit from quiescence. mTORC1 further linked glucose metabolism to the initiation of Th2 cell differentiation by orchestrating cytokine receptor expression and cytokine responsiveness. Activation of Raptor-mTORC1 integrated T cell receptor and CD28 co-stimulatory signals in antigen-stimulated T cells. Our studies identify a Raptor-mTORC1-dependent pathway linking signal-dependent metabolic reprogramming to quiescence exit, and this in turn coordinates lymphocyte activation and fate decisions in adaptive immunity. PMID:24315998

Secretomes reveal several novel proteins as well as TGF-β1 as the top upstream regulator of metastatic process in breast cancer.

PubMed

Erin, Nuray; Ogan, Nur; Yerlikaya, Azmi

2018-03-20

Metastatic breast cancer is resistant to many conventional treatments and novel therapeutic targets are needed. We previously isolated subsets of 4T1 murine breast cancer cells which metastasized to liver (4TLM), brain (4TBM), and heart (4THM). Among these cells, 4TLM is the most aggressive one, demonstrating mesenchymal phenotype. Here we compared secreted proteins from 4TLM, 4TBM, and 4THM cells and compared with that of hardly metastatic 67NR cells to detect differentially secreted factors involved in organ-specific metastasis. Label-free LC-MS/MS proteomic technique was used to detect the differentially secreted proteins. Eighty-five of over 500 secreted proteins were significantly altered in metastatic breast cancer cells. Differential expression of several proteins such as fibulin-4, Bone Morphogenetic Protein 1, TGF-β1 MMP-3, MMP-9, and Thymic Stromal Lymphopoietin were further verified using ELISA or Western blotting. Many of these identified proteins were also present in human metastatic breast carcinomas. Annexin A1 and A5, laminin beta 1, Neutral alpha-glucosidase AB were commonly found at least in three out of six studies examined here. Ingenuity Pathway Analysis showed that proteins differentially secreted from metastatic cells are involved primarily in carcinogenesis and TGF-β1 is the top upstream regulator in all metastatic cells. Cells metastasized to different organs displayed significant differences in several of secreted proteins. Proteins differentially altered were fibronectin, insulin-like growth factor-binding protein 7, and Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1. On the other hand, many exosomal proteins were also common to all metastatic cells, demonstrating involvement of key universal factors in distant metastatic process.

The cytoskeletal arrangements necessary to neurogenesis

PubMed Central

Compagnucci, Claudia; Piemonte, Fiorella; Sferra, Antonella; Piermarini, Emanuela; Bertini, Enrico

2016-01-01

During the process of neurogenesis, the stem cell committed to the neuronal cell fate starts a series of molecular and morphological changes. The understanding of the physio-pathology of mechanisms controlling the molecular and morphological changes occurring during neuronal differentiation is fundamental to the development of effective therapies for many neurologic diseases. Unfortunately, our knowledge of the biological events occurring in the cell during neuronal differentiation is still poor. In this study, we focus preliminarily on the relevance of the cytoskeletal rearrangements, which earlier drive the morphology of the neuronal precursors, and later the migrating/mature neurons. In fact, neuritogenesis, neurite branching, outgrowth and retraction are seminal to the development of a fully functional nervous system. With this in mind, we highlight the importance of iPSC technology to study the processes of cytoskeletal-driven morphological changes during neuronal differentiation. PMID:26760504

Vinpocetine Attenuates the Osteoblastic Differentiation of Vascular Smooth Muscle Cells.

PubMed

Ma, Yun-Yun; Sun, Lin; Chen, Xiu-Juan; Wang, Na; Yi, Peng-Fei; Song, Min; Zhang, Bo; Wang, Yu-Zhong; Liang, Qiu-Hua

2016-01-01

Vascular calcification is an active process of osteoblastic differentiation of vascular smooth muscle cells; however, its definite mechanism remains unknown. Vinpocetine, a derivative of the alkaloid vincamine, has been demonstrated to inhibit the high glucose-induced proliferation of vascular smooth muscle cells; however, it remains unknown whether vinpocetine can affect the osteoblastic differentiation of vascular smooth muscle cells. We hereby investigated the effect of vinpocetine on vascular calcification using a beta-glycerophosphate-induced cell model. Our results showed that vinpocetine significantly reduced the osteoblast-like phenotypes of vascular smooth muscle cells including ALP activity, osteocalcin, collagen type I, Runx2 and BMP-2 expression as well as the formation of mineralized nodule. Vinpocetine, binding to translocation protein, induced phosphorylation of extracellular signal-related kinase and Akt and thus inhibited the translocation of nuclear factor-kappa B into the nucleus. Silencing of translocator protein significantly attenuated the inhibitory effect of vinpocetine on osteoblastic differentiation of vascular smooth muscle cells. Taken together, vinpocetine may be a promising candidate for the clinical therapy of vascular calcification.

Developing defined substrates for stem cell culture and differentiation.

PubMed

Hagbard, Louise; Cameron, Katherine; August, Paul; Penton, Christopher; Parmar, Malin; Hay, David C; Kallur, Therése

2018-07-05

Over the past few decades, a variety of different reagents for stem cell maintenance and differentiation have been commercialized. These reagents share a common goal in facilitating the manufacture of products suitable for cell therapy while reducing the amount of non-defined components. Lessons from developmental biology have identified signalling molecules that can guide the differentiation process in vitro , but less attention has been paid to the extracellular matrix used. With the introduction of more biologically relevant and defined matrices, that better mimic specific cell niches, researchers now have powerful resources to fine-tune their in vitro differentiation systems, which may allow the manufacture of therapeutically relevant cell types. In this review article, we revisit the basics of the extracellular matrix, and explore the important role of the cell-matrix interaction. We focus on laminin proteins because they help to maintain pluripotency and drive cell fate specification.This article is part of the theme issue 'Designer human tissue: coming to a lab near you'. © 2018 The Authors.

Reversible immortalization of Nestin-positive precursor cells from pancreas and differentiation into insulin-secreting cells

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

Wei, Pei; Li, Li; Qi, Hui

2012-02-10

Highlights: Black-Right-Pointing-Pointer The NPPCs from mouse pancreas were isolated. Black-Right-Pointing-Pointer Tet-on system for SV40 large in NPPCs was used to get RINPPCs. Black-Right-Pointing-Pointer The RINPPCs can undergo at least 80 population doublings without senescence. Black-Right-Pointing-Pointer The RINPPCs can be induced to differentiate into insulin-producing cells. Black-Right-Pointing-Pointer The combination of GLP-1 and sodium butyrate promoted the differentiation process. -- Abstract: Pancreatic stem cells or progenitor cells posses the ability of directed differentiation into pancreatic {beta} cells. However, these cells usually have limited proliferative capacity and finite lifespan in vitro. In the present study, Nestin-positive progenitor cells (NPPCs) from mouse pancreas thatmore » expressed the pancreatic stem cells or progenitor cell marker Nestin were isolated to obtain a sufficient number of differentiated pancreatic {beta} cells. Tet-on system for SV40 large T-antigen expression in NPPCs was used to achieve reversible immortalization. The reversible immortal Nestin-positive progenitor cells (RINPPCs) can undergo at least 80 population doublings without senescence in vitro while maintaining their biological and genetic characteristics. RINPPCs can be efficiently induced to differentiate into insulin-producing cells that contain a combination of glucagon-like peptide-1 (GLP-1) and sodium butyrate. The results of the present study can be used to explore transplantation therapy of type I diabetes mellitus.« less

Dynamic Network-Based Relevance Score Reveals Essential Proteins and Functional Modules in Directed Differentiation

PubMed Central

Wu, Chia-Chou; Lin, Che

2015-01-01

The induction of stem cells toward a desired differentiation direction is required for the advancement of stem cell-based therapies. Despite successful demonstrations of the control of differentiation direction, the effective use of stem cell-based therapies suffers from a lack of systematic knowledge regarding the mechanisms underlying directed differentiation. Using dynamic modeling and the temporal microarray data of three differentiation stages, three dynamic protein-protein interaction networks were constructed. The interaction difference networks derived from the constructed networks systematically delineated the evolution of interaction variations and the underlying mechanisms. A proposed relevance score identified the essential components in the directed differentiation. Inspection of well-known proteins and functional modules in the directed differentiation showed the plausibility of the proposed relevance score, with the higher scores of several proteins and function modules indicating their essential roles in the directed differentiation. During the differentiation process, the proteins and functional modules with higher relevance scores also became more specific to the neuronal identity. Ultimately, the essential components revealed by the relevance scores may play a role in controlling the direction of differentiation. In addition, these components may serve as a starting point for understanding the systematic mechanisms of directed differentiation and for increasing the efficiency of stem cell-based therapies. PMID:25977693

Death penalty for keratinocytes: apoptosis versus cornification.

PubMed

Lippens, S; Denecker, G; Ovaere, P; Vandenabeele, P; Declercq, W

2005-11-01

Homeostasis implies a balance between cell growth and cell death. This balance is essential for the development and maintenance of multicellular organisms. Homeostasis is controlled by several mechanisms including apoptosis, a process by which cells condemned to death are completely eliminated. However, in some cases, total destruction and removal of dead cells is not desirable, as when they fulfil a specific function such as formation of the skin barrier provided by corneocytes, also known as terminally differentiated keratinocytes. In this case, programmed cell death results in accumulation of functional cell corpses. Previously, this process has been associated with apoptotic cell death. In this overview, we discuss differences and similarities in the molecular regulation of epidermal programmed cell death and apoptosis. We conclude that despite earlier confusion, apoptosis and cornification occur through distinct molecular pathways, and that possibly antiapoptotic mechanisms are implicated in the terminal differentiation of keratinocytes.

A zinc finger protein Zfp521 directs neural differentiation and beyond

PubMed Central

2011-01-01

Neural induction is largely considered a default process, whereas little is known about intrinsic factors that drive neural differentiation. Kamiya and colleagues now demonstrate that a transcription factor, Zfp521, is capable of directing embryonic stem (ES) cells into neural progenitors. They discovered that Zfp521 transcripts were enriched in early neural lineage of ES cell differentiation. Forced expression of Zfp521 turned ES cells into neural progenitors in culture conditions that would normally inhibit neural differentiation. Zfp521 was expressed in mouse embryos during gastrulation. The protein was shown to associate with a co-activator p300 and directly induce expression of early neural genes. Knockdown of the Zfp521 by shRNA halted cells at the epiblast stage and suppressed neural differentiation. Zfp521 is a nuclear protein with 30 Krüppel-like zinc fingers mediating multiple protein-protein interactions, and regulates transcription in diverse tissues and organs. The protein promotes proliferation, delays differentiation and reduces apoptosis. The findings by Kamiya and colleagues that Zfp521 directs and sustains early neural differentiation now opens up a series of studies to investigate roles of Zfp521 in stem cells and brain development of mice and men. PMID:21539723

Establishment of a translational endothelial cell model using directed differentiation of induced pluripotent stem cells from Cynomolgus monkey.

PubMed

Thoma, Eva C; Heckel, Tobias; Keller, David; Giroud, Nicolas; Leonard, Brian; Christensen, Klaus; Roth, Adrian; Bertinetti-Lapatki, Cristina; Graf, Martin; Patsch, Christoph

2016-10-25

Due to their broad differentiation potential, pluripotent stem cells (PSCs) offer a promising approach for generating relevant cellular models for various applications. While human PSC-based cellular models are already advanced, similar systems for non-human primates (NHPs) are still lacking. However, as NHPs are the most appropriate animals for evaluating the safety of many novel pharmaceuticals, the availability of in vitro systems would be extremely useful to bridge the gap between cellular and animal models. Here, we present a NHP in vitro endothelial cell system using induced pluripotent stem cells (IPSCs) from Cynomolgus monkey (Macaca fascicularis). Based on an adapted protocol for human IPSCs, we directly differentiated macaque IPSCs into endothelial cells under chemically defined conditions. The resulting endothelial cells can be enriched using immuno-magnetic cell sorting and display endothelial marker expression and function. RNA sequencing revealed that the differentiation process closely resembled vasculogenesis. Moreover, we showed that endothelial cells derived from macaque and human IPSCs are highly similar with respect to gene expression patterns and key endothelial functions, such as inflammatory responses. These data demonstrate the power of IPSC differentiation technology to generate defined cell types for use as translational in vitro models to compare cell type-specific responses across species.

Hanging drop culture enhances differentiation of human adipose-derived stem cells into anterior neuroectodermal cells using small molecules.

PubMed

Amirpour, Noushin; Razavi, Shahnaz; Esfandiari, Ebrahim; Hashemibeni, Batoul; Kazemi, Mohammad; Salehi, Hossein

2017-06-01

Inspired by in vivo developmental process, several studies were conducted to design a protocol for differentiating of mesenchymal stem cells into neural cells in vitro. Human adipose-derived stem cells (hADSCs) as mesenchymal stem cells are a promising source for this purpose. At current study, we applied a defined neural induction medium by using small molecules for direct differentiation of hADSCs into anterior neuroectodermal cells. Anterior neuroectodermal differentiation of hADSCs was performed by hanging drop and monolayer protocols. At these methods, three small molecules were used to suppress the BMP, Nodal, and Wnt signaling pathways in order to obtain anterior neuroectodermal (eye field) cells from hADSCs. After two and three weeks of induction, the differentiated cells with neural morphology expressed anterior neuroectodermal markers such as OTX2, SIX3, β-TUB III and PAX6. The protein expression of such markers was confirmed by real time, RT-PCR and immunocytochemistry methods According to our data, it seems that the hanging drop method is a proper approach for neuroectodermal induction of hADSCs. Considering wide availability and immunosuppressive properties of hADSCs, these cells may open a way for autologous cell therapy of neurodegenerative disorders. Copyright © 2017 ISDN. Published by Elsevier Ltd. All rights reserved.

p53-Regulated Apoptosis Is Differentiation Dependent in Ultraviolet B-Irradiated Mouse Keratinocytes

PubMed Central

Tron, Victor A.; Trotter, Martin J.; Tang, Liren; Krajewska, Maryla; Reed, John C.; Ho, Vincent C.; Li, Gang

1998-01-01

Previous studies from our laboratory, using p53 transgenic mice, have suggested that ultraviolet (UV) light-induced keratinocyte apoptosis in the skin is not affected by overexpression of mutant p53 protein. To further elucidate a possible role for p53 in UV-induced keratinocyte cell death, we now examine apoptosis in skin and isolated keratinocytes from p53 null (−/−) mice and assess the influence of cell differentiation on this process. In vivo, using this knockout model, epidermal keratinocytes in p53−/− mice exhibited only a 5.2-fold increase in apoptosis after 2000 J/m2 UVB irradiation compared with a 26.3-fold increase in normal control animals. If this p53-dependent apoptosis is important in elimination of precancerous, UV-damaged keratinocytes, then it should be active in the undifferentiated cells of the epidermal basal layer. To test this hypothesis, we examined the effect of differentiation on UV-induced apoptosis in primary cultures of murine and human keratinocytes. Apoptosis was p53-independent in undifferentiated murine keratinocytes, which exhibited relative resistance to UVB-induced killing with only a 1.5-fold increase in apoptosis in p53+/+ cells and a 1.4-fold increase in p53−/− cells. Differentiated keratinocytes, in contrast, showed a 9.4-fold UVB induction of apoptosis in p53+/+ cells, almost three times the induction observed in p53−/− cells. This UV-induced difference in apoptosis was observed when keratinocytes were cultured on type IV collagen substrate, but not on plastic alone. Western blotting of UV-irradiated, differentiated keratinocytes did not support a role for either Bax or Bcl-2 in this process. In support of these findings in mice, cell death in human cultured keratinocytes also occurred in a differentiation-associated fashion. We conclude that p53-induced apoptosis eliminates damaged keratinocytes in the differentiated cell compartment, but this mechanism is not active in the basal, undifferentiated cells and is therefore of questionable significance in protection against skin cancer induction. PMID:9708817

Neuropeptides and epitheliopeptides: structural and functional diversity in an ancestral metazoan Hydra.

PubMed

Takahashi, Toshio

2013-06-01

Peptides are known to play important developmental and physiological roles in signaling. The rich diversity of peptides, with functions as diverse as intercellular communication, neurotransmission and signaling that spatially and temporally controls axis formation and cell differentiation, hints at the wealth of information passed between interacting cells. Little is known about peptides that control developmental processes such as cell differentiation and pattern formation in metazoans. The cnidarian Hydra is one of the most basic metazoans and is a key model system for study of the peptides involved in these processes. We developed a novel peptidomic approach for the isolation and identification of functional peptide signaling molecules from Hydra (the Hydra Peptide Project). Over the course of this project, a wide variety of novel neuropeptides were identified. Most of these peptides act directly on muscle cells and their functions include induction of contraction and relaxation. Some peptides are involved in cell differentiation and morphogenesis. Moreover, epitheliopeptides that are produced by epithelial cells were originally identified in Hydra. Some of these epitheliopeptides exhibit morphogen-like activities, whereas others are involved in regulating neuron differentiation, possibly through neuron-epithelial cell interactions. We also describe below our high-throughput reverse-phase nano-flow LCMALDI- TOF-MS/MS approach, which has proved a powerful tool for the discovery of novel peptide signaling molecules in Hydra.

Dissecting Stages of Human Kidney Development and Tumorigenesis with Surface Markers Affords Simple Prospective Purification of Nephron Stem Cells.

PubMed

Pode-Shakked, Naomi; Pleniceanu, Oren; Gershon, Rotem; Shukrun, Rachel; Kanter, Itamar; Bucris, Efrat; Pode-Shakked, Ben; Tam, Gal; Tam, Hadar; Caspi, Revital; Pri-Chen, Sara; Vax, Einav; Katz, Guy; Omer, Dorit; Harari-Steinberg, Orit; Kalisky, Tomer; Dekel, Benjamin

2016-03-29

When assembling a nephron during development a multipotent stem cell pool becomes restricted as differentiation ensues. A faulty differentiation arrest in this process leads to transformation and initiation of a Wilms' tumor. Mapping these transitions with respective surface markers affords accessibility to specific cell subpopulations. NCAM1 and CD133 have been previously suggested to mark human renal progenitor populations. Herein, using cell sorting, RNA sequencing, in vitro studies with serum-free media and in vivo xenotransplantation we demonstrate a sequential map that links human kidney development and tumorigenesis; In nephrogenesis, NCAM1(+)CD133(-) marks SIX2(+) multipotent renal stem cells transiting to NCAM1(+)CD133(+) differentiating segment-specific SIX2(-) epithelial progenitors and NCAM1(-)CD133(+) differentiated nephron cells. In tumorigenesis, NCAM1(+)CD133(-) marks SIX2(+) blastema that includes the ALDH1(+) WT cancer stem/initiating cells, while NCAM1(+)CD133(+) and NCAM1(-)CD133(+) specifying early and late epithelial differentiation, are severely restricted in tumor initiation capacity and tumor self-renewal. Thus, negative selection for CD133 is required for defining NCAM1(+) nephron stem cells in normal and malignant nephrogenesis.

A Rapid Filter Insert-based 3D Culture System for Primary Prostate Cell Differentiation

PubMed Central

Tricoli, Lucas; Berry, Deborah L.; Albanese, Chris

2018-01-01

Conditionally reprogrammed cells (CRCs) provide a sustainable method for primary cell culture and the ability to develop extensive “living biobanks” of patient derived cell lines. For many types of epithelial cells, various three dimensional (3D) culture approaches have been described that support an improved differentiated state. While CRCs retain their lineage commitment to the tissue from which they are isolated, they fail to express many of the differentiation markers associated with the tissue of origin when grown under normal two dimensional (2D) culture conditions. To enhance the application of patient-derived CRCs for prostate cancer research, a 3D culture format has been defined that enables a rapid (2 weeks total) luminal cell differentiation in both normal and tumor-derived prostate epithelial cells. Herein, a filter insert-based format is described for the culturing and differentiation of both normal and malignant prostate CRCs. A detailed description of the procedures required for cell collection and processing for immunohistochemical and immunofluorescent staining are provided. Collectively the 3D culture format described, combined with the primary CRC lines, provides an important medium- to high- throughput model system for biospecimen-based prostate research. PMID:28287583

AN ELECTRON MICROSCOPE STUDY OF MATURE AND DIFFERENTIATING PANETH CELLS IN THE RAT, ESPECIALLY OF THEIR ENDOPLASMIC RETICULUM AND LYSOSOMES

PubMed Central

Behnke, O.; Moe, H.

1964-01-01

In an electron microsope study, the morphology of mature Paneth cells from the small intestine of adult rats is compared with that of differentiating Paneth cells from young rats 2 to 4 weeks old. All mature cells exhibit a marked polarity similar to that of other exocrine gland cells and contain a well developed endoplasmic reticulum, an elaborate Golgi complex, and numerous large secretory granules; they also possess an abundance of lysosomes. The most conspicuous occurrence in the process of differentiation is the development of the endoplasmic reticulum. The most immature Paneth cells possess an endoplasmic reticulum of the vesicular type, which, during maturation, is replaced by the characteristic lamellated ergastoplasm of the mature cell. At a certain stage of differentiation the cavities of the developing cisternae show numerous communications with the perinuclear space, suggesting an outgrowth of the ergastoplasm from the nuclear envelope. Furthermore, the cavities and the perinuclear space at this particular stage contain a material which shows a remarkable intrinsic periodicity. An identical periodicity was exhibited by material contained in Golgi cisternae and secretory granules. Lysosomes are also present in the differentiating cells. PMID:14206428

Stochastic cellular automata model of cell migration, proliferation and differentiation: validation with in vitro cultures of muscle satellite cells.

PubMed

Garijo, N; Manzano, R; Osta, R; Perez, M A

2012-12-07

Cell migration and proliferation has been modelled in the literature as a process similar to diffusion. However, using diffusion models to simulate the proliferation and migration of cells tends to create a homogeneous distribution in the cell density that does not correlate to empirical observations. In fact, the mechanism of cell dispersal is not diffusion. Cells disperse by crawling or proliferation, or are transported in a moving fluid. The use of cellular automata, particle models or cell-based models can overcome this limitation. This paper presents a stochastic cellular automata model to simulate the proliferation, migration and differentiation of cells. These processes are considered as completely stochastic as well as discrete. The model developed was applied to predict the behaviour of in vitro cell cultures performed with adult muscle satellite cells. Moreover, non homogeneous distribution of cells has been observed inside the culture well and, using the above mentioned stochastic cellular automata model, we have been able to predict this heterogeneous cell distribution and compute accurate quantitative results. Differentiation was also incorporated into the computational simulation. The results predicted the myotube formation that typically occurs with adult muscle satellite cells. In conclusion, we have shown how a stochastic cellular automata model can be implemented and is capable of reproducing the in vitro behaviour of adult muscle satellite cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

Early transcriptional and epigenetic regulation of CD8+ T cell differentiation revealed by single-cell RNA-seq

PubMed Central

Kakaradov, Boyko; Arsenio, Janilyn; Widjaja, Christella E.; He, Zhaoren; Aigner, Stefan; Metz, Patrick J.; Yu, Bingfei; Wehrens, Ellen J.; Lopez, Justine; Kim, Stephanie H.; Zuniga, Elina I.; Goldrath, Ananda W.; Chang, John T.; Yeo, Gene W.

2017-01-01

SUMMARY During microbial infection, responding CD8+ T lymphocytes differentiate into heterogeneous subsets that together provide immediate and durable protection. To elucidate the dynamic transcriptional changes that underlie this process, we applied a single-cell RNA sequencing approach and analyzed individual CD8+ T lymphocytes sequentially throughout the course of a viral infection in vivo. Our analyses revealed a striking transcriptional divergence among cells that had undergone their first division and identified previously unknown molecular determinants controlling CD8+ T lymphocyte fate specification. These findings suggest a model of terminal effector cell differentiation initiated by an early burst of transcriptional activity and subsequently refined by epigenetic silencing of transcripts associated with memory lymphocytes, highlighting the power and necessity of single-cell approaches. PMID:28218746

MYB36 regulates the transition from proliferation to differentiation in the Arabidopsis root

PubMed Central

Liberman, Louisa M.; Sparks, Erin E.; Moreno-Risueno, Miguel A.; Petricka, Jalean J.; Benfey, Philip N.

2015-01-01

Stem cells are defined by their ability to self-renew and produce daughter cells that proliferate and mature. These maturing cells transition from a proliferative state to a terminal state through the process of differentiation. In the Arabidopsis thaliana root the transcription factors SCARECROW and SHORTROOT regulate specification of the bipotent stem cell that gives rise to cortical and endodermal progenitors. Subsequent progenitor proliferation and differentiation generate mature endodermis, marked by the Casparian strip, a cell-wall modification that prevents ion diffusion into and out of the vasculature. We identified a transcription factor, MYB DOMAIN PROTEIN 36 (MYB36), that regulates the transition from proliferation to differentiation in the endodermis. We show that SCARECROW directly activates MYB36 expression, and that MYB36 likely acts in a feed-forward loop to regulate essential Casparian strip formation genes. We show that myb36 mutants have delayed and defective barrier formation as well as extra divisions in the meristem. Our results demonstrate that MYB36 is a critical positive regulator of differentiation and negative regulator of cell proliferation. PMID:26371322

Cell-Cell Contact Area Affects Notch Signaling and Notch-Dependent Patterning.

PubMed

Shaya, Oren; Binshtok, Udi; Hersch, Micha; Rivkin, Dmitri; Weinreb, Sheila; Amir-Zilberstein, Liat; Khamaisi, Bassma; Oppenheim, Olya; Desai, Ravi A; Goodyear, Richard J; Richardson, Guy P; Chen, Christopher S; Sprinzak, David

2017-03-13

During development, cells undergo dramatic changes in their morphology. By affecting contact geometry, these morphological changes could influence cellular communication. However, it has remained unclear whether and how signaling depends on contact geometry. This question is particularly relevant for Notch signaling, which coordinates neighboring cell fates through direct cell-cell signaling. Using micropatterning with a receptor trans-endocytosis assay, we show that signaling between pairs of cells correlates with their contact area. This relationship extends across contact diameters ranging from micrometers to tens of micrometers. Mathematical modeling predicts that dependence of signaling on contact area can bias cellular differentiation in Notch-mediated lateral inhibition processes, such that smaller cells are more likely to differentiate into signal-producing cells. Consistent with this prediction, analysis of developing chick inner ear revealed that ligand-producing hair cell precursors have smaller apical footprints than non-hair cells. Together, these results highlight the influence of cell morphology on fate determination processes. Copyright © 2017 Elsevier Inc. All rights reserved.

Effect of aging on stem cells

PubMed Central

Ahmed, Abu Shufian Ishtiaq; Sheng, Matilda HC; Wasnik, Samiksha; Baylink, David J; Lau, Kin-Hing William

2017-01-01

Pluripotent stem cells have the remarkable self-renewal ability and are capable of differentiating into multiple diverse cells. There is increasing evidence that the aging process can have adverse effects on stem cells. As stem cells age, their renewal ability deteriorates and their ability to differentiate into the various cell types is altered. Accordingly, it is suggested aging-induced deterioration of stem cell functions may play a key role in the pathophysiology of the various aging-associated disorders. Understanding the role of the aging process in deterioration of stem cell function is crucial, not only in understanding the pathophysiology of aging-associated disorders, but also in future development of novel effective stem cell-based therapies to treat aging-associated diseases. This review article first focuses on the basis of the various aging disease-related stem cell dysfunction. It then addresses the several concepts on the potential mechanism that causes aging-related stem cell dysfunction. It also briefly discusses the current potential therapies under development for aging-associated stem cell defects. PMID:28261550

Cell-cell contact area affects Notch signaling and Notch-dependent patterning

PubMed Central

Shaya, Oren; Binshtok, Udi; Hersch, Micha; Rivkin, Dmitri; Weinreb, Sheila; Amir-Zilberstein, Liat; Khamaisi, Bassma; Oppenheim, Olya; Desai, Ravi A.; Goodyear, Richard J.; Richardson, Guy P.; Chen, Christopher S.; Sprinzak, David

2017-01-01

Summary During development, cells undergo dramatic changes in their morphology. By affecting contact geometry, these morphological changes could influence cellular communication. However, it has remained unclear whether and how signaling depends on contact geometry. This question is particularly relevant for Notch signaling, which coordinates neighboring cell fates through direct cell-cell signaling. Using micropatterning with a receptor trans-endocytosis assay, we show that signaling between pairs of cells correlates with their contact area. This relationship extends across contact diameters ranging from microns to tens of microns. Mathematical modeling predicts that dependence of signaling on contact area can bias cellular differentiation in Notch-mediated lateral inhibition processes, such that smaller cells are more likely to differentiate into signal-producing cells. Consistent with this prediction, analysis of developing chick inner ear revealed that ligand-producing hair cell precursors have smaller apical footprints than non-hair cells. Together, these results highlight the influence of cell morphology on fate determination processes. PMID:28292428

Role of fibroblast growth factor receptors (FGFR) and FGFR like-1 (FGFRL1) in mesenchymal stromal cell differentiation to osteoblasts and adipocytes.

PubMed

Kähkönen, T E; Ivaska, K K; Jiang, M; Büki, K G; Väänänen, H K; Härkönen, P L

2018-02-05

Fibroblast growth factors (FGF) and their receptors (FGFRs) regulate many developmental processes including differentiation of mesenchymal stromal cells (MSC). We developed two MSC lines capable of differentiating to osteoblasts and adipocytes and studied the role of FGFRs in this process. We identified FGFR2 and fibroblast growth factor receptor like-1 (FGFRL1) as possible actors in MSC differentiation with gene microarray and qRT-PCR. FGFR2 and FGFRL1 mRNA expression strongly increased during MSC differentiation to osteoblasts. FGF2 treatment, resulting in downregulation of FGFR2, or silencing FGFR2 expression with siRNAs inhibited osteoblast differentiation. During adipocyte differentiation expression of FGFR1 and FGFRL1 increased and was down-regulated by FGF2. FGFR1 knockdown inhibited adipocyte differentiation. Silencing FGFR2 and FGFR1 in MSCs was associated with decreased FGFRL1 expression in osteoblasts and adipocytes, respectively. Our results suggest that FGFR1 and FGFR2 regulate FGFRL1 expression. FGFRL1 may mediate or modulate FGFR regulation of MSC differentiation together with FGFR2 in osteoblastic and FGFR1 in adipocytic lineage. Copyright © 2017 Elsevier B.V. All rights reserved.

Mesenchymal stem cell-derived exosomes have altered microRNA profiles and induce osteogenic differentiation depending on the stage of differentiation

PubMed Central

Wang, Xiaoqin; Omar, Omar; Vazirisani, Forugh; Thomsen, Peter

2018-01-01

Human mesenchymal stem cell (hMSC)-derived exosomes have shown regenerative effects, but their role in osteogenesis and the underlying mechanism are yet to be determined. In this study, we examined the time-course secretion of exosomes by hMSCs during the entire process of osteogenic differentiation. Exosomes derived from hMSCs in various stages of osteogenic differentiation committed homotypic cells to differentiate towards osteogenic lineage, but only exosomes from late stages of osteogenic differentiation induced extracellular matrix mineralisation. Exosomes from expansion and early and late stages of osteogenic differentiation were internalised by a subpopulation of hMSCs. MicroRNA profiling revealed a set of differentially expressed exosomal microRNAs from the late stage of osteogenic differentiation, which were osteogenesis related. Target prediction demonstrated that these microRNAs enriched pathways involved in regulation of osteogenic differentiation and general mechanisms how exosomes exert their functions, such as “Wnt signalling pathway” and “endocytosis”. Taken together, the results show that MSCs secrete exosomes with different biological properties depending on differentiation stage of their parent cells. The exosomal cargo transferred from MSCs in the late stage of differentiation induces osteogenic differentiation and mineralisation. Moreover, it is suggested that the regulatory effect on osteogenesis by exosomes is at least partly exerted by exosomal microRNA. PMID:29447276

Induction of hepatocyte-like cells from mouse embryonic stem cells by lentivirus-mediated constitutive expression of Foxa2/Hnf4a.

PubMed

Liu, Tao; Zhang, Shichang; Xiang, Dedong; Wang, Yingjie

2013-11-01

Hepatocytes can be generated from embryonic stem cells (ESCs) using inducers such as chemical compounds and cytokines, but issues related to low differentiation efficiencies remain to be resolved. Recent work has shown that overexpression of lineage-specific transcription factors can directly cause cells phenotypic changes, including differentiation, trans-differentiation, and de-differentiation. We hypothesized that lentivirus-mediated constitutive expression of forkhead box A2 (Foxa2) and hepatocyte nuclear factor 4 alpha (Hnf4a) could promote inducing mouse ESCs to hepatocyte-likes cells. First, ESC lines that stably expressed Foxa2, Hnf4a, or Foxa2/Hnf4a were constructed via lentiviral expression vectors. Second, observations of cell morphology changes were made during the cell culture process, followed by experiments examining teratoma formation. Then, the effects of constitutive expression of Foxa2 and Hnf4a on hepatic differentiation and maturation were determined by measuring the marker gene expression levels of Albumin, α-fetoprotein, Cytokeratin18, and α1-antitrypsin. The results indicate that constitutive expression of Foxa2 and Hnf4a does not affect ESCs culture, teratoma formation, or the expression levels of the specific hepatocyte genes under autonomous differentiation. However, with some assistance from inducing factors, Foxa2 significantly increased the hepatic differentiation of ESCs, whereas the expression of Hnf4a alone or Foxa2/Hnf4a could not. Differentiated CCE-Foxa2 cells were more superior in expressing several liver-specific markers and protein, storing glycogen than differentiated CCE cells. Therefore, our method employing the transduction of Foxa2 would be a valuable tool for the efficient generation of functional hepatocytes derived from ESCs. © 2013 Wiley Periodicals, Inc.

Tracking the Spatial and Functional Gradient of Monocyte-To-Macrophage Differentiation in Inflamed Lung.

PubMed

Sen, Debasish; Jones, Stephen M; Oswald, Erin M; Pinkard, Henry; Corbin, Kaitlin; Krummel, Matthew F

2016-01-01

Myeloid-derived cells such as monocytes, dendritic cells (DCs), and macrophages are at the heart of the immune effector function in an inflammatory response. But because of the lack of an efficient imaging system to trace these cells live during their migration and maturation in their native environment at sub-cellular resolution, our knowledge is limited to data available from specific time-points analyzed by flow cytometry, histology, genomics and other immunological methods. Here, we have developed a ratiometric imaging method for measuring monocyte maturation in inflamed mouse lungs in situ using real-time using 2-photon imaging and complementary methods. We visualized that while undifferentiated monocytes were predominantly found only in the vasculature, a semi-differentiated monocyte/macrophage population could enter the tissue and resembled more mature and differentiated populations by morphology and surface phenotype. As these cells entered and differentiated, they were already selectively localized near inflamed airways and their entry was associated with changes in motility and morphology. We were able to visualize these during the act of differentiation, a process that can be demonstrated in this way to be faster on a per-cell basis under inflammatory conditions. Finally, our in situ analyses demonstrated increases, in the differentiating cells, for both antigen uptake and the ability to mediate interactions with T cells. This work, while largely confirming proposed models for in situ differentiation, provides important in situ data on the coordinated site-specific recruitment and differentiation of these cells and helps elaborate the predominance of immune pathology at the airways. Our novel imaging technology to trace immunogenic cell maturation in situ will complement existing information available on in situ differentiation deduced from other immunological methods, and assist better understanding of the spatio-temporal cellular behavior during an inflammatory response.

Cell growth and differentiation on feeder layers is predicted to be influenced by bioreactor geometry.

PubMed

Peng, C A; Palsson, B Ø

1996-06-05

Tissue function is comprised of a complex interplay between biological and physicochemical rate processes. The design of bioreactors for tissue engineering must account for these processes simultaneously in order to obtain a bioreactor that provides a uniform environment for tissue growth and development. In the present study we consider the effects of fluid flow and mass transfer on the growth of a tissue in a parallel-plate bioreactor configuration. The parenchymal cells grow on a preformed stromal (feeder) layer that secretes a growth factor that stimulates parenchymal stem cell replication and differentiation. The biological dynamics are described by a unilineage model that describes the replication and differentiation of the tissue stem cell. The physicochemical rates are described by the Navier-Stokes and convective-diffusion equations. The model equations are solved by a finite element method. Two dimensionless groups govern the behavior of the solution. One is the Graetz number (Gz) that describes the relative rates of convection and diffusion, and the other a new dimensionless ratio (designated by P) that describes the interplay of the growth factor production, diffusion, and stimulation. Four geometries (slab, gondola, diamond, and radial shapes) for the parallel-plate bioreactor are analyzed. The uniformity of cell growth is measured by a two-dimensional coefficient of variance. The concentration distribution of the stroma-derived growth factor was computed first based on fluid flow and bioreactor geometry. Then the concomitant cell density distribution was obtained by integrating the calculated growth factor concentration with the parenchymal cell growth and unilineage differentiation process. The spatiotemporal cell growth patterns in four different bioreactor configurations were investigated under a variety of combinations of Gz (10(-1), 10(0), and 10(1)) and P(10(-2), 10(-1), 10(0), 10(1), and 10(2)). The results indicate high cell density and uniformity can be achieved for parameter values of P = 0.01, ..., 0.1 and Gz = 0.1, ..., 1.0. Among the four geometries investigated the radial-flow-type bioreactor provides the most uniform environment in which parenchymal cells can grow and differentiate ex vivo due to the absence of walls that are parallel to the flow paths creating slow flowing regions.

Identification of genes involved in the ACC-mediated control of root cell elongation in Arabidopsis thaliana

PubMed Central

2012-01-01

Background Along the root axis of Arabidopsis thaliana, cells pass through different developmental stages. In the apical meristem repeated cycles of division increase the numbers of cells. Upon leaving the meristem, these cells pass the transition zone where they are physiologically and mechanically prepared to undergo subsequent rapid elongation. During the process of elongation epidermal cells increase their length by 300% in a couple of hours. When elongation ceases, the cells acquire their final size, shape and functions (in the differentiation zone). Ethylene administered as its precursor 1-aminocyclopropane-1-carboxylic acid (ACC) is capable of inhibiting elongation in a concentration-dependent way. Using a microarray analysis, genes and/or processes involved in this elongation arrest are identified. Results Using a CATMA-microarray analysis performed on control and 3h ACC-treated roots, 240 differentially expressed genes were identified. Quantitative Real-Time RT-PCR analysis of the 10 most up and down regulated genes combined with literature search confirmed the accurateness of the analysis. This revealed that inhibition of cell elongation is, at least partly, caused by restricting the events that under normal growth conditions initiate elongation and by increasing the processes that normally stop cellular elongation at the end of the elongation/onset of differentiation zone. Conclusions ACC interferes with cell elongation in the Arabidopsis thaliana roots by inhibiting cells from entering the elongation process and by immediately stimulating the formation of cross-links in cell wall components, diminishing the remaining elongation capacity. From the analysis of the differentially expressed genes, it becomes clear that many genes identified in this response, are also involved in several other kind of stress responses. This suggests that many responses originate from individual elicitors, but that somewhere in the downstream signaling cascade, these are converged to a ’common pathway’. Furthermore, several potential keyplayers, such as transcription factors and auxin-responsive genes, were identified by the microarray analysis. They await further analysis to reveal their exact role in the control of cell elongation. PMID:23134674

Review: Transcriptional Regulation of CD4+ T Cell Differentiation in Experimentally Induced Arthritis and Rheumatoid Arthritis.

PubMed

Kondo, Yuya; Yokosawa, Masahiro; Kaneko, Shunta; Furuyama, Kotona; Segawa, Seiji; Tsuboi, Hiroto; Matsumoto, Isao; Sumida, Takayuki

2018-05-01

Rheumatoid arthritis (RA) is an autoimmune disorder characterized by chronic inflammation of the joint synovium and infiltration by activated inflammatory cells. CD4+ T cells form a large proportion of the inflammatory cells invading the synovial tissue, and are involved in the RA pathologic process. In general, CD4+ T cells differentiate into various T helper cell subsets and acquire the functional properties to respond to specific pathogens, and also mediate some autoimmune disorders such as RA. Because the differentiation of T helper cell subsets is determined by the expression of specific transcription factors in response to the cytokine environment, these transcription factors are considered to have a role in the pathology of RA. Treg cells control an excess of T cell-mediated immune response, and the transcription factor FoxP3 is critical for the differentiation and function of Treg cells. Treg cell dysfunction can result in the development of systemic autoimmunity. In this review, we summarize how the expression of transcription factors modulates T helper cell immune responses and the development of autoimmune diseases, especially in RA. Understanding the role of transcription factors in the pathogenesis of autoimmunity may lead to novel therapeutic strategies to control the differentiation and function of both T helper cells and Treg cells. © 2017 The Authors. Arthritis & Rheumatology published by Wiley Periodicals, Inc. on behalf of American College of Rheumatology.

Regenerative effects of human embryonic stem cell-derived neural crest cells for treatment of peripheral nerve injury.

PubMed

Jones, Iwan; Novikova, Liudmila N; Novikov, Lev N; Renardy, Monika; Ullrich, Andreas; Wiberg, Mikael; Carlsson, Leif; Kingham, Paul J

2018-04-01

Surgical intervention is the current gold standard treatment following peripheral nerve injury. However, this approach has limitations, and full recovery of both motor and sensory modalities often remains incomplete. The development of artificial nerve grafts that either complement or replace current surgical procedures is therefore of paramount importance. An essential component of artificial grafts is biodegradable conduits and transplanted cells that provide trophic support during the regenerative process. Neural crest cells are promising support cell candidates because they are the parent population to many peripheral nervous system lineages. In this study, neural crest cells were differentiated from human embryonic stem cells. The differentiated cells exhibited typical stellate morphology and protein expression signatures that were comparable with native neural crest. Conditioned media harvested from the differentiated cells contained a range of biologically active trophic factors and was able to stimulate in vitro neurite outgrowth. Differentiated neural crest cells were seeded into a biodegradable nerve conduit, and their regeneration potential was assessed in a rat sciatic nerve injury model. A robust regeneration front was observed across the entire width of the conduit seeded with the differentiated neural crest cells. Moreover, the up-regulation of several regeneration-related genes was observed within the dorsal root ganglion and spinal cord segments harvested from transplanted animals. Our results demonstrate that the differentiated neural crest cells are biologically active and provide trophic support to stimulate peripheral nerve regeneration. Differentiated neural crest cells are therefore promising supporting cell candidates to aid in peripheral nerve repair. © 2018 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons, Ltd.

The rise and fall of long-lived humoral immunity: terminal differentiation of plasma cells in health and disease

PubMed Central

O'Connor, Brian P.; Gleeson, Michael W.; Noelle, Randolph J.; Erickson, Loren D.

2010-01-01

Summary Long-lived humoral immune responses are a hallmark of thymus-dependent immunity. The cellular basis for enduring antibody-mediated immunity is long-lived memory B cells and plasma cells (PCs). Both of these cell populations acquire longevity as a result of antigen-specific, CD40–dependent, cognate interactions with helper T cells within germinal centers (GCs). At the molecular level, defined functional domains of CD40 control the post-GC fate of B cells. PC precursors that emerge from these GC reactions are highly proliferative and terminally differentiate to end-stage cells within the bone marrow (BM). The striking phenotypic similarities between the PC precursors and the putative malignant cell in multiple myeloma (MM) suggests that MM may result from the transformation of PC precursors. Within the domain of autoimmune disease, recent studies have shown that dysregulated migration of PCs to the BM may impact immune homeostasis and the development of lupus. Understanding the processes of normal PC differentiation will provide strategic insights into identifying therapeutic targets for the treatment of differentiated B-cell disorders. PMID:12846808

Single-cell RNA sequencing identifies diverse roles of epithelial cells in idiopathic pulmonary fibrosis

PubMed Central

Mizuno, Takako; Sridharan, Anusha; Du, Yina; Guo, Minzhe; Wikenheiser-Brokamp, Kathryn A.; Perl, Anne-Karina T.; Funari, Vincent A.; Gokey, Jason J.; Stripp, Barry R.; Whitsett, Jeffrey A.

2016-01-01

Idiopathic pulmonary fibrosis (IPF) is a lethal interstitial lung disease characterized by airway remodeling, inflammation, alveolar destruction, and fibrosis. We utilized single-cell RNA sequencing (scRNA-seq) to identify epithelial cell types and associated biological processes involved in the pathogenesis of IPF. Transcriptomic analysis of normal human lung epithelial cells defined gene expression patterns associated with highly differentiated alveolar type 2 (AT2) cells, indicated by enrichment of RNAs critical for surfactant homeostasis. In contrast, scRNA-seq of IPF cells identified 3 distinct subsets of epithelial cell types with characteristics of conducting airway basal and goblet cells and an additional atypical transitional cell that contributes to pathological processes in IPF. Individual IPF cells frequently coexpressed alveolar type 1 (AT1), AT2, and conducting airway selective markers, demonstrating “indeterminate” states of differentiation not seen in normal lung development. Pathway analysis predicted aberrant activation of canonical signaling via TGF-β, HIPPO/YAP, P53, WNT, and AKT/PI3K. Immunofluorescence confocal microscopy identified the disruption of alveolar structure and loss of the normal proximal-peripheral differentiation of pulmonary epithelial cells. scRNA-seq analyses identified loss of normal epithelial cell identities and unique contributions of epithelial cells to the pathogenesis of IPF. The present study provides a rich data source to further explore lung health and disease. PMID:27942595

Intracellular fragment of NLRR3 (NLRR3-ICD) stimulates ATRA-dependent neuroblastoma differentiation.

PubMed

Akter, Jesmin; Takatori, Atsushi; Islam, Md Sazzadul; Nakazawa, Atsuko; Ozaki, Toshinori; Nagase, Hiroki; Nakagawara, Akira

2014-10-10

We have previously identified neuronal leucine-rich repeat protein-3 (NLRR3) gene which is preferentially expressed in favorable human neuroblastomas as compared with unfavorable ones. In this study, we have found for the first time that NLRR3 is proteolytically processed by secretases and its intracellular domain (NLRR3-ICD) is then released to translocate into cell nucleus during ATRA-mediated neuroblastoma differentiation. According to our present observations, NLRR3-ICD was induced to accumulate in cell nucleus of neuroblastoma SH-SY5Y cells following ATRA treatment. Since the proteolytic cleavage of NLRR3 was blocked by α- or γ-secretase inhibitor, it is likely that NLRR3-ICD is produced through the secretase-mediated processing of NLRR3. Intriguingly, forced expression of NLRR3-ICD in neuroblastoma SK-N-BE cells significantly suppressed their proliferation as examined by a live-cell imaging system and colony formation assay. Similar results were also obtained in neuroblastoma TGW cells. Furthermore, overexpression of NLRR3-ICD stimulated ATRA-dependent neurite elongation in SK-N-BE cells. Together, our present results strongly suggest that NLRR3-ICD produced by the secretase-mediated proteolytic processing of NLRR3 plays a crucial role in ATRA-mediated neuronal differentiation, and provide a clue to develop a novel therapeutic strategy against aggressive neuroblastomas. Copyright © 2014 Elsevier Inc. All rights reserved.

Mirna biogenesis pathway is differentially regulated during adipose derived stromal/stem cell differentiation.

PubMed

Martin, E C; Qureshi, A T; Llamas, C B; Burow, M E; King, A G; Lee, O C; Dasa, V; Freitas, M A; Forsberg, J A; Elster, E A; Davis, T A; Gimble, J M

2018-02-07

Stromal/stem cell differentiation is controlled by a vast array of regulatory mechanisms. Included within these are methods of mRNA gene regulation that occur at the level of epigenetic, transcriptional, and/or posttranscriptional modifications. Current studies that evaluate the posttranscriptional regulation of mRNA demonstrate microRNAs (miRNAs) as key mediators of stem cell differentiation through the inhibition of mRNA translation. miRNA expression is enhanced during both adipogenic and osteogenic differentiation; however, the mechanism by which miRNA expression is altered during stem cell differentiation is less understood. Here we demonstrate for the first time that adipose-derived stromal/stem cells (ASCs) induced to an adipogenic or osteogenic lineage have differences in strand preference (-3p and -5p) for miRNAs originating from the same primary transcript. Furthermore, evaluation of miRNA expression in ASCs demonstrates alterations in both miRNA strand preference and 5'seed site heterogeneity. Additionally, we show that during stem cell differentiation there are alterations in expression of genes associated with the miRNA biogenesis pathway. Quantitative RT-PCR demonstrated changes in the Argonautes (AGO1-4), Drosha, and Dicer at intervals of ASC adipogenic and osteogenic differentiation compared to untreated ASCs. Specifically, we demonstrated altered expression of the AGOs occurring during both adipogenesis and osteogenesis, with osteogenesis increasing AGO1-4 expression and adipogenesis decreasing AGO1 gene and protein expression. These data demonstrate changes to components of the miRNA biogenesis pathway during stromal/stem cell differentiation. Identifying regulatory mechanisms for miRNA processing during ASC differentiation may lead to novel mechanisms for the manipulation of lineage differentiation of the ASC through the global regulation of miRNA as opposed to singular regulatory mechanisms.

A Contact-Based Method for Differentiation of Human Mesenchymal Stem Cells into an Endothelial Cell-Phenotype.

PubMed

Joddar, Binata; Kumar, Shweta Anil; Kumar, Alok

2018-06-01

Adult stem cells such as mesenchymal stem cells (MSC) are known to possess the ability to augment neovascularization processes and are thus widely popular as an autologous source of progenitor cells. However there is a huge gap in our current knowledge of mechanisms involved in differentiating MSC into endothelial cells (EC), essential for lining engineered blood vessels. To fill up this gap, we attempted to differentiate human MSC into EC, by culturing the former onto chemically fixed layers of EC or its ECM, respectively. We expected direct contact of MSC when cultured atop fixed EC or its ECM, would coax the former to differentiate into EC. Results showed that human MSC cultured atop chemically fixed EC or its ECM using EC-medium showed enhanced expression of CD31, a marker for EC, compared to other cases. Further in all human MSC cultured using EC-medium, typically characteristic cobble stone shaped morphologies were noted in comparison to cells cultured using MSC medium, implying that the differentiated cells were sensitive to soluble VEGF supplementation present in the EC-medium. Results will enhance and affect therapies utilizing autologous MSC as a cell source for generating vascular cells to be used in a variety of tissue engineering applications.

Effects of transforming growth factor-beta1 on cell motility, collagen gel contraction, myofibroblastic differentiation, and extracellular matrix expression of human adipose-derived stem cell.

PubMed

Kakudo, Natsuko; Kushida, Satoshi; Suzuki, Kenji; Ogura, Tsunetaka; Notodihardjo, Priscilla Valentin; Hara, Tomoya; Kusumoto, Kenji

2012-12-01

Human adipose-derived stem cells (ASCs) are adult pluripotent stem cells, and their usefulness in plastic surgery has garnered attention in recent years. Although, there have been expectations that ASCs might function in wound repair and regeneration, no studies to date have examined the role of ASCs in the mechanism that promotes wound-healing. Transforming growth factor-beta1 (TGF-β1) is a strong candidate cytokine for the triggering of mesenchymal stem cell migration, construction of extracellular matrices, and differentiation of ASCs into myofibroblasts. Cell proliferation, motility, and differentiation, as well as extracellular matrix production, play an important role in wound-healing. We have evaluated the capacity of ASCs to proliferate and their potential to differentiate into phenotypic myofibroblasts, as well as their cell motility and collagen gel contraction ability, when cultured with TGF-β1. Cell motility was analyzed using a wound-healing assay. ASCs that differentiated into myofibroblasts expressed the gene for alpha-smooth muscle actin, and its protein expression was detected immunohistochemically. The extracellular matrix expression in ASCs was evaluated using real-time RT-PCR. Based on the results, we conclude that human ASCs have the potential for cell motility, extracellular matrix gene expression, gel contraction, and differentiation into myofibroblasts and, therefore, may play an important role in the wound-healing process.

Development of an in vitro culture method for stepwise differentiation of mouse embryonic stem cells and induced pluripotent stem cells into mature osteoclasts.

PubMed

Nishikawa, Keizo; Iwamoto, Yoriko; Ishii, Masaru

2014-05-01

The development of methods for differentiation of embryonic stem cells (ESCs) and induced pluripotent stem cell (iPSCs) into functional cells have helped to analyze the mechanism regulating cellular processes and to explore cell-based assays for drug discovery. Although several reports have demonstrated methods for differentiation of mouse ESCs into osteoclast-like cells, it remains unclear whether these methods are applicable for differentiation of iPSCs to osteoclasts. In this study, we developed a simple method for stepwise differentiation of mouse ESCs and iPSCs into bone-resorbing osteoclasts based upon a monoculture approach consisting of three steps. First, based on conventional hanging-drop methods, embryoid bodies (EBs) were produced from mouse ESCs or iPSCs. Second, EBs were cultured in medium supplemented with macrophage colony-stimulating factor (M-CSF), and differentiated to osteoclast precursors, which expressed CD11b. Finally, ESC- or iPSC-derived osteoclast precursors stimulated with receptor activator of nuclear factor-B ligand (RANKL) and M-CSF formed large multinucleated osteoclast-like cells that expressed tartrate-resistant acid phosphatase and were capable of bone resorption. Molecular analysis showed that the expression of osteoclast marker genes such as Nfatc1, Ctsk, and Acp5 are increased in a RANKL-dependent manner. Thus, our procedure is simple and easy and would be helpful for stem cell-based bone research.

DD-RT-PCR identifies 7-dehydrocholesterol reductase as a key marker of early Leydig cell steroidogenesis.

PubMed

Anbalagan, M; Yashwanth, R; Jagannadha Rao, A

2004-04-30

Postnatal Leydig cell development in rat involves an initial phase of proliferation of progenitor Leydig cells (PLCs) and subsequent differentiation of these cells into immature Leydig cells (ILCs) and adult Leydig cells (ALCs). With an objective to identify the molecular changes associated with Leydig cell differentiation, the mRNA population in PLCs and ILCs were analyzed by the technique of differential display reverse transcription polymerase chain reaction (DD-RT-PCR). Results revealed differential expression of several transcripts in PLCs and ILCs. Of the several differentially expressed transcripts, the expression of transcripts corresponding to collagen IV alpha6 (Col IV alpha6) and ribosomal protein L 41 (RpL41) decreased during the differentiation of PLC to ILC. Also there was an increase in the expression of transcripts encoding enzymes such as microsomal glutathione-S-transferase (mGST 1) and 7-dehydrocholesterol reductase (7-DHCR) during this process. While Col IV alpha6 and RpL41 are known to be involved in cellular proliferation, mGST 1 and 7-DHCR are essential for normal Leydig cell steroidogenesis. A detailed study on 7-DHCR expression in Leydig cells revealed that this enzyme plays a crucial role in steroidogenesis. Interestingly expression of this enzyme is not under acute regulation by Luteinizing hormone (LH). Copyright 2004 Elsevier Ireland Ltd.

Interleukin-3 plays dual roles in osteoclastogenesis by promoting the development of osteoclast progenitors but inhibiting the osteoclastogenic process

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

Hong, Huixian; Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294; Shi, Zhenqi

2013-11-01

Highlights: •IL-3 treatment of bone marrow cells generates a population of hematopoietic cells. •IL-3-dependent hematopoietic cells are capable of differentiating into osteoclasts. •Osteoclasts derived from IL-3-dependent hematopoietic cells are functional. •IL-3 promotes the development of osteoclast progenitors. •IL-3 inhibits the osteoclastogenic process. -- Abstract: Interleukin (IL)-3, a multilineage hematopoietic growth factor, is implicated in the regulation of osteoclastogenesis. However, the role of IL-3 in osteoclastogenesis remains controversial; whereas early studies showed that IL-3 stimulates osteoclastogenesis, recent investigations demonstrated that IL-3 inhibits osteoclast formation. The objective of this work is to further address the role of IL-3 in osteoclastogenesis. We foundmore » that IL-3 treatment of bone marrow cells generated a population of cells capable of differentiating into osteoclasts in tissue culture dishes in response to the stimulation of the monocyte/macrophage-colony stimulating factor (M-CSF) and the receptor activator of nuclear factor kappa B ligand (RANKL). The IL-3-dependent hematopoietic cells were able to further proliferate and differentiate in response to M-CSF stimulation and the resulting cells were also capable of forming osteoclasts with M-CSF and RANKL treatment. Interestingly, IL-3 inhibits M-CSF-/RANKL-induced differentiation of the IL-3-dependent hematopoietic cells into osteoclasts. The flow cytometry analysis indicates that while IL-3 treatment of bone marrow cells slightly affected the percentage of osteoclast precursors in the surviving populations, it considerably increased the percentage of osteoclast precursors in the populations after subsequent M-CSF treatment. Moreover, osteoclasts derived from IL-3-dependent hematopoietic cells were fully functional. Thus, we conclude that IL-3 plays dual roles in osteoclastogenesis by promoting the development of osteoclast progenitors but inhibiting the osteoclastogenic process. These findings provide a better understanding of the role of IL-3 in osteoclastogenesis.« less

Specific Biomimetic Hydroxyapatite Nanotopographies Enhance Osteoblastic Differentiation and Bone Graft Osteointegration

PubMed Central

Loiselle, Alayna E.; Wei, Lai; Faryad, Muhammad; Paul, Emmanuel M.; Lewis, Gregory S.; Gao, Jun; Lakhtakia, Akhlesh

2013-01-01

Impaired healing of cortical bone grafts represents a significant clinical problem. Cadaveric bone grafts undergo extensive chemical processing to decrease the risk of disease transmission; however, these processing techniques alter the bone surface and decrease the osteogenic potential of cells at the healing site. Extensive work has been done to optimize the surface of bone grafts, and hydroxyapatite (HAP) and nanotopography both increase osteoblastic differentiation. HAP is the main mineral component of bone and can enhance osteoblastic differentiation and bone implant healing in vivo, while nanotopography can enhance osteoblastic differentiation, adhesion, and proliferation. This is the first study to test the combined effects of HAP and nanotopographies on bone graft healing. With the goal of identifying the optimized surface features to improve bone graft healing, we tested the hypothesis that HAP-based nanotopographic resurfacing of bone grafts improves integration of cortical bone grafts by enhancing osteoblastic differentiation. Here we show that osteoblastic cells cultured on processed bones coated with specific-scale (50–60 nm) HAP nanotopographies display increased osteoblastic differentiation compared to cells on uncoated bone, bones coated with poly-l-lactic acid nanotopographies, or other HAP nanotopographies. Further, bone grafts coated with 50–60-nm HAP exhibited increased formation of new bone and improved healing, with mechanical properties equivalent to live autografts. These data indicate the potential for specific HAP nanotopographies to not only increase osteoblastic differentiation but also improve bone graft incorporation, which could significantly increase patient quality of life after traumatic bone injuries or resection of an osteosarcoma. PMID:23510012

Spontaneous calcium transients in human neural progenitor cells mediated by transient receptor potential channels.

PubMed

Morgan, Peter J; Hübner, Rayk; Rolfs, Arndt; Frech, Moritz J

2013-09-15

Calcium signals affect many developmental processes, including proliferation, migration, survival, and apoptosis, processes that are of particular importance in stem cells intended for cell replacement therapies. The mechanisms underlying Ca(2+) signals, therefore, have a role in determining how stem cells respond to their environment, and how these responses might be controlled in vitro. In this study, we examined the spontaneous Ca(2+) activity in human neural progenitor cells during proliferation and differentiation. Pharmacological characterization indicates that in proliferating cells, most activity is the result of transient receptor potential (TRP) channels that are sensitive to Gd(3+) and La(3+), with the more subtype selective antagonist Ruthenium red also reducing activity, suggesting the involvement of transient receptor potential vanilloid (TRPV) channels. In differentiating cells, Gd(3+) and La(3+)-sensitive TRP channels also appear to underlie the spontaneous activity; however, no sub-type-specific antagonists had any effect. Protein levels of TRPV2 and TRPV3 decreased in differentiated cells, which is demonstrated by western blot. Thus, it appears that TRP channels represent the main route of Ca(2+) entry in human neural progenitor cells (hNPCs), but the responsible channel types are subject to substitution under differentiating conditions. The level of spontaneous activity could be increased and decreased by lowering and raising the extracellular K(+) concentration. Proliferating cells in low K(+) slowed the cell cycle, with a disproportionate increased percentage of cells in G1 phase and a reduction in S phase. Taken together, these results suggest a link between external K(+) concentration, spontaneous Ca(2+) transients, and cell cycle distribution, which is able to influence the fate of stem and progenitor cells.

Enforced expression of the c-myc oncogene inhibits cell differentiation by precluding entry into a distinct predifferentiation state in G/sub 0//G/sub 1/

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

Freytag, S.O.

1988-04-01

A broad base of data has implicated a role for the c-myc proto-oncogene in the control of the cell cycle and cell differentiation. To further define the role of myc in these processes, the authors examined the effect of enforced myc expression on several events that are thought to be important steps leading to the terminally differentiated state: (i) the ability to arrest growth in G/sub 0//G/sub 1/, (ii) the ability to replicate the genome upon initiation of the differentiation program, and (iii) the ability to loose responsiveness to mitogens and withdraw from the cell cycle. 3T3-L1 preadipocyte cell linesmore » expressing various levels of myc mRNA were established by transfection with a recombinant myc gene under the transcriptional control of the Rous sarcoma virus (RSV) promoter. Cells that expressed high constitutive levels of pRSV myc mRNA arrested in G/sub 0//G/sub 1/ at densities similar to those of normal cells at confluence. Upon initiation of the differentiation program, such cells traversed the cell cycle with kinetics similar to those of normal cells and subsequently arrested in G/sub 0//G/sub 1/. Thus, enforced expression of myc had no effect on the ability of cells to arrest growth in G/sub 0//G/sub 1/ or to replicate the genome upon initiation of the differentiation program. Cells were then tested for their ability to reenter the cell cycle upon exposure to high concentrations of serum and for their capacity to differentiate. In contrast to normal cells, cells expressing high constitutive levels of myc RNA reentered the cell cycle when challenged with 30% serum and failed to terminally differentiate.« less

MicroRNA, miR-374b, directly targets Myf6 and negatively regulates C2C12 myoblasts differentiation

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

Ma, Zhiyuan; Sun, Xiaorui; Xu, Dequan

Myogenesis is a complex process including myoblast proliferation, differentiation and myotube formation and is controlled by myogenic regulatory factors (MRFs), MyoD, MyoG, Myf5 and Myf6 (also known as MRF4). MicroRNA is a kind of ∼22 nt-long non-coding small RNAs, and act as key transcriptional or post-transcriptional regulators of gene expression. Identification of miRNAs involved in the regulation of muscle genes could improve our understanding of myogenesis process. In this study, we investigated the regulation of Myf6 gene by miRNAs. We showed that miR-374b specifically bound to the 3'untranslated region (UTR) of Myf6 and down-regulated the expression of Myf6 gene at bothmore » mRNA and protein level. Furthermore, miR-374b is ubiquitously expressed in the tissues of adult C57BL6 mouse, and the mRNA abundance increases first and then decreases during C2C12 myoblasts differentiation. Over-expression of miR-374b impaired C2C12 cell differentiation, while inhibiting miR-374b expression by 2′-O-methyl antisense oligonucleotides promoted C2C12 cell differentiation. Taken together, our findings identified miR-374b directly targets Myf6 and negatively regulates myogenesis. - Highlights: • MiR-374b directly targets 3′UTR of Myf6. • MiR-374b negatively regulates Myf6 in C2C12 cells. • MiR-374b abundance significiently changes during C2C12 cells differentiation. • MiR-374b negatively regulates C2C12 cells differentiation.« less

Decline in c-myc mRNA expression but not the induction of c-fos mRNA expression is associated with differentiation of SH-SY5Y human neuroblastoma cells

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

Jalava, A.M.; Heikkilae, J.E.; Akerman, K.E.O.

1988-11-01

The induction of differentiation in SH-SY5Y human neuroblastoma cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is accompanied by a rapid and a transient expression of c-fos mRNA and a down-regulation of c-myc RNA. The TPA-induced expression of c-fos mRNA was inhibited by H-7, a specific inhibitor of protein kinase C (PK-C). Dioctanoylglycerol (DiC{sub 8}) failed to induce differentiation of SH-SY5Y cells or to down-regulate c-myc mRNA but it did induce the expression of c-fos mRNA. Treatment of IMR-32 human neuroblastoma cells with TPA did not cause differentiation although c-fos mRNA was induced. Since PK-C in SH-SY5Y cells was activated by both TPA andmore » DiC{sub 8} it is suggested that the activation of PK-C alone is not sufficient to induce differentiation in SH-SY5Y cells. The down-regulation of c-myc mRNA rather than the induction of c-fos mRNA seems to be associated with differentiation process in SH-SY5Y cells.« less

Differential growth of wrinkled biofilms

NASA Astrophysics Data System (ADS)

Espeso, D. R.; Carpio, A.; Einarsson, B.

2015-02-01

Biofilms are antibiotic-resistant bacterial aggregates that grow on moist surfaces and can trigger hospital-acquired infections. They provide a classical example in biology where the dynamics of cellular communities may be observed and studied. Gene expression regulates cell division and differentiation, which affect the biofilm architecture. Mechanical and chemical processes shape the resulting structure. We gain insight into the interplay between cellular and mechanical processes during biofilm development on air-agar interfaces by means of a hybrid model. Cellular behavior is governed by stochastic rules informed by a cascade of concentration fields for nutrients, waste, and autoinducers. Cellular differentiation and death alter the structure and the mechanical properties of the biofilm, which is deformed according to Föppl-Von Kármán equations informed by cellular processes and the interaction with the substratum. Stiffness gradients due to growth and swelling produce wrinkle branching. We are able to reproduce wrinkled structures often formed by biofilms on air-agar interfaces, as well as spatial distributions of differentiated cells commonly observed with B. subtilis.

Exploiting single-cell variability to infer the dynamics of immune responses

NASA Astrophysics Data System (ADS)

Höfer, Thomas

Cell division, differentiation, migration and death determine the dynamics of immune responses. These processes are regulated by a multitude of biochemical signals which, at present, cannot faithfully be reconstituted outside the living organism. However, quantitative measurements in living organisms have been limited. In recent years experimental techniques for the ``fate mapping'' of single immune cells have been developed that allow performing parallel single-cell experiments in an experimental animal. The resulting data are more informative about underlying biological processes than traditional measurements. I will show how the theory of stochastic dynamical systems can be used to infer the topology and dynamics of cell differentiation pathways from such data. The focus will be on joint theoretical and experimental work addressing: (i) the development of immune cells during hematopoiesis, and (ii) T cell responses to diverse pathogens. I will discuss questions on the nature of cellular variability that are posed by these new findings.

The neuroregenerative mechanism mediated by the Hsp90-binding immunophilin FKBP52 resembles the early steps of neuronal differentiation

PubMed Central

Quintá, HR; Galigniana, MD

2012-01-01

BACKGROUND AND PURPOSE The immunosuppressive macrolide FK506 (tacrolimus) shows neuroregenerative action by a mechanism that appears to involve the Hsp90-binding immunophilin FKBP52. This study analyses some aspects of the early steps of neuronal differentiation and neuroregeneration. EXPERIMENTAL APPROACH Undifferentiated murine neuroblastoma cells and hippocampal neurones isolated from embryonic day-17 rat embryos were induced to differentiate with FK506. Subcellular relocalization of FKBP52, Hsp90 and its co-chaperone p23 was analysed by indirect immunofluorescence confocal microscopy and by Western blots of axonal fractions isolated from cells grown on a porous transwell cell culture chamber. Neuroregeneration was evaluated using a scratch-wound assay. KEY RESULTS In undifferentiated cells, FKBP52, Hsp90 and p23 are located in the cell nucleus, forming an annular structure that disassembles when the differentiation process is triggered by FK506. This was observed in the N2a cell line and in hippocampal neurones. More importantly, the annular structure of chaperones is reassembled after damaging the neurones, whereas FK506 prompts their rapid regeneration, a process linked to the subcellular redistribution of the heterocomplex. CONCLUSIONS AND IMPLICATIONS There is a direct relationship between the disassembly of the chaperone complex and the progression of neuronal differentiation upon stimulation with the immunophilin ligand FK506. Both neuronal differentiation and neuroregeneration appear to be mechanistically linked, so the elucidation of one mechanism may lead to unravel the properties of the other. This study also implies that the discovery of FK506 derivatives, devoid of immunosuppressive action, would be therapeutically significant for neurotrophic use. PMID:22091865

Differentiation-dependent expression of hypothetical proteins in the neuroblastoma cell line N1E-115.

PubMed

Oh, Ji-eun; Karlmark, Karlin Raja; Shin, Jooho; Hengstschläger, Markus; Lubec, Gert

2006-05-15

Several protein cascades, including signaling, cytoskeletal, chaperones, metabolic, and antioxidant proteins, have been shown to be involved in the process of neuronal differentiation (ND) of neuroblastoma cell lines. No systematic approach to detect hitherto unknown and unnamed proteins or structures that have been predicted upon nucleic acid sequences in ND has been published so far. We therefore decided to screen hypothetical protein (HP) expression by protein profiling. Two-dimensional gel electrophoresis with subsequent matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/TOF) identification was used for expression analysis of undifferentiated and dimethylsulfoxide-induced neuronally differentiated N1E-115 cells. We unambiguously identified six HPs: Q8C520, Q99LF4, Q9CXS1, Q9DAF8, Q91WT0, and Q8C5G2. A prefoldin domain in Q91WT0, a t-SNARE domain in Q9CXS1, and a bromodomain were observed in Q8C5G2. For the three remaining proteins, no putative function using Pfam, BLOCKS, PROSITE, PRINTS, InterPro, Superfamily, CoPS, and ExPASy could be assigned. While two proteins were present in both cell lines, Q9CXS1 was switched off (i.e., undetectably low) in differentiated cells only, and Q9DAF8, Q91WT0, and Q8C5G2 were switched on in differentiated cells exclusively. Herein, using a proteomic approach suitable for screening and identification of HP, we present HP structures that have been only predicted so far based upon nucleic acid sequences. The four differentially regulated HPs may play a putative role in the process of ND. (c) 2006 Wiley-Liss, Inc.

Indian hedgehog signaling promotes chondrocyte differentiation in enchondral ossification in human cervical ossification of the posterior longitudinal ligament.

PubMed

Sugita, Daisuke; Yayama, Takafumi; Uchida, Kenzo; Kokubo, Yasuo; Nakajima, Hideaki; Yamagishi, Atsushi; Takeura, Naoto; Baba, Hisatoshi

2013-10-15

Histological, immunohistochemical, and immunoblot analyses of the expression of Indian hedgehog (Ihh) signaling in human cervical ossification of the posterior longitudinal ligament (OPLL). To examine the hypothesis that Ihh signaling in correlation with Sox9 and parathyroid-related peptide hormone (PTHrP) facilitates chondrocyte differentiation in enchondral ossification process in human cervical OPLL. In enchondral ossification, certain transcriptional factors regulate cell differentiation. OPLL is characterized by overexpression of these factors and disturbance of the normal cell differentiation process. Ihh signaling is essential for enchondral ossification, especially in chondrocyte hypertrophy. Samples of ossified ligaments were harvested from 45 patients who underwent anterior cervical decompressive surgery for symptomatic OPLL, and 6 control samples from patients with cervical spondylotic myelopathy/radiculopathy without OPLL. The harvested sections were stained with hematoxylin-eosin and toluidine blue, examined by transmission electron microscopy, and immunohistochemically stained for Ihh, PTHrP, Sox9, type X, XI collagen, and alkaline phosphatase. Immunoblot analysis was performed in cultured cells derived from the posterior longitudinal ligaments in the vicinity of the ossified plaque and examined for the expression of these factors. The ossification front in OPLL contained chondrocytes at various differentiation stages, including proliferating chondrocytes in fibrocartilaginous area, hypertrophic chondrocytes around the calcification front, and apoptotic chondrocytes near the ossified area. Immunoreactivity for Ihh and Sox9 was evident in proliferating chondrocytes and was strongly positive for PTHrP in hypertrophic chondrocytes. Mesenchymal cells with blood vessel formation were positive for Ihh, PTHrP, and Sox9. Cultured cells from OPLL tissues expressed significantly higher levels of Ihh, PTHrP, and Sox9 than those in non-OPLL cells. Our results indicated that overexpression of Ihh signaling promotes abnormal chondrocyte differentiation in enchondral ossification and enhances bone formation in OPLL.

A Voltage-Sensitive Dye-Based Assay for the Identification of Differentiated Neurons Derived from Embryonic Neural Stem Cell Cultures

PubMed Central

Emirandetti, Amanda; Lewicka, Michalina; Hermanson, Ola; Fisahn, André

2010-01-01

Background Pluripotent and multipotent stem cells hold great therapeutical promise for the replacement of degenerated tissue in neurological diseases. To fulfill that promise we have to understand the mechanisms underlying the differentiation of multipotent cells into specific types of neurons. Embryonic stem cell (ESC) and embryonic neural stem cell (NSC) cultures provide a valuable tool to study the processes of neural differentiation, which can be assessed using immunohistochemistry, gene expression, Ca2+-imaging or electrophysiology. However, indirect methods such as protein and gene analysis cannot provide direct evidence of neuronal functionality. In contrast, direct methods such as electrophysiological techniques are well suited to produce direct evidence of neural functionality but are limited to the study of a few cells on a culture plate. Methodology/Principal Findings In this study we describe a novel method for the detection of action potential-capable neurons differentiated from embryonic NSC cultures using fast voltage-sensitive dyes (VSD). We found that the use of extracellularly applied VSD resulted in a more detailed labeling of cellular processes compared to calcium indicators. In addition, VSD changes in fluorescence translated precisely to action potential kinetics as assessed by the injection of simulated slow and fast sodium currents using the dynamic clamp technique. We further demonstrate the use of a finite element model of the NSC culture cover slip for optimizing electrical stimulation parameters. Conclusions/Significance Our method allows for a repeatable fast and accurate stimulation of neurons derived from stem cell cultures to assess their differentiation state, which is capable of monitoring large amounts of cells without harming the overall culture. PMID:21079795

Amyloid precursor protein expression and processing are differentially regulated during cortical neuron differentiation.

PubMed

Bergström, Petra; Agholme, Lotta; Nazir, Faisal Hayat; Satir, Tugce Munise; Toombs, Jamie; Wellington, Henrietta; Strandberg, Joakim; Bontell, Thomas Olsson; Kvartsberg, Hlin; Holmström, Maria; Boreström, Cecilia; Simonsson, Stina; Kunath, Tilo; Lindahl, Anders; Blennow, Kaj; Hanse, Eric; Portelius, Erik; Wray, Selina; Zetterberg, Henrik

2016-07-07

Amyloid precursor protein (APP) and its cleavage product amyloid β (Aβ) have been thoroughly studied in Alzheimer's disease. However, APP also appears to be important for neuronal development. Differentiation of induced pluripotent stem cells (iPSCs) towards cortical neurons enables in vitro mechanistic studies on human neuronal development. Here, we investigated expression and proteolytic processing of APP during differentiation of human iPSCs towards cortical neurons over a 100-day period. APP expression remained stable during neuronal differentiation, whereas APP processing changed. α-Cleaved soluble APP (sAPPα) was secreted early during differentiation, from neuronal progenitors, while β-cleaved soluble APP (sAPPβ) was first secreted after deep-layer neurons had formed. Short Aβ peptides, including Aβ1-15/16, peaked during the progenitor stage, while processing shifted towards longer peptides, such as Aβ1-40/42, when post-mitotic neurons appeared. This indicates that APP processing is regulated throughout differentiation of cortical neurons and that amyloidogenic APP processing, as reflected by Aβ1-40/42, is associated with mature neuronal phenotypes.

Chemical Activation of the Hypoxia-Inducible Factor Reversibly Reduces Tendon Stem Cell Proliferation, Inhibits Their Differentiation, and Maintains Cell Undifferentiation.

PubMed

Menon, Alessandra; Creo, Pasquale; Piccoli, Marco; Bergante, Sonia; Conforti, Erika; Banfi, Giuseppe; Randelli, Pietro; Anastasia, Luigi

2018-01-01

Adult stem cell-based therapeutic approaches for tissue regeneration have been proposed for several years. However, adult stem cells are usually limited in number and difficult to be expanded in vitro, and they usually tend to quickly lose their potency with passages, as they differentiate and become senescent. Culturing stem cells under reduced oxygen tensions (below 21%) has been proposed as a tool to increase cell proliferation, but many studies reported opposite effects. In particular, cell response to hypoxia seems to be very stem cell type specific. Nonetheless, it is clear that a major role in this process is played by the hypoxia inducible factor (HIF), the master regulator of cell response to oxygen deprivation, which affects cell metabolism and differentiation. Herein, we report that a chemical activation of HIF in human tendon stem cells reduces their proliferation and inhibits their differentiation in a reversible and dose-dependent manner. These results support the notion that hypoxia, by activating HIF, plays a crucial role in preserving stem cells in an undifferentiated state in the "hypoxic niches" present in the tissue in which they reside before migrating in more oxygenated areas to heal a damaged tissue.

5-Azacytidine delivered by mesoporous silica nanoparticles regulates the differentiation of P19 cells into cardiomyocytes

NASA Astrophysics Data System (ADS)

Cheng, Jin; Ding, Qian; Wang, Jia; Deng, Lin; Yang, Lu; Tao, Lei; Lei, Haihong; Lu, Shaoping

2016-01-01

Heart disease is one of the deadliest diseases causing mortality due to the limited regenerative capability of highly differentiated cardiomyocytes. Stem cell-based therapy in tissue engineering is one of the most exciting and rapidly growing areas and raises promising prospects for cardiac repair. In this study, we have synthesized FITC-mesoporous silica nanoparticles (FMSNs) based on a sol-gel method (known as Stöber's method) as a drug delivery platform to transport 5-azacytidine in P19 embryonic carcinoma stem cells. The surfactant CTAB is utilized as a liquid crystal template to self-aggregate into micelles, resulting in the synthesis of MSNs. Based on the cell viability assay, treatment with FMSNs + 5-azacytidine resulted in much more significant inhibition of the proliferation than 5-azacytidine alone. To study the mechanism, we have tested the differentiation genes and cardiac marker genes in P19 cells and found that these genes have been up-regulated in P19 embryonic carcinoma stem cells treated with FMSNs + 5-azacytidine + poly(allylamine hydrochloride) (PAH), with the changes of histone modifications on the regulatory region. In conclusion, with FMSNs as drug delivery platforms, 5-azacytidine can be more efficiently delivered into stem cells and can be used to monitor and track the transfection process in situ to clarify their effects on stem cell functions and the differentiation process, which can serve as a promising tool in tissue engineering and other biomedical fields.

Tuberin and PRAS40 are anti-apoptotic gatekeepers during early human amniotic fluid stem-cell differentiation.

PubMed

Fuchs, Christiane; Rosner, Margit; Dolznig, Helmut; Mikula, Mario; Kramer, Nina; Hengstschläger, Markus

2012-03-01

Embryoid bodies (EBs) are three-dimensional multicellular aggregates allowing the in vitro investigation of stem-cell differentiation processes mimicking early embryogenesis. Human amniotic fluid stem (AFS) cells harbor high proliferation potential, do not raise the ethical issues of embryonic stem cells, have a lower risk for tumor development, do not need exogenic induction of pluripotency and are chromosomal stable. Starting from a single human AFS cell, EBs can be formed accompanied by the differentiation into cells of all three embryonic germ layers. Here, we report that siRNA-mediated knockdown of the endogenous tuberous sclerosis complex-2 (TSC2) gene product tuberin or of proline-rich Akt substrate of 40 kDa (PRAS40), the two major negative regulators of mammalian target of rapamycin (mTOR), leads to massive apoptotic cell death during EB development of human AFS cells without affecting the endodermal, mesodermal and ectodermal cell differentiation spectrum. Co-knockdown of endogenous mTOR demonstrated these effects to be mTOR-dependent. Our findings prove this enzyme cascade to be an essential anti-apoptotic gatekeeper of stem-cell differentiation during EB formation. These data allow new insights into the regulation of early stem-cell maintenance and differentiation and identify a new role of the tumor suppressor tuberin and the oncogenic protein PRAS40 with the relevance for a more detailed understanding of the pathogenesis of diseases associated with altered activities of these gene products.

In vitro study on bone formation and surface topography from the standpoint of biomechanics.

PubMed

Kawahara, H; Soeda, Y; Niwa, K; Takahashi, M; Kawahara, D; Araki, N

2004-12-01

Effect of surface topography upon cell-adhesion, -orientation and -differentiation was investigated by in vitro study on cellular responses to titanium substratum with different surface roughness. Cell-shape, -function and -differentiation depending upon the surface topography were clarified by use of bone formative group cells (BFGCs) derived from bone marrow of beagle's femur. BFGCs consisted of hematopoietic stem cells (HSC) and osteogenetic stem cells (OSC). Cell differentiation of BFGCs was expressed and promoted by structural changes of cytoskeleton, and cell-organella, which was caused by mechanical stress with cytoplasmic stretching of cell adhesions to the substratum. Phagocytic monocytes of HSC differentiated to osteomediator cells (OMC) by cytoplasmic stretching with cell adhesion to the substratum. The OMC mediated and promoted cell differentiation from OSC to osteoblast through osteoblastic phenotype cell (OBC) by cell-aggregation of nodules with "pile up" phenomenon of OBC onto OMC. The osteogenesis might be performed by coupling work of both cells, OMC originated from monocyte of HSC and OBC originated from OSC, which were explained by SEM, TEM and fluorescent probe investigation on BFGCs on the test plate of cp titanium plates with different topographies. This osteogenetic process was proved by investigating cell proliferation, DNA contents, cell-adhesion, alkaline phosphatase activity and osteocalcine productivity for cells on the titanium plates with different topographies. The study showed increased osteogenic effects for cells cultured on Ti with increased surface roughness. Possible mechanisms were discussed from a biomechanical perspective.

Macrophages control vascular stem/progenitor cell plasticity through tumor necrosis factor-α-mediated nuclear factor-κB activation.

PubMed

Wong, Mei Mei; Chen, Yikuan; Margariti, Andriani; Winkler, Bernhard; Campagnolo, Paola; Potter, Claire; Hu, Yanhua; Xu, Qingbo

2014-03-01

Vascular lineage differentiation of stem/progenitor cells can contribute to both tissue repair and exacerbation of vascular diseases such as in vein grafts. The role of macrophages in controlling vascular progenitor differentiation is largely unknown and may play an important role in graft development. This study aims to identify the role of macrophages in vascular stem/progenitor cell differentiation and thereafter elucidate the mechanisms that are involved in the macrophage- mediated process. We provide in vitro evidence that macrophages can induce endothelial cell (EC) differentiation of the stem/progenitor cells while simultaneously inhibiting their smooth muscle cell differentiation. Mechanistically, both effects were mediated by macrophage-derived tumor necrosis factor-α (TNF-α) via TNF-α receptor 1 and canonical nuclear factor-κB activation. Although the overexpression of p65 enhanced EC (or attenuated smooth muscle cell) differentiation, p65 or TNF-α receptor 1 knockdown using lentiviral short hairpin RNA inhibited EC (or rescued smooth muscle cell) differentiation in response to TNF-α. Furthermore, TNF-α-mediated EC differentiation was driven by direct binding of nuclear factor-κB (p65) to specific VE-cadherin promoter sequences. Subsequent experiments using an ex vivo decellularized vessel scaffold confirmed an increase in the number of ECs and reduction in smooth muscle cell marker expression in the presence of TNF-α. The lack of TNF-α in a knockout mouse model of vein graft decreased endothelialization and significantly increased thrombosis formation. Our study highlights the role of macrophages in directing vascular stem/progenitor cell lineage commitment through TNF-α-mediated TNF-α receptor 1 and nuclear factor-κB activation that is likely required for endothelial repair in vascular diseases such as vein graft.

Effective myotube formation in human adipose tissue-derived stem cells expressing dystrophin and myosin heavy chain by cellular fusion with mouse C2C12 myoblasts

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

Eom, Young Woo; Biomedical Research Institute, Lifeliver Co., Ltd., Suwon; Lee, Jong Eun

2011-04-29

Highlights: {yields} hASCs were differentiated into skeletal muscle cells by treatment with 5-azacytidine, FGF-2, and the supernatant of cultured hASCs. {yields} Dystrophin and MyHC were expressed in late differentiation step by treatment with the supernatant of cultured hASCs. {yields} hASCs expressing dystrophin and MyHC contributed to myotube formation during co-culture with mouse myoblast C2C12 cells. -- Abstract: Stem cell therapy for muscular dystrophies requires stem cells that are able to participate in the formation of new muscle fibers. However, the differentiation steps that are the most critical for this process are not clear. We investigated the myogenic phases of humanmore » adipose tissue-derived stem cells (hASCs) step by step and the capability of myotube formation according to the differentiation phase by cellular fusion with mouse myoblast C2C12 cells. In hASCs treated with 5-azacytidine and fibroblast growth factor-2 (FGF-2) for 1 day, the early differentiation step to express MyoD and myogenin was induced by FGF-2 treatment for 6 days. Dystrophin and myosin heavy chain (MyHC) expression was induced by hASC conditioned medium in the late differentiation step. Myotubes were observed only in hASCs undergoing the late differentiation step by cellular fusion with C2C12 cells. In contrast, hASCs that were normal or in the early stage were not involved in myotube formation. Our results indicate that stem cells expressing dystrophin and MyHC are more suitable for myotube formation by co-culture with myoblasts than normal or early differentiated stem cells expressing MyoD and myogenin.« less

Differential biological effects of dehydroepiandrosterone (DHEA) between mouse (B16F10) and human melanoma (BLM) cell lines.

PubMed

Joshi, Kumud; Hassan, Sherif S; Ramaraj, Pandurangan

2017-01-01

Dehydroepiandrosterone (DHEA) is a weak androgen and had been shown to have anti-cancer, anti-adipogenic and anti-inflammatory effects on mouse and other rodent models, but not on humans, suggesting a systemic level difference between mouse and human. Our previous study on DHEA biological functions involving a variety of cell lines, suggested that the functional differences between mouse and human existed even at the cellular level. Hence, using mouse and human melanoma cell models, in-vitro effects of DHEA on cell growth, mechanism of cell death and mechanism of DHEA action were studied. Results indicated a differential biological effects of DHEA between mouse and human melanoma cell lines. These in-vitro studies also suggested that the differential biological effects observed between these two cell lines could be due to the difference in the way DHEA was processed or metabolized inside the cell.

Efficient differentiation of mouse embryonic stem cells into motor neurons.

PubMed

Wu, Chia-Yen; Whye, Dosh; Mason, Robert W; Wang, Wenlan

2012-06-09

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb9. Using this robust protocol, we achieved 51 ± 0.8% of differentiation efficiency (n = 3; p < 0.01, Student's t-test). Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

The effect of chemically modified electrospun silica nanofiber on the mRNA and miRNA expression profile of neural stem cell differentiation.

PubMed

Mercado, Augustus T; Yeh, Jui-Ming; Chin, Ting Yu; Chen, Wen Shuo; Chen-Yang, Yui Whei; Chen, Chung-Yung

2016-11-01

A detailed genomic and epigenomic analyses of neural stem cells (NSCs) differentiation in synthetic microenvironments is essential for the advancement of regenerative medicine and therapeutic treatment of diseases. This study identified the changes in mRNA and miRNA expression profile during NSC differentiation on an artificial matrix. NSCs were grown on a surface-modified, electrospun tetraethyl-orthosilicate nanofiber (designated as SNF-AP) by providing a 3D-environment for cell growth and differentiation. Differentially expressed mRNAs and miRNAs of NSC differentiated in this microenvironment were identified through microarray analysis. The genes and miRNA targets responsible for the differentiation fate of NSCs and neuron development process were determined using Ingenuity Pathway Analysis (IPA). SNF-AP enhanced the expression of genes that activates the proliferation, development, and outgrowth of neurons, differentiation and generation of cells, neuritogenesis, outgrowth of neurites, microtubule dynamics, formation of cellular protrusions, and long-term potentiation during NSC differentiation. On the other hand, PDL inhibited neuritogenesis, microtubule dynamics, and proliferation and differentiation of cells and activated the apoptosis function. Moreover, the nanomaterial promoted the expression of more let-7 miRNAs, which have vital roles in NSC differentiation. Overall, SNF-AP is biocompatible and applicable scaffold for NSC differentiation in the development of neural tissue engineering. These findings are useful in enhancing in vitro NSC differentiation potential for preclinical studies and future clinical applications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2730-2743, 2016. © 2016 Wiley Periodicals, Inc.

Differential effects of retinoic acid (RA) and N-(4-hydroxyphenyl) retinamide (4-HPR) on cell growth, induction of differentiation, and changes in p34cdc2, Bcl-2, and actin expression in the human promyelocytic HL-60 leukemic cells.

PubMed

Dipietrantonio, A; Hsieh, T C; Wu, J M

1996-07-25

Incubation of the HL-60 cells with 3 microM of RA and 4-HPR resulted in suppression of cell growth and decrease in cell viability. A significant percentage of the RA-treated cells also displayed differentiation towards neutrophils, as assayed by changes in nitroblue tetrazolium reduction (NBT) and alpha-naphthyl-acetate esterase (ANAE) activities, whereas the 4-HPR treated cells remained essentially undifferentiated. Flow cytometric analysis showed 4-HPR to cause partial cell arrest in the G2/M phase after a 3-day treatment and an additional G1 phase arrest after a 7-day treatment. With RA-treated cells, a reduction in the percentage of cells in the G1 phase was observed after 7 days of treatment. In 4-HPR-treated cells an extra peak, characteristic of cells undergoing apoptosis, was found in the cell cycle phase distribution analysis. Determination of specific protein expression changes by Western blot analysis showed that the p34cdc2 was down-regulated by both chemicals. Furthermore, RA induced bcl-2 but prevented the processing of actin, whereas 4-HPR had little effect on bcl-2 but increased the specific processing of actin. These results suggest that RA promotes neutrophil differentiation and the establishment of a semi apoptosis-resistant state, possibly through the overexpression of the bcl-2 gene. By contrast, 4-HPR may trigger apoptosis by inducing overall cyto-architectural changes and specific DNA fragmentation subsequent to increased turnover of the protein actin.

In vitro differentiation of adipose-tissue-derived mesenchymal stem cells into neural retinal cells through expression of human PAX6 (5a) gene.

PubMed

Rezanejad, Habib; Soheili, Zahra-Soheila; Haddad, Farhang; Matin, Maryam M; Samiei, Shahram; Manafi, Ali; Ahmadieh, Hamid

2014-04-01

The neural retina is subjected to various degenerative conditions. Regenerative stem-cell-based therapy holds great promise for treating severe retinal degeneration diseases, although many drawbacks remain to be overcome. One important problem is to gain authentically differentiated cells for replacement. Paired box 6 protein (5a) (PAX6 (5a)) is a highly conserved master control gene that has an essential role in the development of the vertebrate visual system. Human adipose-tissue-derived stem cell (hADSC) isolation was performed by using fat tissues and was confirmed by the differentiation potential of the cells into adipocytes and osteocytes and by their surface marker profile. The coding region of the human PAX6 (5a) gene isoform was cloned and lentiviral particles were propagated in HEK293T. The differentiation of hADSCs into retinal cells was characterized by morphological characteristics, quantitative real-time reverse transcription plus the polymerase chain reaction (qPCR) and immunocytochemistry (ICC) for some retinal cell-specific and retinal pigmented epithelial (RPE) cell-specific markers. hADSCs were successfully isolated. Flow cytometric analysis of surface markers indicated the high purity (~97 %) of isolated hADSCs. After 30 h of post-transduction, cells gradually showed the characteristic morphology of neuronal cells and small axon-like processes emerged. qPCR and ICC confirmed the differentiation of some neural retinal cells and RPE cells. Thus, PAX6 (5a) transcription factor expression, together with medium supplemented with fibronectin, is able to induce the differentiation of hADSCs into retinal progenitors, RPE cells and photoreceptors.

Prion potency in stem cells biology.

PubMed

Lopes, Marilene H; Santos, Tiago G

2012-01-01

Prion protein (PrP) can be considered a pivotal molecule because it interacts with several partners to perform a diverse range of critical biological functions that might differ in embryonic and adult cells. In recent years, there have been major advances in elucidating the putative role of PrP in the basic biology of stem cells in many different systems. Here, we review the evidence indicating that PrP is a key molecule involved in driving different aspects of the potency of embryonic and tissue-specific stem cells in self-perpetuation and differentiation in many cell types. It has been shown that PrP is involved in stem cell self-renewal, controlling pluripotency gene expression, proliferation, and neural and cardiomyocyte differentiation. PrP also has essential roles in distinct processes that regulate tissue-specific stem cell biology in nervous and hematopoietic systems and during muscle regeneration. Results from our own investigations have shown that PrP is able to modulate self-renewal and proliferation in neural stem cells, processes that are enhanced by PrP interactions with stress inducible protein 1 (STI1). Thus, the available data reveal the influence of PrP in acting upon the maintenance of pluripotent status or the differentiation of stem cells from the early embryogenesis through adulthood.

The role of cannabinoids in adult neurogenesis

PubMed Central

Prenderville, Jack A; Kelly, Áine M; Downer, Eric J

2015-01-01

The processes underpinning post-developmental neurogenesis in the mammalian brain continue to be defined. Such processes involve the proliferation of neural stem cells and neural progenitor cells (NPCs), neuronal migration, differentiation and integration into a network of functional synapses within the brain. Both intrinsic (cell signalling cascades) and extrinsic (neurotrophins, neurotransmitters, cytokines, hormones) signalling molecules are intimately associated with adult neurogenesis and largely dictate the proliferative activity and differentiation capacity of neural cells. Cannabinoids are a unique class of chemical compounds incorporating plant-derived cannabinoids (the active components of Cannabis sativa), the endogenous cannabinoids and synthetic cannabinoid ligands, and these compounds are becoming increasingly recognized for their roles in neural developmental processes. Indeed, cannabinoids have clear modulatory roles in adult neurogenesis, probably through activation of both CB1 and CB2 receptors. In recent years, a large body of literature has deciphered the signalling networks involved in cannabinoid-mediated regulation of neurogenesis. This timely review summarizes the evidence that the cannabinoid system is intricately associated with neuronal differentiation and maturation of NPCs and highlights intrinsic/extrinsic signalling mechanisms that are cannabinoid targets. Overall, these findings identify the central role of the cannabinoid system in adult neurogenesis in the hippocampus and the lateral ventricles and hence provide insight into the processes underlying post-developmental neurogenesis in the mammalian brain. PMID:25951750

Syndecan-4 Regulates Muscle Differentiation and Is Internalized from the Plasma Membrane during Myogenesis.

PubMed

Rønning, Sissel B; Carlson, Cathrine R; Stang, Espen; Kolset, Svein O; Hollung, Kristin; Pedersen, Mona E

2015-01-01

The cell surface proteoglycan syndecan-4 has been reported to be crucial for muscle differentiation, but the molecular mechanisms still remain to be fully understood. During in vitro differentiation of bovine muscle cells immunocytochemical analyses showed strong labelling of syndecan-4 intracellularly, in close proximity with Golgi structures, in membranes of intracellular vesicles and finally, in the nuclear area including the nuclear envelope. Chase experiments showed that syndecan-4 was internalized from the plasma membrane during this process. Furthermore, when syndecan-4 was knocked down by siRNA more myotubes were formed, and the expression of myogenic transcription factors, β1-integrin and actin was influenced. However, when bovine muscle cells were treated with a cell-penetrating peptide containing the cytoplasmic region of syndecan-4, myoblast fusion and thus myotube formation was blocked, both in normal cells and in syndecan-4 knock down cells. Altogether this suggests that the cytoplasmic domain of syndecan-4 is important in regulation of myogenesis. The internalization of syndecan-4 from the plasma membrane during muscle differentiation and the nuclear localization of syndecan-4 in differentiated muscle cells may be part of this regulation, and is a novel aspect of syndecan biology which merits further studies.

High Efficient Differentiation of Functional Hepatocytes from Porcine Induced Pluripotent Stem Cells

PubMed Central

Ao, Ying; Mich-Basso, Jocelyn Danielle; Lin, Bo; Yang, Lei

2014-01-01

Hepatocyte transplantation is considered to be a promising therapy for patients with liver diseases. Induced pluripotent stem cells (iPSCs) provide an unlimited source for the generation of functional hepatocytes. In this study, we generated iPSCs from porcine ear fibroblasts (PEFs) by overexpressing Sox2, Klf4, Oct4, and c-Myc (SKOM), and developed a novel strategy for the efficient differentiation of hepatocyte-like cells from porcine iPSCs by following the processes of early liver development. The differentiated cells displayed the phenotypes of hepatocytes, exhibited classic hepatocyte-associated bio-functions, such as LDL uptake, glycogen storage and urea secretion, as well as possessed the metabolic activities of cytochrome P-450 (CYP) 3A and 2C. Furthermore, we compared the hepatocyte differentiation efficacy of our protocol with another published method, and the results demonstrated that our differentiation strategy could significantly improve the generation of morphological and functional hepatocyte-like cells from porcine iPSCs. In conclusion, this study establishes an efficient method for in vitro generation of functional hepatocytes from porcine iPSCs, which could represent a promising cell source for preclinical testing of cell-based therapeutics for liver failure and for pharmacological applications. PMID:24949734

Induced pluripotent stem cells with NOTCH1 gene mutation show impaired differentiation into smooth muscle and endothelial cells: Implications for bicuspid aortic valve-related aortopathy.

PubMed

Jiao, Jiao; Tian, Weihua; Qiu, Ping; Norton, Elizabeth L; Wang, Michael M; Chen, Y Eugene; Yang, Bo

2018-03-12

The NOTCH1 gene mutation has been identified in bicuspid aortic valve patients. We developed an in vitro model with human induced pluripotent stem cells (iPSCs) to evaluate the role of NOTCH1 in smooth muscle and endothelial cell (EC) differentiation. The iPSCs were derived from a patient with a normal tricuspid aortic valve and aorta. The NOTCH1 gene was targeted in iPSCs with the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 nuclease (Cas9) system. The NOTCH1 -/- (NOTCH1 homozygous knockout) and isogenic control iPSCs (wild type) were differentiated into neural crest stem cells (NCSCs) and into cardiovascular progenitor cells (CVPCs). The NCSCs were differentiated into smooth muscle cells (SMCs). The CVPCs were differentiated into ECs. The differentiations of SMCs and ECs were compared between NOTCH1 -/- and wild type cells. The expression of NCSC markers (SRY-related HMG-box 10 and transcription factor AP-2 alpha) was significantly lower in NOTCH1 -/- NCSCs than in wild type NCSCs. The SMCs derived from NOTCH1 -/- NCSCs showed immature morphology with smaller size and decreased expression of all SMC-specific contractile proteins. In NOTCH1 -/- CVPCs, the expression of ISL1, NKX2.5, and MYOCD was significantly lower than that in isogenic control CVPCs, indicating impaired differentiation from iPSCs to CVPCs. The NOTCH1 -/- ECs derived from CVPCs showed significantly lower expression of cluster of differentiation 105 and cluster of differentiation 31 mRNA and protein, indicating a defective differentiation process. NOTCH1 is critical in SMC and EC differentiation of iPSCs through NCSCs and CVPCs, respectively. NOTCH1 gene mutations might potentially contribute to the development of thoracic aortic aneurysms by affecting SMC differentiation in some patients with bicuspid aortic valve. Copyright © 2018 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.

Pipette-based Method to Study Embryoid Body Formation Derived from Mouse and Human Pluripotent Stem Cells Partially Recapitulating Early Embryonic Development Under Simulated Microgravity Conditions

NASA Astrophysics Data System (ADS)

Shinde, Vaibhav; Brungs, Sonja; Hescheler, Jürgen; Hemmersbach, Ruth; Sachinidis, Agapios

2016-06-01

The in vitro differentiation of pluripotent stem cells partially recapitulates early in vivo embryonic development. More recently, embryonic development under the influence of microgravity has become a primary focus of space life sciences. In order to integrate the technique of pluripotent stem cell differentiation with simulated microgravity approaches, the 2-D clinostat compatible pipette-based method was experimentally investigated and adapted for investigating stem cell differentiation processes under simulated microgravity conditions. In order to keep residual accelerations as low as possible during clinorotation, while also guaranteeing enough material for further analysis, stem cells were exposed in 1-mL pipettes with a diameter of 3.5 mm. The differentiation of mouse and human pluripotent stem cells inside the pipettes resulted in the formation of embryoid bodies at normal gravity (1 g) after 24 h and 3 days. Differentiation of the mouse pluripotent stem cells on a 2-D pipette-clinostat for 3 days also resulted in the formation of embryoid bodies. Interestingly, the expression of myosin heavy chain was downregulated when cultivation was continued for an additional 7 days at normal gravity. This paper describes the techniques for culturing and differentiation of pluripotent stem cells and exposure to simulated microgravity during culturing or differentiation on a 2-D pipette clinostat. The implementation of these methodologies along with -omics technologies will contribute to understand the mechanisms regulating how microgravity influences early embryonic development.

Roles of autophagy in controlling stem cell identity: a perspective of self-renewal and differentiation.

PubMed

Sotthibundhu, Areechun; Promjuntuek, Wilasinee; Liu, Min; Shen, Sanbing; Noisa, Parinya

2018-04-25

Autophagy is crucial for the removal of dysfunctional organelles and protein aggregates and for maintaining stem cell homeostasis, which includes self-renewal, cell differentiation and somatic reprogramming. Loss of self-renewal capacity and pluripotency is a major obstacle to stem cell-based therapies. It has been reported that autophagy regulates stem cells under biological stimuli, starvation, hypoxia, generation of reactive oxygen species (ROS) and cellular senescence. On the one hand, autophagy is shown to play roles in self-renewal by co-function with the ubiquitin-proteasome system (UPS) to promote pluripotency-associated proteins (NANOG, OCT4 and SOX2) in human embryonic stem cells (hESCs). On the other hand, autophagy activity acts as cell reprogramming processes that play an important role for clearance fate determination and upregulates neural and cardiac differentiation. Deregulation of autophagy triggers protein disorders such as neurodegenerative cardiac/muscle diseases and cancer. Therefore, understanding of the roles of the autophagy in stem cell renewal and differentiation may benefit therapeutic development for a range of human diseases.

Classification of C2C12 cells at differentiation by convolutional neural network of deep learning using phase contrast images.

PubMed

Niioka, Hirohiko; Asatani, Satoshi; Yoshimura, Aina; Ohigashi, Hironori; Tagawa, Seiichi; Miyake, Jun

2018-01-01

In the field of regenerative medicine, tremendous numbers of cells are necessary for tissue/organ regeneration. Today automatic cell-culturing system has been developed. The next step is constructing a non-invasive method to monitor the conditions of cells automatically. As an image analysis method, convolutional neural network (CNN), one of the deep learning method, is approaching human recognition level. We constructed and applied the CNN algorithm for automatic cellular differentiation recognition of myogenic C2C12 cell line. Phase-contrast images of cultured C2C12 are prepared as input dataset. In differentiation process from myoblasts to myotubes, cellular morphology changes from round shape to elongated tubular shape due to fusion of the cells. CNN abstract the features of the shape of the cells and classify the cells depending on the culturing days from when differentiation is induced. Changes in cellular shape depending on the number of days of culture (Day 0, Day 3, Day 6) are classified with 91.3% accuracy. Image analysis with CNN has a potential to realize regenerative medicine industry.

The Extracellular Environment's Effect on Cellular Processes: An In Vitro Study of Mechanical and Chemical Cues on Human Mesenchymal Stem Cells and C17.2 Neural Stem Cells

NASA Astrophysics Data System (ADS)

Casey, Meghan E.

Stem cells are widely used in the area of tissue engineering. The ability of cells to interact with materials on the nano- and micro- level is important in the success of the biomaterial. It is well-known that cells respond to their micro- and nano-environments through a process termed chemo-mechanotransduction. It is important to establish standard protocols for cellular experiments, as chemical modifications to maintenance environments can alter long-term research results. In this work, the effects of different media compositions on human mesenchymal stem cells (hMSCs) throughout normal in vitro maintenance are investigated. Changes in RNA regulation, protein expression and proliferation are studied via quantitative polymerase chain reaction (qPCR), immunocytochemistry (ICC) and cell counts, respectively. Morphological differences are also observed throughout the experiment. Results of this study illustrate the dynamic response of hMSC maintenance to differences in growth medium and passage number. These experiments highlight the effect growth medium has on in vitro experiments and the need of consistent protocols in hMSC research. A substantial opportunity exists in neuronal research to develop a material platform that allows for both the proliferation and differentiation of stem cells into neurons and the ability to quantify the secretome of neuronal cells. Anodic aluminum oxide (AAO) membranes are fabricated in a two-step anodization procedure where voltage is varied to control the pore size and morphology of the membranes. C17.2 neural stem cells are differentiated on the membranes via serum-withdrawal. Cellular growth is characterized by scanning electron microscopy (SEM), ICC and qPCR. ImageJ software is used to obtain phenotypic cell counts and neurite outgrowth lengths. Results indicate a highly tunable correlation between AAO nanopore sizes and differentiated cell populations. By selecting AAO membranes with specific pore size ranges, control of neuronal network density and neurite outgrowth length is achievable. To understand differentiation marker expressions in C17.2 NSCs and how material stiffness affects differentiation, cells are cultured on substrates of varying stiffness. qPCR is used to analyze neural stem cell, neural progenitor cell, neuron-restricted progenitor and differentiated post-mitotic neuronal cell RNA expression. Results suggest a relationship between material stiffness and neuronal development in C17.2 neural stem cells.

High content analysis of differentiation and cell death in human adipocytes.

PubMed

Doan-Xuan, Quang Minh; Sarvari, Anitta K; Fischer-Posovszky, Pamela; Wabitsch, Martin; Balajthy, Zoltan; Fesus, Laszlo; Bacso, Zsolt

2013-10-01

Understanding adipocyte biology and its homeostasis is in the focus of current obesity research. We aimed to introduce a high-content analysis procedure for directly visualizing and quantifying adipogenesis and adipoapoptosis by laser scanning cytometry (LSC) in a large population of cell. Slide-based image cytometry and image processing algorithms were used and optimized for high-throughput analysis of differentiating cells and apoptotic processes in cell culture at high confluence. Both preadipocytes and adipocytes were simultaneously scrutinized for lipid accumulation, texture properties, nuclear condensation, and DNA fragmentation. Adipocyte commitment was found after incubation in adipogenic medium for 3 days identified by lipid droplet formation and increased light absorption, while terminal differentiation of adipocytes occurred throughout day 9-14 with characteristic nuclear shrinkage, eccentric nuclei localization, chromatin condensation, and massive lipid deposition. Preadipocytes were shown to be more prone to tumor necrosis factor alpha (TNFα)-induced apoptosis compared to mature adipocytes. Importantly, spontaneous DNA fragmentation was observed at early stage when adipocyte commitment occurs. This DNA damage was independent from either spontaneous or induced apoptosis and probably was part of the differentiation program. © 2013 International Society for Advancement of Cytometry. Copyright © 2013 International Society for Advancement of Cytometry.

Hybrid cell adhesive material for instant dielectrophoretic cell trapping and long-term cell function assessment.

PubMed

Reyes, Darwin R; Hong, Jennifer S; Elliott, John T; Gaitan, Michael

2011-08-16

Dielectrophoresis (DEP) for cell manipulation has focused, for the most part, on approaches for separation/enrichment of cells of interest. Advancements in cell positioning and immobilization onto substrates for cell culture, either as single cells or as cell aggregates, has benefited from the intensified research efforts in DEP (electrokinetic) manipulation. However, there has yet to be a DEP approach that provides the conditions for cell manipulation while promoting cell function processes such as cell differentiation. Here we present the first demonstration of a system that combines DEP with a hybrid cell adhesive material (hCAM) to allow for cell entrapment and cell function, as demonstrated by cell differentiation into neuronlike cells (NLCs). The hCAM, comprised of polyelectrolytes and fibronectin, was engineered to function as an instantaneous cell adhesive surface after DEP manipulation and to support long-term cell function (cell proliferation, induction, and differentiation). Pluripotent P19 mouse embryonal carcinoma cells flowing within a microchannel were attracted to the DEP electrode surface and remained adhered onto the hCAM coating under a fluid flow field after the DEP forces were removed. Cells remained viable after DEP manipulation for up to 8 d, during which time the P19 cells were induced to differentiate into NLCs. This approach could have further applications in areas such as cell-cell communication, three-dimensional cell aggregates to create cell microenvironments, and cell cocultures.

MicroRNA-127 Promotes Mesendoderm Differentiation of Mouse Embryonic Stem Cells by Targeting Left-Right Determination Factor 2.

PubMed

Ma, Haixia; Lin, Yu; Zhao, Zhen-Ao; Lu, Xukun; Yu, Yang; Zhang, Xiaoxin; Wang, Qiang; Li, Lei

2016-06-03

Specification of the three germ layers is a fundamental process and is essential for the establishment of organ rudiments. Multiple genetic and epigenetic factors regulate this dynamic process; however, the function of specific microRNAs in germ layer differentiation remains unknown. In this study, we established that microRNA-127 (miR-127) is related to germ layer specification via microRNA array analysis of isolated three germ layers of E7.5 mouse embryos and was verified through differentiation of mouse embryonic stem cells. miR-127 is highly expressed in endoderm and primitive streak. Overexpression of miR-127 increases and inhibition of miR-127 decreases the expression of mesendoderm markers. We further show that miR-127 promotes mesendoderm differentiation through the nodal pathway, a determinative signaling pathway in early embryogenesis. Using luciferase reporter assay, left-right determination factor 2 (Lefty2), an antagonist of nodal, is identified to be a novel target of miR-127. Furthermore, the role of miR-127 in mesendoderm differentiation is attenuated by Lefty2 overexpression. Altogether, our results indicate that miR-127 accelerates mesendoderm differentiation of mouse embryonic stem cells through nodal signaling by targeting Lefty2. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Regulation of Mesenchymal Stem Cell Differentiation by Nanopatterning of Bulk Metallic Glass.

PubMed

Loye, Ayomiposi M; Kinser, Emily R; Bensouda, Sabrine; Shayan, Mahdis; Davis, Rose; Wang, Rui; Chen, Zheng; Schwarz, Udo D; Schroers, Jan; Kyriakides, Themis R

2018-06-08

Mesenchymal stem cell (MSC) differentiation is regulated by surface modification including texturing, which is applied to materials to enhance tissue integration. Here, we used Pt 57.5 Cu 14.7 Ni 5.3 P 22.5 bulk metallic glass (Pt-BMG) with nanopatterned surfaces achieved by thermoplastic forming to influence differentiation of human MSCs. Pt-BMGs are a unique class of amorphous metals with high strength, elasticity, corrosion resistance, and an unusual plastic-like processability. It was found that flat and nanopattened Pt-BMGs induced osteogenic and adipogenic differentiation, respectively. In addition, osteogenic differentiation on flat BMG exceeded that observed on medical grade titanium and was associated with increased formation of focal adhesions and YAP nuclear localization. In contrast, cells on nanopatterned BMGs exhibited rounded morphology, formed less focal adhesions and had mostly cytoplasmic YAP. These changes were preserved on nanopatterns made of nanorods with increased stiffness due to shorter aspect ratios, suggesting that MSC differentiation was primarily influenced by topography. These observations indicate that both elemental composition and nanotopography can modulate biochemical cues and influence MSCs. Moreover, the processability and highly tunable nature of Pt-BMGs enables the creation of a wide range of surface topographies that can be reproducibly and systematically studied, leading to the development of implants capable of engineering MSC functions.

Increased numbers of preexisting memory CD8 T cells and decreased T-bet expression can restrain terminal differentiation of secondary effector and memory CD8 T cells.

PubMed

Joshi, Nikhil S; Cui, Weiguo; Dominguez, Claudia X; Chen, Jonathan H; Hand, Timothy W; Kaech, Susan M

2011-10-15

Memory CD8 T cells acquire effector memory cell properties after reinfection and may reach terminally differentiated, senescent states ("Hayflick limit") after multiple infections. The signals controlling this process are not well understood, but we found that the degree of secondary effector and memory CD8 T cell differentiation was intimately linked to the amount of T-bet expressed upon reactivation and preexisting memory CD8 T cell number (i.e., primary memory CD8 T cell precursor frequency) present during secondary infection. Compared with naive cells, memory CD8 T cells were predisposed toward terminal effector (TE) cell differentiation because they could immediately respond to IL-12 and induce T-bet, even in the absence of Ag. TE cell formation after secondary (2°) or tertiary infections was dependent on increased T-bet expression because T-bet(+/-) cells were resistant to these phenotypic changes. Larger numbers of preexisting memory CD8 T cells limited the duration of 2° infection and the amount of IL-12 produced, and consequently, this reduced T-bet expression and the proportion of 2° TE CD8 T cells that formed. Together, these data show that over repeated infections, memory CD8 T cell quality and proliferative fitness is not strictly determined by the number of serial encounters with Ag or cell divisions, but is a function of the CD8 T cell differentiation state, which is genetically controlled in a T-bet-dependent manner. This differentiation state can be modulated by preexisting memory CD8 T cell number and the intensity of inflammation during reinfection. These results have important implications for vaccinations involving prime-boost strategies.

Megakaryocytic differentiation induced by constitutive activation of mitogen-activated protein kinase kinase.

PubMed Central

Whalen, A M; Galasinski, S C; Shapiro, P S; Nahreini, T S; Ahn, N G

1997-01-01

The K562 erythroleukemia cell line was used to study the molecular mechanisms regulating lineage commitment of hematopoietic stem cells. Phorbol esters, which initiate megakaryocyte differentiation in this cell line, caused a rapid increase in extracellular-signal-regulated kinase (ERK), which remained elevated for 2 h and returned to near-basal levels by 24 h. In the absence of extracellular stimuli, ERK could be activated by expression of constitutively active mutants of mitogen-activated protein (MAP) kinase kinase (MKK), resulting in cell adhesion and spreading, increased cell size, inhibition of cell growth, and induction of the platelet-specific integrin alphaIIb beta3, all hallmarks of megakaryocytic differentiation. In contrast, expression of wild-type MKK had little effect. In addition, constitutively active MKK suppressed the expression of an erythroid marker, alpha-globin, indicating the ability to suppress cellular responses necessary for alternative cell lineages. The MKK inhibitor PD98059 blocked MKK/ERK activation and cellular responses to phorbol ester, demonstrating that activation of MKK is necessary and sufficient to induce a differentiation program along the megakaryocyte lineage. Thus, the MAP kinase cascade, which promotes cell growth and proliferation in many cell types, instead inhibits cell proliferation and initiates lineage-specific differentiation in K562 cells, establishing a model system to investigate the mechanisms by which this signal transduction pathway specifies cell fate and developmental processes. PMID:9121442

Megakaryocytic differentiation induced by constitutive activation of mitogen-activated protein kinase kinase.

PubMed

Whalen, A M; Galasinski, S C; Shapiro, P S; Nahreini, T S; Ahn, N G

1997-04-01

The K562 erythroleukemia cell line was used to study the molecular mechanisms regulating lineage commitment of hematopoietic stem cells. Phorbol esters, which initiate megakaryocyte differentiation in this cell line, caused a rapid increase in extracellular-signal-regulated kinase (ERK), which remained elevated for 2 h and returned to near-basal levels by 24 h. In the absence of extracellular stimuli, ERK could be activated by expression of constitutively active mutants of mitogen-activated protein (MAP) kinase kinase (MKK), resulting in cell adhesion and spreading, increased cell size, inhibition of cell growth, and induction of the platelet-specific integrin alphaIIb beta3, all hallmarks of megakaryocytic differentiation. In contrast, expression of wild-type MKK had little effect. In addition, constitutively active MKK suppressed the expression of an erythroid marker, alpha-globin, indicating the ability to suppress cellular responses necessary for alternative cell lineages. The MKK inhibitor PD98059 blocked MKK/ERK activation and cellular responses to phorbol ester, demonstrating that activation of MKK is necessary and sufficient to induce a differentiation program along the megakaryocyte lineage. Thus, the MAP kinase cascade, which promotes cell growth and proliferation in many cell types, instead inhibits cell proliferation and initiates lineage-specific differentiation in K562 cells, establishing a model system to investigate the mechanisms by which this signal transduction pathway specifies cell fate and developmental processes.

Inhibition of Ape1 Redox Activity Promotes Odonto/osteogenic Differentiation of Dental Papilla Cells.

PubMed

Chen, Tian; Liu, Zhi; Sun, Wenhua; Li, Jingyu; Liang, Yan; Yang, Xianrui; Xu, Yang; Yu, Mei; Tian, Weidong; Chen, Guoqing; Bai, Ding

2015-12-07

Dentinogenesis is the formation of dentin, a substance that forms the majority of teeth, and this process is performed by odontoblasts. Dental papilla cells (DPCs), as the progenitor cells of odontoblasts, undergo the odontogenic differentiation regulated by multiple cytokines and paracrine signal molecules. Ape1 is a perfect paradigm of the function complexity of a biological macromolecule with two major functional regions for DNA repair and redox regulation, respectively. To date, it remains unclear whether Ape1 can regulate the dentinogenesis in DPCs. In the present study, we firstly examed the spatio-temporal expression of Ape1 during tooth germ developmental process, and found the Ape1 expression was initially high and then gradually reduced along with the tooth development. Secondly, the osteo/odontogenic differentiation capacity of DPCs was up-regulated when treated with either Ape1-shRNA or E3330 (a specific inhibitor of the Ape1 redox function), respectively. Moreover, we found that the canonical Wnt signaling pathway was activated in this process, and E3330 reinforced-osteo/odontogenic differentiation capacity was suppressed by Dickkopf1 (DKK1), a potent antagonist of canonical Wnt signaling pathway. Taken together, we for the first time showed that inhibition of Ape1 redox regulation could promote the osteo/odontogenic differentiation capacity of DPCs via canonical Wnt signaling pathway.

Analysing intracellular deformation of polymer capsules using structured illumination microscopy

NASA Astrophysics Data System (ADS)

Chen, Xi; Cui, Jiwei; Sun, Huanli; Müllner, Markus; Yan, Yan; Noi, Ka Fung; Ping, Yuan; Caruso, Frank

2016-06-01

Understanding the behaviour of therapeutic carriers is important in elucidating their mechanism of action and how they are processed inside cells. Herein we examine the intracellular deformation of layer-by-layer assembled polymer capsules using super-resolution structured illumination microscopy (SIM). Spherical- and cylindrical-shaped capsules were studied in three different cell lines, namely HeLa (human epithelial cell line), RAW264.7 (mouse macrophage cell line) and differentiated THP-1 (human monocyte-derived macrophage cell line). We observed that the deformation of capsules was dependent on cell line, but independent of capsule shape. This suggests that the mechanical forces, which induce capsule deformation during cell uptake, vary between cell lines, indicating that the capsules are exposed to higher mechanical forces in HeLa cells, followed by RAW264.7 and then differentiated THP-1 cells. Our study demonstrates the use of super-resolution SIM in analysing intracellular capsule deformation, offering important insights into the cellular processing of drug carriers in cells and providing fundamental knowledge of intracellular mechanobiology. Furthermore, this study may aid in the design of novel drug carriers that are sensitive to deformation for enhanced drug release properties.Understanding the behaviour of therapeutic carriers is important in elucidating their mechanism of action and how they are processed inside cells. Herein we examine the intracellular deformation of layer-by-layer assembled polymer capsules using super-resolution structured illumination microscopy (SIM). Spherical- and cylindrical-shaped capsules were studied in three different cell lines, namely HeLa (human epithelial cell line), RAW264.7 (mouse macrophage cell line) and differentiated THP-1 (human monocyte-derived macrophage cell line). We observed that the deformation of capsules was dependent on cell line, but independent of capsule shape. This suggests that the mechanical forces, which induce capsule deformation during cell uptake, vary between cell lines, indicating that the capsules are exposed to higher mechanical forces in HeLa cells, followed by RAW264.7 and then differentiated THP-1 cells. Our study demonstrates the use of super-resolution SIM in analysing intracellular capsule deformation, offering important insights into the cellular processing of drug carriers in cells and providing fundamental knowledge of intracellular mechanobiology. Furthermore, this study may aid in the design of novel drug carriers that are sensitive to deformation for enhanced drug release properties. Electronic supplementary information (ESI) available: Additional figures. See DOI: 10.1039/c6nr02151d

Characteristics of hepatic stem/progenitor cells in the fetal and adult liver.

PubMed

Koike, Hiroyuki; Taniguchi, Hideki

2012-11-01

The liver is an essential organ that maintains vital activity through its numerous important functions. It has a unique capability of fully regenerating after injury. Regulating a balance between self-renewal and differentiation of hepatic stem cells that are resources for functional mature liver cells is required for maintenance of tissue homeostasis. This review describes the characteristics of hepatic stem/progenitor cells and the regulatory mechanism of their self-renewal and differentiation capacity. In liver organogenesis, undifferentiated hepatic stem/progenitor cells expand their pool by repeated self-renewal in the early stage of liver development and then differentiate into two different types of cell lineage, namely hepatocytes and cholangiocytes. Liver development is regulated by expression of stem cell transcription factors in a complex multistep process. Recent studies suggest that stem cells are maintained by integrative regulation of gene expression patterns related to self-renewal and differentiation by epigenetic mechanisms such as histone modification and DNA methylation. Analysis of the proper regulatory mechanism of hepatic stem/progenitor cells is important for regenerative medicine that utilizes hepatic stem cells and for preventing liver cancer through clarification of the carcinogenetic mechanism involved in stem cell system failure.

(beta)-catenin mediates the specification of endoderm cells in ascidian embryos.

PubMed

Imai, K; Takada, N; Satoh, N; Satou, Y

2000-07-01

In the present study, we addressed the role of (beta)-catenin in the specification of embryonic cells of the ascidians Ciona intestinalis and C. savignyi and obtained the following results: (1) During cleavages, (beta)-catenin accumulated in the nuclei of vegetal blastomeres, suggesting that it plays a role in the specification of endoderm. (2) Mis- and/or overexpression of (beta)-catenin induced the development of an endoderm-specific alkaline phosphatase (AP) in presumptive notochord cells and epidermis cells without affecting differentiation of primary lineage muscle cells. (3) Downregulation of (beta)-catenin induced by the overexpression of cadherin resulted in the suppression of endoderm cell differentiation. This suppression was compensated for by the differentiation of extra epidermis cells. (4) Specification of notochord cells did not take place in the absence of endoderm differentiation. Both the overexpression of (beta)-catenin in presumptive notochord cells and the downregulation of (beta)-catenin in presumptive endoderm cells led to the suppression of Brachyury gene expression, resulting in the failure of notochord specification. These results suggest that the accumulation of (beta)-catenin in the nuclei of endoderm progenitor cells is the first step in the process of ascidian endoderm specification.

Progress research of non-Cz silicon material

NASA Technical Reports Server (NTRS)

Campbell, R. B.

1983-01-01

The simultaneous diffusion of liquid boron and liquid phosphorus dopants into N-type dendritic silicon web for solar cells was investigated. It is planned that the diffusion parameters required to achieve the desired P(+)NN(+) cell structure be determined and the resultant cell properties be compared to cells produced in a sequential differential process. A cost analysis of the simultaneous junction formation process is proposed.

Early In Vitro Differentiation of Mouse Definitive Endoderm Is Not Correlated with Progressive Maturation of Nuclear DNA Methylation Patterns

PubMed Central

Tajbakhsh, Jian; Gertych, Arkadiusz; Fagg, W. Samuel; Hatada, Seigo; Fair, Jeffrey H.

2011-01-01

The genome organization in pluripotent cells undergoing the first steps of differentiation is highly relevant to the reprogramming process in differentiation. Considering this fact, chromatin texture patterns that identify cells at the very early stage of lineage commitment could serve as valuable tools in the selection of optimal cell phenotypes for regenerative medicine applications. Here we report on the first-time use of high-resolution three-dimensional fluorescence imaging and comprehensive topological cell-by-cell analyses with a novel image-cytometrical approach towards the identification of in situ global nuclear DNA methylation patterns in early endodermal differentiation of mouse ES cells (up to day 6), and the correlations of these patterns with a set of putative markers for pluripotency and endodermal commitment, and the epithelial and mesenchymal character of cells. Utilizing this in vitro cell system as a model for assessing the relationship between differentiation and nuclear DNA methylation patterns, we found that differentiating cell populations display an increasing number of cells with a gain in DNA methylation load: first within their euchromatin, then extending into heterochromatic areas of the nucleus, which also results in significant changes of methylcytosine/global DNA codistribution patterns. We were also able to co-visualize and quantify the concomitant stochastic marker expression on a per-cell basis, for which we did not measure any correlation to methylcytosine loads or distribution patterns. We observe that the progression of global DNA methylation is not correlated with the standard transcription factors associated with endodermal development. Further studies are needed to determine whether the progression of global methylation could represent a useful signature of cellular differentiation. This concept of tracking epigenetic progression may prove useful in the selection of cell phenotypes for future regenerative medicine applications. PMID:21779341

Comprehensive analysis of alternative splicing and functionality in neuronal differentiation of P19 cells.

PubMed

Suzuki, Hitoshi; Osaki, Ken; Sano, Kaori; Alam, A H M Khurshid; Nakamura, Yuichiro; Ishigaki, Yasuhito; Kawahara, Kozo; Tsukahara, Toshifumi

2011-02-18

Alternative splicing, which produces multiple mRNAs from a single gene, occurs in most human genes and contributes to protein diversity. Many alternative isoforms are expressed in a spatio-temporal manner, and function in diverse processes, including in the neural system. The purpose of the present study was to comprehensively investigate neural-splicing using P19 cells. GeneChip Exon Array analysis was performed using total RNAs purified from cells during neuronal cell differentiation. To efficiently and readily extract the alternative exon candidates, 9 filtering conditions were prepared, yielding 262 candidate exons (236 genes). Semiquantitative RT-PCR results in 30 randomly selected candidates suggested that 87% of the candidates were differentially alternatively spliced in neuronal cells compared to undifferentiated cells. Gene ontology and pathway analyses suggested that many of the candidate genes were associated with neural events. Together with 66 genes whose functions in neural cells or organs were reported previously, 47 candidate genes were found to be linked to 189 events in the gene-level profile of neural differentiation. By text-mining for the alternative isoform, distinct functions of the isoforms of 9 candidate genes indicated by the result of Exon Array were confirmed. Alternative exons were successfully extracted. Results from the informatics analyses suggested that neural events were primarily governed by genes whose expression was increased and whose transcripts were differentially alternatively spliced in the neuronal cells. In addition to known functions in neural cells or organs, the uninvestigated alternative splicing events of 11 genes among 47 candidate genes suggested that cell cycle events are also potentially important. These genes may help researchers to differentiate the roles of alternative splicing in cell differentiation and cell proliferation.

Neurogenic transdifferentiation of human adipose-derived stem cells? A critical protocol reevaluation with special emphasis on cell proliferation and cell cycle alterations.

PubMed

Kompisch, Kai Michael; Lange, Claudia; Steinemann, Doris; Skawran, Britta; Schlegelberger, Brigitte; Müller, Reinhard; Schumacher, Udo

2010-11-01

Adipose-derived stem cells (ASCs) are reported to display multilineage differentiation potential, including neuroectodermal pathways. The aim of the present study was to critically re-evaluate the potential neurogenic (trans-)differentiation capacity of ASCs using a neurogenic induction protocol based on the combination of isobutylmethylxanthine (IBMX), indomethacin and insulin. ASCs isolated from lipo-aspirate samples of five healthy female donors were characterized and potential neurogenic (trans-)differentiation was assessed by means of immunohistochemistry and gene expression analyses. Cell proliferation and cell cycle alterations were studied, and the expression of CREB/ATF transcription factors was analyzed. ASCs expressed CD59, CD90 and CD105, and were tested negative for CD34 and CD45. Under neurogenic induction, ASCs adopted a characteristic morphology comparable to neur(on)al progenitors and expressed musashi1, β-III-tubulin and nestin. Gene expression analyses revealed an increased expression of β-III-tubulin, GFAP, vimentin and BDNF, as well as SOX4 in induced ASCs. Cell proliferation was significantly reduced under neurogenic induction; cell cycle analyses showed a G2-cell cycle arrest accompanied by differential expression of key regulators of cell cycle progression. Differential expression of CREB/ATF transcription factors could be observed on neurogenic induction, pointing to a decisive role of the cAMP-CREB/ATF system. Our findings may point to a potential neurogenic (trans-)differentiation of ASCs into early neur(on)al progenitors, but do not present definite evidence for it. Especially, the adoption of a neural progenitor cell-like morphology must not automatically be misinterpreted as a specific characteristic of a respective (trans-)differentiation process, as this may as well be caused by alterations of cell cycle progression.

Coding of auditory temporal and pitch information by hippocampal individual cells and cell assemblies in the rat.

PubMed

Sakurai, Y

2002-01-01

This study reports how hippocampal individual cells and cell assemblies cooperate for neural coding of pitch and temporal information in memory processes for auditory stimuli. Each rat performed two tasks, one requiring discrimination of auditory pitch (high or low) and the other requiring discrimination of their duration (long or short). Some CA1 and CA3 complex-spike neurons showed task-related differential activity between the high and low tones in only the pitch-discrimination task. However, without exception, neurons which showed task-related differential activity between the long and short tones in the duration-discrimination task were always task-related neurons in the pitch-discrimination task. These results suggest that temporal information (long or short), in contrast to pitch information (high or low), cannot be coded independently by specific neurons. The results also indicate that the two different behavioral tasks cannot be fully differentiated by the task-related single neurons alone and suggest a model of cell-assembly coding of the tasks. Cross-correlation analysis among activities of simultaneously recorded multiple neurons supported the suggested cell-assembly model.Considering those results, this study concludes that dual coding by hippocampal single neurons and cell assemblies is working in memory processing of pitch and temporal information of auditory stimuli. The single neurons encode both auditory pitches and their temporal lengths and the cell assemblies encode types of tasks (contexts or situations) in which the pitch and the temporal information are processed.

Ultrastructural study on the differentiation and the fate of M cells in follicle-associated epithelium of rat Peyer's patch.

PubMed

Onishi, Sachiko; Yokoyama, Toshifumi; Chin, Keigi; Yuji, Midori; Inamoto, Tetsurou; Qi, Wang-Mei; Warita, Katsuhiko; Hoshi, Nobuhiko; Kitagawa, Hiroshi

2007-05-01

The differentiation process of immature microvillous epithelial cells to M cells and the fate of M cells in the follicle-associated epithelium (FAE) of the mucosa-associated lymphoid tissues are still unclear. In this study, the differentiation process and the fate of M cells were clarified in rat Peyer's patches under a transmission electron microscope. Almost all immature epithelial cells were found to possess long, slender microvilli, which gradually shortened, thickened and dispersed as the immature epithelial cells migrated away from the crypt orifices. These morphological changes started in the centers and moved to the peripheries of the apical surfaces of epithelial cells, accompanied by the protrusion of apical cytoplasm out of the terminal web. During these changes, the bundles of microfilaments of microvilli never shortened, and both small vesicles in the apical cytoplasm and tiny invaginations of the apical membranes were found. The intraepithelial migrating cells gradually accumulated to form typical intraepithelial pockets. In all FAE, there was no morphological sign of cell death in M cells. The rearrangement of microfilament bundles, the reconstruction of microvilli and the disappearance of pockets resulted in the transformation of M cells into microvillous epithelial cells. These serial ultrastructural changes suggest that M cells are a temporal and transitional cell type caused by the active engulfment of luminal substances and that when the engulfment ceases, the M cells transform into mature microvillous epithelial cells.

Differential gene expression analysis in glioblastoma cells and normal human brain cells based on GEO database.

PubMed

Wang, Anping; Zhang, Guibin

2017-11-01

The differentially expressed genes between glioblastoma (GBM) cells and normal human brain cells were investigated to performed pathway analysis and protein interaction network analysis for the differentially expressed genes. GSE12657 and GSE42656 gene chips, which contain gene expression profile of GBM were obtained from Gene Expression Omniub (GEO) database of National Center for Biotechnology Information (NCBI). The 'limma' data packet in 'R' software was used to analyze the differentially expressed genes in the two gene chips, and gene integration was performed using 'RobustRankAggreg' package. Finally, pheatmap software was used for heatmap analysis and Cytoscape, DAVID, STRING and KOBAS were used for protein-protein interaction, Gene Ontology (GO) and KEGG analyses. As results: i) 702 differentially expressed genes were identified in GSE12657, among those genes, 548 were significantly upregulated and 154 were significantly downregulated (p<0.01, fold-change >1), and 1,854 differentially expressed genes were identified in GSE42656, among the genes, 1,068 were significantly upregulated and 786 were significantly downregulated (p<0.01, fold-change >1). A total of 167 differentially expressed genes including 100 upregulated genes and 67 downregulated genes were identified after gene integration, and the genes showed significantly different expression levels in GBM compared with normal human brain cells (p<0.05). ii) Interactions between the protein products of 101 differentially expressed genes were identified using STRING and expression network was established. A key gene, called CALM3, was identified by Cytoscape software. iii) GO enrichment analysis showed that differentially expressed genes were mainly enriched in 'neurotransmitter:sodium symporter activity' and 'neurotransmitter transporter activity', which can affect the activity of neurotransmitter transportation. KEGG pathway analysis showed that the differentially expressed genes were mainly enriched in 'protein processing in endoplasmic reticulum', which can affect protein processing in endoplasmic reticulum. The results showed that: i) 167 differentially expressed genes were identified from two gene chips after integration; and ii) protein interaction network was established, and GO and KEGG pathway analyses were successfully performed to identify and annotate the key gene, which provide new insights for the studies on GBN at gene level.

Reserve stem cells: Reprogramming of differentiated cells fuels repair, metaplasia, and neoplasia in the adult gastrointestinal tract

PubMed Central

Mills, Jason C.; Sansom, Owen J.

2016-01-01

It has long been known that differentiated cells can switch fates, especially in vitro, but only recently has there been a critical mass of publications describing the mechanisms adult, post-mitotic cells use in vivo to reverse their differentiation state. We propose that this sort of cellular reprogramming is a fundamental cellular process akin to apoptosis or mitosis. Because reprogramming can invoke regenerative cells from mature cells, it is critical to the longterm maintenance of tissues like the pancreas, which encounter large insults during adulthood but lack constitutively active adult stem cells to repair the damage. However, even in tissues with adult stem cells, like stomach and intestine, reprogramming may allow mature cells to serve as reserve (“quiescent”) stem cells when normal stem cells are compromised. We propose that the potential downside to reprogramming is that it increases risk for cancers that occur late in adulthood. Mature, long-lived cells may have years of exposure to mutagens. Mutations that affect the physiological function of differentiated, post-mitotic cells may lead to apoptosis, but mutations in genes that govern proliferation might not be selected against. Hence, reprogramming with reentry into the cell cycle might unmask those mutations, causing an irreversible progenitor-like, proliferative state. We review recent evidence showing that reprogramming fuels irreversible metaplastic and precancerous proliferations in stomach and pancreas. Finally, we illustrate how we think reprogrammed differentiated cells are likely candidates as cells of origin for cancers of the intestine. PMID:26175494

Hydroxyapatite coatings deposited by liquid precursor plasma spraying: controlled dense and porous microstructures and osteoblastic cell responses.

PubMed

Huang, Yi; Song, Lei; Liu, Xiaoguang; Xiao, Yanfeng; Wu, Yao; Chen, Jiyong; Wu, Fang; Gu, Zhongwei

2010-12-01

Hydroxyapatite coatings were deposited on Ti-6Al-4V substrates by a novel plasma spraying process, the liquid precursor plasma spraying (LPPS) process. X-ray diffraction results showed that the coatings obtained by the LPPS process were mainly composed of hydroxyapatite. The LPPS process also showed excellent control on the coating microstructure, and both nearly fully dense and highly porous hydroxyapatite coatings were obtained by simply adjusting the solid content of the hydroxyapatite liquid precursor. Scanning electron microscope observations indicated that the porous hydroxyapatite coatings had pore size in the range of 10-200 µm and an average porosity of 48.26 ± 0.10%. The osteoblastic cell responses to the dense and porous hydroxyapatite coatings were evaluated with human osteoblastic cell MG-63, in respect of the cell morphology, proliferation and differentiation, with the hydroxyapatite coatings deposited by the atmospheric plasma spraying (APS) process as control. The cell experiment results indicated that the heat-treated LPPS coatings with a porous structure showed the best cell proliferation and differentiation among all the hydroxyapatite coatings. Our results suggest that the LPPS process is a promising plasma spraying technique for fabricating hydroxyapatite coatings with a controllable microstructure, which has great potential in bone repair and replacement applications.

The regulation of autophagy differentially affects Trypanosoma cruzi metacyclogenesis.

PubMed

Vanrell, María Cristina; Losinno, Antonella Denisse; Cueto, Juan Agustín; Balcazar, Darío; Fraccaroli, Laura Virginia; Carrillo, Carolina; Romano, Patricia Silvia

2017-11-01

Autophagy is a cellular process required for the removal of aged organelles and cytosolic components through lysosomal degradation. All types of eukaryotic cells from yeasts to mammalian cells have the machinery to activate autophagy as a result of many physiological and pathological situations. The most frequent stimulus of autophagy is starvation and the result, in this case, is the fast generation of utilizable food (e.g. amino acids and basic nutrients) to maintain the vital biological processes. In some organisms, starvation also triggers other associated processes such as differentiation. The protozoan parasite Trypanosoma cruzi undergoes a series of differentiation processes throughout its complex life cycle. Although not all autophagic genes have been identified in the T. cruzi genome, previous works have demonstrated the presence of essential autophagic-related proteins. Under starvation conditions, TcAtg8, which is the parasite homolog of Atg8/LC3 in other organisms, is located in autophagosome-like vesicles. In this work, we have characterized the autophagic pathway during T. cruzi differentiation from the epimastigote to metacyclic trypomastigote form, a process called metacyclogenesis. We demonstrated that autophagy is stimulated during metacyclogenesis and that the induction of autophagy promotes this process. Moreover, with exception of bafilomycin, other classical autophagy modulators have similar effects on T. cruzi autophagy. We also showed that spermidine and related polyamines can positively regulate parasite autophagy and differentiation. We concluded that both polyamine metabolism and autophagy are key processes during T. cruzi metacyclogenesis that could be exploited as drug targets to avoid the parasite cycle progression.

Systematically labeling developmental stage-specific genes for the study of pancreatic β-cell differentiation from human embryonic stem cells.

PubMed

Liu, Haisong; Yang, Huan; Zhu, Dicong; Sui, Xin; Li, Juan; Liang, Zhen; Xu, Lei; Chen, Zeyu; Yao, Anzhi; Zhang, Long; Zhang, Xi; Yi, Xing; Liu, Meng; Xu, Shiqing; Zhang, Wenjian; Lin, Hua; Xie, Lan; Lou, Jinning; Zhang, Yong; Xi, Jianzhong; Deng, Hongkui

2014-10-01

The applications of human pluripotent stem cell (hPSC)-derived cells in regenerative medicine has encountered a long-standing challenge: how can we efficiently obtain mature cell types from hPSCs? Attempts to address this problem are hindered by the complexity of controlling cell fate commitment and the lack of sufficient developmental knowledge for guiding hPSC differentiation. Here, we developed a systematic strategy to study hPSC differentiation by labeling sequential developmental genes to encompass the major developmental stages, using the directed differentiation of pancreatic β cells from hPSCs as a model. We therefore generated a large panel of pancreas-specific mono- and dual-reporter cell lines. With this unique platform, we visualized the kinetics of the entire differentiation process in real time for the first time by monitoring the expression dynamics of the reporter genes, identified desired cell populations at each differentiation stage and demonstrated the ability to isolate these cell populations for further characterization. We further revealed the expression profiles of isolated NGN3-eGFP(+) cells by RNA sequencing and identified sushi domain-containing 2 (SUSD2) as a novel surface protein that enriches for pancreatic endocrine progenitors and early endocrine cells both in human embryonic stem cells (hESC)-derived pancreatic cells and in the developing human pancreas. Moreover, we captured a series of cell fate transition events in real time, identified multiple cell subpopulations and unveiled their distinct gene expression profiles, among heterogeneous progenitors for the first time using our dual reporter hESC lines. The exploration of this platform and our new findings will pave the way to obtain mature β cells in vitro.

Single-cell RNA-seq and computational analysis using temporal mixture modelling resolves Th1/Tfh fate bifurcation in malaria

PubMed Central

Lönnberg, Tapio; Svensson, Valentine; James, Kylie R.; Fernandez-Ruiz, Daniel; Sebina, Ismail; Montandon, Ruddy; Soon, Megan S. F.; Fogg, Lily G.; Nair, Arya Sheela; Liligeto, Urijah; Stubbington, Michael J. T.; Ly, Lam-Ha; Bagger, Frederik Otzen; Zwiessele, Max; Lawrence, Neil D.; Souza-Fonseca-Guimaraes, Fernando; Bunn, Patrick T.; Engwerda, Christian R.; Heath, William R.; Billker, Oliver; Stegle, Oliver; Haque, Ashraful; Teichmann, Sarah A.

2017-01-01

Differentiation of naïve CD4+ T cells into functionally distinct T helper subsets is crucial for the orchestration of immune responses. Due to extensive heterogeneity and multiple overlapping transcriptional programs in differentiating T cell populations, this process has remained a challenge for systematic dissection in vivo. By using single-cell transcriptomics and computational analysis using a temporal mixtures of Gaussian processes model, termed GPfates, we reconstructed the developmental trajectories of Th1 and Tfh cells during blood-stage Plasmodium infection in mice. By tracking clonality using endogenous TCR sequences, we first demonstrated that Th1/Tfh bifurcation had occurred at both population and single-clone levels. Next, we identified genes whose expression was associated with Th1 or Tfh fates, and demonstrated a T-cell intrinsic role for Galectin-1 in supporting a Th1 differentiation. We also revealed the close molecular relationship between Th1 and IL-10-producing Tr1 cells in this infection. Th1 and Tfh fates emerged from a highly proliferative precursor that upregulated aerobic glycolysis and accelerated cell cycling as cytokine expression began. Dynamic gene expression of chemokine receptors around bifurcation predicted roles for cell-cell in driving Th1/Tfh fates. In particular, we found that precursor Th cells were coached towards a Th1 but not a Tfh fate by inflammatory monocytes. Thus, by integrating genomic and computational approaches, our study has provided two unique resources, a database www.PlasmoTH.org, which facilitates discovery of novel factors controlling Th1/Tfh fate commitment, and more generally, GPfates, a modelling framework for characterizing cell differentiation towards multiple fates. PMID:28345074

From Understanding the Development Landscape of the Canonical Fate-Switch Pair to Constructing a Dynamic Landscape for Two-Step Neural Differentiation

PubMed Central

2012-01-01

Abstract Recent progress in stem cell biology, notably cell fate conversion, calls for novel theoretical understanding for cell differentiation. The existing qualitative concept of Waddington’s “epigenetic landscape” has attracted particular attention because it captures subsequent fate decision points, thus manifesting the hierarchical (“tree-like”) nature of cell fate diversification. Here, we generalized a recent work and explored such a developmental landscape for a two-gene fate decision circuit by integrating the underlying probability landscapes with different parameters (corresponding to distinct developmental stages). The change of entropy production rate along the parameter changes indicates which parameter changes can represent a normal developmental process while other parameters’ change can not. The transdifferentiation paths over the landscape under certain conditions reveal the possibility of a direct and reversible phenotypic conversion. As the intensity of noise increases, we found that the landscape becomes flatter and the dominant paths more straight, implying the importance of biological noise processing mechanism in development and reprogramming. We further extended the landscape of the one-step fate decision to that for two-step decisions in central nervous system (CNS) differentiation. A minimal network and dynamic model for CNS differentiation was firstly constructed where two three-gene motifs are coupled. We then implemented the SDEs (Stochastic Differentiation Equations) simulation for the validity of the network and model. By integrating the two landscapes for the two switch gene pairs, we constructed the two-step development landscape for CNS differentiation. Our work provides new insights into cellular differentiation and important clues for better reprogramming strategies. PMID:23300518

β-Cell Replacement in Mice Using Human Type 1 Diabetes Nuclear Transfer Embryonic Stem Cells.

PubMed

Sui, Lina; Danzl, Nichole; Campbell, Sean R; Viola, Ryan; Williams, Damian; Xing, Yuan; Wang, Yong; Phillips, Neil; Poffenberger, Greg; Johannesson, Bjarki; Oberholzer, Jose; Powers, Alvin C; Leibel, Rudolph L; Chen, Xiaojuan; Sykes, Megan; Egli, Dieter

2018-01-01

β-Cells derived from stem cells hold great promise for cell replacement therapy for diabetes. Here we examine the ability of nuclear transfer embryonic stem cells (NT-ESs) derived from a patient with type 1 diabetes to differentiate into β-cells and provide a source of autologous islets for cell replacement. NT-ESs differentiate in vitro with an average efficiency of 55% into C-peptide-positive cells, expressing markers of mature β-cells, including MAFA and NKX6.1. Upon transplantation in immunodeficient mice, grafted cells form vascularized islet-like structures containing MAFA/C-peptide-positive cells. These β-cells adapt insulin secretion to ambient metabolite status and show normal insulin processing. Importantly, NT-ES-β-cells maintain normal blood glucose levels after ablation of the mouse endogenous β-cells. Cystic structures, but no teratomas, were observed in NT-ES-β-cell grafts. Isogenic induced pluripotent stem cell lines showed greater variability in β-cell differentiation. Even though different methods of somatic cell reprogramming result in stem cell lines that are molecularly indistinguishable, full differentiation competence is more common in ES cell lines than in induced pluripotent stem cell lines. These results demonstrate the suitability of NT-ES-β-cells for cell replacement for type 1 diabetes and provide proof of principle for therapeutic cloning combined with cell therapy. © 2017 by the American Diabetes Association.

bigSCale: an analytical framework for big-scale single-cell data.

PubMed

Iacono, Giovanni; Mereu, Elisabetta; Guillaumet-Adkins, Amy; Corominas, Roser; Cuscó, Ivon; Rodríguez-Esteban, Gustavo; Gut, Marta; Pérez-Jurado, Luis Alberto; Gut, Ivo; Heyn, Holger

2018-06-01

Single-cell RNA sequencing (scRNA-seq) has significantly deepened our insights into complex tissues, with the latest techniques capable of processing tens of thousands of cells simultaneously. Analyzing increasing numbers of cells, however, generates extremely large data sets, extending processing time and challenging computing resources. Current scRNA-seq analysis tools are not designed to interrogate large data sets and often lack sensitivity to identify marker genes. With bigSCale, we provide a scalable analytical framework to analyze millions of cells, which addresses the challenges associated with large data sets. To handle the noise and sparsity of scRNA-seq data, bigSCale uses large sample sizes to estimate an accurate numerical model of noise. The framework further includes modules for differential expression analysis, cell clustering, and marker identification. A directed convolution strategy allows processing of extremely large data sets, while preserving transcript information from individual cells. We evaluated the performance of bigSCale using both a biological model of aberrant gene expression in patient-derived neuronal progenitor cells and simulated data sets, which underlines the speed and accuracy in differential expression analysis. To test its applicability for large data sets, we applied bigSCale to assess 1.3 million cells from the mouse developing forebrain. Its directed down-sampling strategy accumulates information from single cells into index cell transcriptomes, thereby defining cellular clusters with improved resolution. Accordingly, index cell clusters identified rare populations, such as reelin ( Reln )-positive Cajal-Retzius neurons, for which we report previously unrecognized heterogeneity associated with distinct differentiation stages, spatial organization, and cellular function. Together, bigSCale presents a solution to address future challenges of large single-cell data sets. © 2018 Iacono et al.; Published by Cold Spring Harbor Laboratory Press.

Modulation of CD4(+) T cell-dependent specific cytotoxic CD8(+) T cells differentiation and proliferation by the timing of increase in the pathogen load.

PubMed

Tzelepis, Fanny; Persechini, Pedro M; Rodrigues, Mauricio M

2007-04-25

Following infection with viruses, bacteria or protozoan parasites, naïve antigen-specific CD8(+) T cells undergo a process of differentiation and proliferation to generate effector cells. Recent evidences suggest that the timing of generation of specific effector CD8(+) T cells varies widely according to different pathogens. We hypothesized that the timing of increase in the pathogen load could be a critical parameter governing this process. Using increasing doses of the protozoan parasite Trypanosoma cruzi to infect C57BL/6 mice, we observed a significant acceleration in the timing of parasitemia without an increase in mouse susceptibility. In contrast, in CD8 deficient mice, we observed an inverse relationship between the parasite inoculum and the timing of death. These results suggest that in normal mice CD8(+) T cells became protective earlier, following the accelerated development of parasitemia. The evaluation of specific cytotoxic responses in vivo to three distinct epitopes revealed that increasing the parasite inoculum hastened the expansion of specific CD8(+) cytotoxic T cells following infection. The differentiation and expansion of T. cruzi-specific CD8(+) cytotoxic T cells is in fact dependent on parasite multiplication, as radiation-attenuated parasites were unable to activate these cells. We also observed that, in contrast to most pathogens, the activation process of T. cruzi-specific CD8(+) cytotoxic T cells was dependent on MHC class II restricted CD4(+) T cells. Our results are compatible with our initial hypothesis that the timing of increase in the pathogen load can be a critical parameter governing the kinetics of CD4(+) T cell-dependent expansion of pathogen-specific CD8(+) cytotoxic T cells.

Differentiation of Symbiotic Cells and Endosymbionts in Medicago truncatula Nodulation Are Coupled to Two Transcriptome-Switches

PubMed Central

Maunoury, Nicolas; Redondo-Nieto, Miguel; Bourcy, Marie; Van de Velde, Willem; Alunni, Benoit; Laporte, Philippe; Durand, Patricia; Agier, Nicolas; Marisa, Laetitia; Vaubert, Danièle; Delacroix, Hervé; Duc, Gérard; Ratet, Pascal; Aggerbeck, Lawrence; Kondorosi, Eva; Mergaert, Peter

2010-01-01

The legume plant Medicago truncatula establishes a symbiosis with the nitrogen-fixing bacterium Sinorhizobium meliloti which takes place in root nodules. The formation of nodules employs a complex developmental program involving organogenesis, specific cellular differentiation of the host cells and the endosymbiotic bacteria, called bacteroids, as well as the specific activation of a large number of plant genes. By using a collection of plant and bacterial mutants inducing non-functional, Fix− nodules, we studied the differentiation processes of the symbiotic partners together with the nodule transcriptome, with the aim of unravelling links between cell differentiation and transcriptome activation. Two waves of transcriptional reprogramming involving the repression and the massive induction of hundreds of genes were observed during wild-type nodule formation. The dominant features of this “nodule-specific transcriptome” were the repression of plant defense-related genes, the transient activation of cell cycle and protein synthesis genes at the early stage of nodule development and the activation of the secretory pathway along with a large number of transmembrane and secretory proteins or peptides throughout organogenesis. The fifteen plant and bacterial mutants that were analyzed fell into four major categories. Members of the first category of mutants formed non-functional nodules although they had differentiated nodule cells and bacteroids. This group passed the two transcriptome switch-points similarly to the wild type. The second category, which formed nodules in which the plant cells were differentiated and infected but the bacteroids did not differentiate, passed the first transcriptome switch but not the second one. Nodules in the third category contained infection threads but were devoid of differentiated symbiotic cells and displayed a root-like transcriptome. Nodules in the fourth category were free of bacteria, devoid of differentiated symbiotic cells and also displayed a root-like transcriptome. A correlation thus exists between the differentiation of symbiotic nodule cells and the first wave of nodule specific gene activation and between differentiation of rhizobia to bacteroids and the second transcriptome wave in nodules. The differentiation of symbiotic cells and of bacteroids may therefore constitute signals for the execution of these transcriptome-switches. PMID:20209049

Cellular Factors Targeting APCs to Modulate Adaptive T Cell Immunity

PubMed Central

Do, Jeongsu; Min, Booki

2014-01-01

The fate of adaptive T cell immunity is determined by multiple cellular and molecular factors, among which the cytokine milieu plays the most important role in this process. Depending on the cytokines present during the initial T cell activation, T cells become effector cells that produce different effector molecules and execute adaptive immune functions. Studies thus far have primarily focused on defining how these factors control T cell differentiation by targeting T cells themselves. However, other non-T cells, particularly APCs, also express receptors for the factors and are capable of responding to them. In this review, we will discuss how APCs, by responding to those cytokines, influence T cell differentiation and adaptive immunity. PMID:25126585

Evaluating Cell Processes, Quality, and Biomarkers in Pluripotent Stem Cells Using Video Bioinformatics

PubMed Central

Lin, Sabrina C.; Bays, Brett C.; Omaiye, Esther; Bhanu, Bir; Talbot, Prue

2016-01-01

There is a foundational need for quality control tools in stem cell laboratories engaged in basic research, regenerative therapies, and toxicological studies. These tools require automated methods for evaluating cell processes and quality during in vitro passaging, expansion, maintenance, and differentiation. In this paper, an unbiased, automated high-content profiling toolkit, StemCellQC, is presented that non-invasively extracts information on cell quality and cellular processes from time-lapse phase-contrast videos. Twenty four (24) morphological and dynamic features were analyzed in healthy, unhealthy, and dying human embryonic stem cell (hESC) colonies to identify those features that were affected in each group. Multiple features differed in the healthy versus unhealthy/dying groups, and these features were linked to growth, motility, and death. Biomarkers were discovered that predicted cell processes before they were detectable by manual observation. StemCellQC distinguished healthy and unhealthy/dying hESC colonies with 96% accuracy by non-invasively measuring and tracking dynamic and morphological features over 48 hours. Changes in cellular processes can be monitored by StemCellQC and predictions can be made about the quality of pluripotent stem cell colonies. This toolkit reduced the time and resources required to track multiple pluripotent stem cell colonies and eliminated handling errors and false classifications due to human bias. StemCellQC provided both user-specified and classifier-determined analysis in cases where the affected features are not intuitive or anticipated. Video analysis algorithms allowed assessment of biological phenomena using automatic detection analysis, which can aid facilities where maintaining stem cell quality and/or monitoring changes in cellular processes are essential. In the future StemCellQC can be expanded to include other features, cell types, treatments, and differentiating cells. PMID:26848582

Evaluating Cell Processes, Quality, and Biomarkers in Pluripotent Stem Cells Using Video Bioinformatics.

PubMed

Zahedi, Atena; On, Vincent; Lin, Sabrina C; Bays, Brett C; Omaiye, Esther; Bhanu, Bir; Talbot, Prue

2016-01-01

There is a foundational need for quality control tools in stem cell laboratories engaged in basic research, regenerative therapies, and toxicological studies. These tools require automated methods for evaluating cell processes and quality during in vitro passaging, expansion, maintenance, and differentiation. In this paper, an unbiased, automated high-content profiling toolkit, StemCellQC, is presented that non-invasively extracts information on cell quality and cellular processes from time-lapse phase-contrast videos. Twenty four (24) morphological and dynamic features were analyzed in healthy, unhealthy, and dying human embryonic stem cell (hESC) colonies to identify those features that were affected in each group. Multiple features differed in the healthy versus unhealthy/dying groups, and these features were linked to growth, motility, and death. Biomarkers were discovered that predicted cell processes before they were detectable by manual observation. StemCellQC distinguished healthy and unhealthy/dying hESC colonies with 96% accuracy by non-invasively measuring and tracking dynamic and morphological features over 48 hours. Changes in cellular processes can be monitored by StemCellQC and predictions can be made about the quality of pluripotent stem cell colonies. This toolkit reduced the time and resources required to track multiple pluripotent stem cell colonies and eliminated handling errors and false classifications due to human bias. StemCellQC provided both user-specified and classifier-determined analysis in cases where the affected features are not intuitive or anticipated. Video analysis algorithms allowed assessment of biological phenomena using automatic detection analysis, which can aid facilities where maintaining stem cell quality and/or monitoring changes in cellular processes are essential. In the future StemCellQC can be expanded to include other features, cell types, treatments, and differentiating cells.

Nuclear organization during in vitro differentiation of porcine mesenchymal stem cells (MSCs) into adipocytes.

PubMed

Stachecka, Joanna; Walczak, Agnieszka; Kociucka, Beata; Ruszczycki, Błażej; Wilczyński, Grzegorz; Szczerbal, Izabela

2018-02-01

Differentiation of progenitor cells into adipocytes is accompanied by remarkable changes in cell morphology, cytoskeletal organization, and gene expression profile. Mature adipocytes are filled with a large lipid droplet and the nucleus tends to move to the cell periphery. It was hypothesized that the differentiation process is also associated with changes of nuclear organization. The aim of this study was to determine the number and distribution of selected components of nuclear architecture during porcine in vitro adipogenesis. The pig is an important animal model sharing many similarities to humans at the anatomical, physiological, and genetic levels and has been recognized as a good model for human obesity. Thus, understanding how cellular structures important for fundamental nuclear processes may be altered during adipocyte differentiation is of great importance. Mesenchymal stem cells (MSCs) were derived from bone marrow (BM-MSCs) and adipose tissue (AD-MSCs) and were cultured for 7 days in the adipogenic medium. A variable differentiation potential of these cell populations towards adipogenic lineage was observed, and for further study, a comparative characteristic of the nuclear organization in BM-MSCs and AD-MSCs was performed. Nuclear substructures were visualized by indirect immunofluorescence (nucleoli, nuclear speckles, PML bodies, lamins, and HP1α) or fluorescence in situ hybridization (telomeres) on fixed cells at 0, 3, 5, and 7 days of differentiation. Comprehensive characterization of these structures, in terms of their number, size, dynamics, and arrangement in three-dimensional space of the nucleus, was performed. It was found that during differentiation of porcine MSCs into adipocytes, changes of nuclear organization occurred and concerned: (1) the nuclear size and shape; (2) reduced lamin A/C expression; and (3) reorganization of chromocenters. Other elements of nuclear architecture such as nucleoli, SC-35 nuclear speckles, and telomeres showed no significant changes when compared to undifferentiated and mature fat cells. In addition, the presence of a low number of PML bodies was characteristic of the studied porcine mesenchymal stem cell adipogenesis system. It has been shown that the arrangement of selected components of nuclear architecture was very similar in MSCs derived from different sources, whereas adipocyte differentiation involves nuclear reorganization. This study adds new data on nuclear organization during adipogenesis using the pig as a model organism.

The association of GSK3 beta with E2F1 facilitates nerve growth factor-induced neural cell differentiation.

PubMed

Zhou, Fangfang; Zhang, Long; Wang, Aijun; Song, Bo; Gong, Kai; Zhang, Lihai; Hu, Min; Zhang, Xiufang; Zhao, Nanming; Gong, Yandao

2008-05-23

It is widely acknowledged that E2F1 and GSK3beta are both involved in the process of cell differentiation. However, the relationship between E2F1 and GSK3beta in cell differentiation has yet to be discovered. Here, we provide evidence that in the differentiation of PC12 cells induced by nerve growth factor (NGF), GSK3beta was increased at both the mRNA and protein levels, whereas E2F1 at these two levels was decreased. Both wild-type GSK3beta and its kinase-defective mutant GSK3beta KM can inhibit E2F1 by promoting its ubiquitination through physical interaction. In addition, the colocalization of GSK3beta and E2F1 and their subcellular distribution, regulated by NGF, were observed in the process of PC12 differentiation. At the tissue level, GSK3beta colocalized and interacted with E2F1 in mouse hippocampus. Furthermore, GSK3beta facilitated neurite outgrowth by rescuing the promoter activities of Cdk inhibitors p21 and p15 from the inhibition caused by E2F1. To summarize, our findings suggest that GSK3beta can promote the ubiquitination of E2F1 via physical interaction and thus inhibit its transcription activity in a kinase activity independent manner, which plays an important role in the NGF-induced PC12 differentiation.

Unraveling the non-senescence phenomenon in Hydra.

PubMed

Dańko, Maciej J; Kozłowski, Jan; Schaible, Ralf

2015-10-07

Unlike other metazoans, Hydra does not experience the distinctive rise in mortality with age known as senescence, which results from an increasing imbalance between cell damage and cell repair. We propose that the Hydra controls damage accumulation mainly through damage-dependent cell selection and cell sloughing. We examine our hypothesis with a model that combines cellular damage with stem cell renewal, differentiation, and elimination. The Hydra individual can be seen as a large single pool of three types of stem cells with some features of differentiated cells. This large stem cell community prevents "cellular damage drift," which is inevitable in complex conglomerate (differentiated) metazoans with numerous and generally isolated pools of stem cells. The process of cellular damage drift is based on changes in the distribution of damage among cells due to random events, and is thus similar to Muller's ratchet in asexual populations. Events in the model that are sources of randomness include budding, cellular death, and cellular damage and repair. Our results suggest that non-senescence is possible only in simple Hydra-like organisms which have a high proportion and number of stem cells, continuous cell divisions, an effective cell selection mechanism, and stem cells with the ability to undertake some roles of differentiated cells. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Repeated whole cigarette smoke exposure alters cell differentiation and augments secretion of inflammatory mediators in air-liquid interface three-dimensional co-culture model of human bronchial tissue.

PubMed

Ishikawa, Shinkichi; Ito, Shigeaki

2017-02-01

In vitro models of human bronchial epithelium are useful for toxicological testing because of their resemblance to in vivo tissue. We constructed a model of human bronchial tissue which has a fibroblast layer embedded in a collagen matrix directly below a fully-differentiated epithelial cell layer. The model was applied to whole cigarette smoke (CS) exposure repeatedly from an air-liquid interface culture while bronchial epithelial cells were differentiating. The effects of CS exposure on differentiation were determined by histological and gene expression analyses on culture day 21. We found a decrease in ciliated cells and perturbation of goblet cell differentiation. We also analyzed the effects of CS exposure on the inflammatory response, and observed a significant increase in secretion of IL-8, GRO-α, IL-1β, and GM-CSF. Interestingly, secretion of these mediators was augmented with repetition of whole CS exposure. Our data demonstrate the usefulness of our bronchial tissue model for in vitro testing and the importance of exposure repetition in perturbing the differentiation and inflammation processes. Copyright © 2016 Elsevier B.V. All rights reserved.

Transcriptome Analysis of Human Injured Meniscus Reveals a Distinct Phenotype of Meniscus Degeneration with Aging

PubMed Central

Rai, Muhammad Farooq; Patra, Debabrata; Sandell, Linda J.; Brophy, Robert H.

2013-01-01

Objective Meniscus tears are associated with a heightened risk for osteoarthritis. We aimed to advance our understanding of the metabolic state of human injured meniscus at the time of arthroscopic partial meniscectomy through transcriptome-wide analysis of gene expression in relation to patient age and degree of cartilage chondrosis. Methods The degree of chondrosis of knee cartilage was recorded at the time of meniscectomy in symptomatic patients without radiographic osteoarthritis. RNA preparations from resected menisci (N=12) were subjected to transcriptome-wide microarray and QuantiGene Plex analyses. The relative changes in gene expression variation with age and chondrosis were analyzed and integrated biological processes were investigated computationally. Results We identified a set of genes in torn meniscus that were differentially expressed with age and chondrosis. There were 866 genes differentially regulated (≥1.5-fold; P<0.05) with age and 49 with chondrosis. In older patients, genes associated with cartilage and skeletal development and extracellular matrix synthesis were repressed while those involved in immune response, inflammation, cell cycle, and cellular proliferation were stimulated. With chondrosis, genes representing cell catabolism (cAMP catabolic process) and tissue and endothelial cell development were repressed and those involved in T cell differentiation and apoptosis were elevated. Conclusion Differences in age-related gene expression suggest that in older adults, meniscal cells might de-differentiate and initiate a proliferative phenotype. Conversely, meniscal cells in younger patients appear to respond to injury, but maintain the differentiated phenotype. Definitive molecular signatures identified in damaged meniscus could be segregated largely with age and, to a lesser extent, with chondrosis. PMID:23658108

Proteomics Identification of Potential Candidates Involved in Cell Proliferation for Early Stage of Brain Regeneration in the Adult Zebrafish.

PubMed

Lim, Fei Tieng; Ogawa, Satoshi; Smith, A Ian; Parhar, Ishwar S

2017-02-01

The central nervous system (CNS) of the non-mammalian vertebrates has better neuroregenerative capability as compared with the mammalian CNS. Regeneration of habenula was observed 40 days after damage in zebrafish. During the early stage of regeneration, we found a significant increase of apoptotic cells on day-1 post-damage and of proliferative cells on day-3 post-damage. To identify the molecular factor(s) involved in the early stages of neuroregeneration, differentially expressed proteins during sham, 20- and 40-h post-habenula damage were investigated by proteomic approach by using two-dimensional differential gel electrophoresis (2D-DIGE) coupled with Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight (MALDI-ToF) and tandem mass spectrometry. Protein profiles revealed 17 differentially (>1.5-fold) expressed proteins: 10 upregulated, 4 downregulated, 2 proteins were found to be downregulated at the early stage but upregulated at a later stage, and 1 protein was found to be upregulated at 2 different time points. All proteins identified can be summarized under few molecular processes involved in the early stages of neuroregeneration in zebrafish CNS: apoptosis regulation (Wnt inhibitory factor 1 [WIF1]), neuroprotection (metallothionein), cell proliferation (Spred2, ependymin, Lhx1, and Wnts), differentiation (Spred2, Lhx9, and Wnts), and morphogenesis (cytoplasmic actins and draculin). These protein profiling results suggest that drastic molecular changes occur in the neuroregenerative process during this period, which includes cell proliferation, differentiation, and protection.

Differential Expression of Adhesion-Related Proteins and MAPK Pathways Lead to Suitable Osteoblast Differentiation of Human Mesenchymal Stem Cells Subpopulations.

PubMed

Leyva-Leyva, Margarita; López-Díaz, Annia; Barrera, Lourdes; Camacho-Morales, Alberto; Hernandez-Aguilar, Felipe; Carrillo-Casas, Erika M; Arriaga-Pizano, Lourdes; Calderón-Pérez, Jaime; García-Álvarez, Jorge; Orozco-Hoyuela, Gabriel; Piña-Barba, Cristina; Rojas-Martínez, Augusto; Romero-Díaz, Víktor; Lara-Arias, Jorge; Rivera-Bolaños, Nancy; López-Camarillo, César; Moncada-Saucedo, Nidia; Galván-De los Santos, Alejandra; Meza-Urzúa, Fátima; Villarreal-Gómez, Luis; Fuentes-Mera, Lizeth

2015-11-01

Cellular adhesion enables communication between cells and their environment. Adhesion can be achieved throughout focal adhesions and its components influence osteoblast differentiation of human mesenchymal stem cells (hMSCs). Because cell adhesion and osteoblast differentiation are closely related, this article aimed to analyze the expression profiles of adhesion-related proteins during osteoblastic differentiation of two hMSCs subpopulations (CD105(+) and CD105(-)) and propose a strategy for assembling bone grafts based on its adhesion ability. In vitro experiments of osteogenic differentiation in CD105(-) cells showed superior adhesion efficiency and 2-fold increase of α-actinin expression compared with CD105(+) cells at the maturation stage. Interestingly, levels of activated β1-integrin increased in CD105(-) cells during the process. Additionally, the CD105(-) subpopulation showed 3-fold increase of phosphorylated FAK(Y397) compared to CD105(+) cells. Results also indicate that ERK1/2 was activated during CD105(-) bone differentiation and participation of mitogen-activated protein kinase (MAPK)-p38 in CD105(+) differentiation through a focal adhesion kinase (FAK)-independent pathway. In vivo trial demonstrated that grafts containing CD105(-) showed osteocytes embedded in a mineralized matrix, promoted adequate graft integration, increased host vascular infiltration, and efficient intramembranous repairing. In contrast, grafts containing CD105(+) showed deficient endochondral ossification and fibrocartilaginous tissue. Based on the expression of α-actinin, FAKy,(397) and ERK1/2 activation, we define maturation stage as critical for bone graft assembling. By in vitro assays, CD105(-) subpopulation showed superior adhesion efficiency compared to CD105(+) cells. Considering in vitro and in vivo assays, this study suggests that integration of a scaffold with CD105(-) subpopulation at the maturation stage represents an attractive strategy for clinical use in orthopedic bioengineering.

Effects of the Transforming Growth Factor Beta Signaling Pathway on the Differentiation of Chicken Embryonic Stem Cells into Male Germ Cells

PubMed Central

Zhang, Yani; Wang, Yingjie; Zuo, Qisheng; Li, Dong; Zhang, Wenhui; Lian, Chao; Tang, Beibei; Xiao, Tianrong; Wang, Man; Wang, Kehua

2016-01-01

Abstract The objectives of the present study were to screen for key gene and signaling pathways involved in the production of male germ cells in poultry and to investigate the effects of the transforming growth factor beta (TGF-β) signaling pathway on the differentiation of chicken embryonic stem cells (ESCs) into male germ cells. The ESCs, primordial germ cells, and spermatogonial stem cells (SSCs) were sorted using flow cytometry for RNA sequencing (RNA-seq) technology. Male chicken ESCs were induced using 40 ng/mL of bone morphogenetic protein 4 (BMP4). The effects of the TGF-β signaling pathway on the production of chicken SSCs were confirmed by morphology, quantitative real-time polymerase chain reaction, and immunocytochemistry. One hundred seventy-three key genes relevant to development, differentiation, and metabolism and 20 signaling pathways involved in cell reproduction, differentiation, and signal transduction were identified by RNA-seq. The germ cells formed agglomerates and increased in number 14 days after induction by BMP4. During the induction process, the ESCs, Nanog, and Sox2 marker gene expression levels decreased, whereas expression of the germ cell-specific genes Stra8, Dazl, integrin-α6, and c-kit increased. The results indicated that the TGF-β signaling pathway participated in the differentiation of chicken ESCs into male germ cells. PMID:27906584

From Histones to RNA: Role of Methylation in Signal Proteins Involved in Adipogenesis.

PubMed

Wang, Xinxia; Wang, Yizhen

2017-01-01

New fat cells originate from a preexisting population of undifferentiated progenitor cells named preadipocytes. The process in which preadipocytes proliferate and differentiate into mature adipocytes under certain circumstances is called adipogenesis. In the past decade, many epigenetic factors have been shown to be pivotal for the appropriate timing of adipogenesis. A large number of coregulators at critical gene promoters set up specific patterns of DNA methylation, histone methylation and RNA methylation, which act as an epigenetic code to modulate the correct progress of adipocyte differentiation and adipogenesis. In this review, we focus on the functions and roles of epigenetic processes in preadipocyte differentiation and adipogenesis. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Adaxial cell migration in the zebrafish embryo is an active cell autonomous property that requires the Prdm1a transcription factor.

PubMed

Ono, Yosuke; Yu, Weimiao; Jackson, Harriet E; Parkin, Caroline A; Ingham, Philip W

2015-01-01

Adaxial cells, the progenitors of slow-twitch muscle fibres in zebrafish, exhibit a stereotypic migratory behaviour during somitogenesis. Although this process is known to be disrupted in various mutants, its precise nature has remained unclear. Here, using in vivo imaging and chimera analysis, we show that adaxial cell migration is a cell autonomous process, during which cells become polarised and extend filopodia at their leading edge. Loss of function of the Prdm1a transcription factor disrupts the polarisation and migration of adaxial cells, reflecting a role that is independent of its repression of sox6 expression. Expression of the M- and N-cadherins, previously implicated in driving adaxial cell migration, is largely unaffected by loss of Prdm1a function, suggesting that differential cadherin expression is not sufficient for adaxial cell migration. Copyright © 2015 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Oxidative Stress, Redox Regulation and Diseases of Cellular Differentiation

PubMed Central

Ye, Zhi-Wei; Zhang, Jie; Townsend, Danyelle M.; Tew, Kenneth D.

2015-01-01

Background Within cells, there is a narrow concentration threshold that governs whether reactive oxygen species (ROS) induce toxicity or act as second messengers. Scope of review We discuss current understanding of how ROS arise, facilitate cell signaling, cause toxicities and disease related to abnormal cell differentiation and those (primarily) sulfur based pathways that provide nucleophilicity to offset these effects. Primary conclusions Cellular redox homeostasis mediates a plethora of cellular pathways that determine life and death events. For example, ROS intersect with GSH based enzyme pathways to influence cell differentiation, a process integral to normal hematopoiesis, but also affecting a number of diverse cell differentiation related human diseases. Recent attempts to manage such pathologies have focused on intervening in some of these pathways, with the consequence that differentiation therapy targeting redox homeostasis has provided a platform for drug discovery and development. General Significance The balance between electrophilic oxidative stress and protective biomolecular nucleophiles predisposes the evolution of modern life forms. Imbalances of the two can produce aberrant redox homeostasis with resultant pathologies. Understanding the pathways involved provides opportunities to consider interventional strategies. PMID:25445706

Roles of mTOR Signaling in Brain Development.

PubMed

Lee, Da Yong

2015-09-01

mTOR is a serine/threonine kinase composed of multiple protein components. Intracellular signaling of mTOR complexes is involved in many of physiological functions including cell survival, proliferation and differentiation through the regulation of protein synthesis in multiple cell types. During brain development, mTOR-mediated signaling pathway plays a crucial role in the process of neuronal and glial differentiation and the maintenance of the stemness of neural stem cells. The abnormalities in the activity of mTOR and its downstream signaling molecules in neural stem cells result in severe defects of brain developmental processes causing a significant number of brain disorders, such as pediatric brain tumors, autism, seizure, learning disability and mental retardation. Understanding the implication of mTOR activity in neural stem cells would be able to provide an important clue in the development of future brain developmental disorder therapies.

Mediating human stem cell behaviour via defined fibrous architectures by melt electrospinning writing.

PubMed

Eichholz, Kian F; Hoey, David A

2018-05-29

The architecture within which cells reside is key to mediating their specific functions within the body. In this study, we use melt electrospinning writing (MEW) to fabricate cell micro-environments with various fibrous architectures to study their effect on human stem cell behaviour. We designed, built and optimised a MEW apparatus and used it to fabricate four different platform designs of 10.4±2μm fibre diameter, with angles between fibres on adjacent layers of 90°, 45°, 10° and R (random). Mechanical characterisation was conducted via tensile testing, and human skeletal stem cells (hSSCs) were seeded to scaffolds to study the effect of architecture on cell morphology and mechanosensing (nuclear YAP). Cell morphology was significantly altered between groups, with cells on 90° scaffolds having a lower aspect ratio, greater spreading, greater cytoskeletal tension and nuclear YAP expression. Long term cell culture studies were then conducted to determine the differentiation potential of scaffolds in terms of alkaline phosphatase activity, collagen and mineral production. Across these studies, an increased cell spreading in 3-dimensions is seen with decreasing alignment of architecture correlated with enhanced osteogenesis. This study therefore highlights the critical role of fibrous architecture in regulating stem cell behaviour with implications for tissue engineering and disease progression. This is the first study which has investigated the effect of controlled fibrous architectures fabricated via melt electrospinning writing on cell behaviour and differentiation. After optimising the process and characterising scaffolds via SEM and tensile testing, cells were seeded to fibrous scaffolds with various micro-architectures and studied in terms of cell morphology. Nuclear YAP expression was further investigated as a marker of cell shape, cytoskeletal tension and differentiation potential. In agreement with these early markers, long term cell culture studies revealed for the first time that a 90° fibrous architecture is optimal for the osteogenic differentiation of skeletal stem cells. This is the first study to investigate the effect of controlled fibrous material architectures fabricated via melt electrospinning writing on cell shape, mechanosignalling and differentiation. After optimising the biofabrication process and characterising scaffolds via SEM and tensile testing, cells were seeded to fibrous scaffolds with various micro-architectures and studied in terms of cell shape. Nuclear YAP expression was further investigated as a marker of cytoskeletal tension and differentiation potential. In agreement with these early markers, long term cell culture studies revealed for the first time that a 90° fibrous architecture is optimal for the osteogenic differentiation of skeletal stem cells, by driving a spread morphology and nuclear translocation of YAP in 3 dimensions . Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

NFIX Regulates Neural Progenitor Cell Differentiation During Hippocampal Morphogenesis

PubMed Central

Heng, Yee Hsieh Evelyn; McLeay, Robert C.; Harvey, Tracey J.; Smith, Aaron G.; Barry, Guy; Cato, Kathleen; Plachez, Céline; Little, Erica; Mason, Sharon; Dixon, Chantelle; Gronostajski, Richard M.; Bailey, Timothy L.; Richards, Linda J.; Piper, Michael

2014-01-01

Neural progenitor cells have the ability to give rise to neurons and glia in the embryonic, postnatal and adult brain. During development, the program regulating whether these cells divide and self-renew or exit the cell cycle and differentiate is tightly controlled, and imbalances to the normal trajectory of this process can lead to severe functional consequences. However, our understanding of the molecular regulation of these fundamental events remains limited. Moreover, processes underpinning development of the postnatal neurogenic niches within the cortex remain poorly defined. Here, we demonstrate that Nuclear factor one X (NFIX) is expressed by neural progenitor cells within the embryonic hippocampus, and that progenitor cell differentiation is delayed within Nfix−/− mice. Moreover, we reveal that the morphology of the dentate gyrus in postnatal Nfix−/− mice is abnormal, with fewer subgranular zone neural progenitor cells being generated in the absence of this transcription factor. Mechanistically, we demonstrate that the progenitor cell maintenance factor Sry-related HMG box 9 (SOX9) is upregulated in the hippocampus of Nfix−/− mice and demonstrate that NFIX can repress Sox9 promoter-driven transcription. Collectively, our findings demonstrate that NFIX plays a central role in hippocampal morphogenesis, regulating the formation of neuronal and glial populations within this structure. PMID:23042739

Extracellular guanosine and GTP promote expression of differentiation markers and induce S-phase cell-cycle arrest in human SH-SY5Y neuroblastoma cells.

PubMed

Guarnieri, S; Pilla, R; Morabito, C; Sacchetti, S; Mancinelli, R; Fanò, G; Mariggiò, M A

2009-04-01

SH-SY5Y neuroblastoma cells, a model for studying neuronal differentiation, are able to differentiate into either cholinergic or dopaminergic/adrenergic phenotypes depending on media conditions. Using this system, we asked whether guanosine (Guo) or guanosine-5'-triphosphate (GTP) are able to drive differentiation towards one particular phenotype. Differentiation was determined by evaluating the frequency of cells bearing neurites and assessing neurite length after exposure to different concentrations of Guo or GTP for different durations. After 6 days, 0.3 mM Guo or GTP induced a significant increase in the number of cells bearing neurites and increased neurite length. Western blot analyses confirmed that purines induced differentiation; cells exposed to purines showed increases in the levels of GAP43, MAP2, and tyrosine hydroxylase. Proliferation assays and cytofluorimetric analyses indicated a significant anti-proliferative effect of purines, and a concentration-dependent accumulation of cells in S-phase, starting after 24 h of purine exposure and extending for up to 6 days. A transcriptional profile analysis using gene arrays showed that an up-regulation of cyclin E2/cdk2 evident after 24 h was responsible for S-phase entry, and a concurrent down-regulation of cell-cycle progression-promoting cyclin B1/B2 prevented S-phase exit. In addition, patch-clamp recordings revealed that 0.3 mM Guo or GTP, after 6 day incubation, significantly decreased Na(+) currents. In conclusion, we showed Guo- and GTP-induced cell-cycle arrest in neuroblastoma cells and suggest that this makes these cells more responsive to differentiation processes that favor the dopaminergic/adrenergic phenotype.

Importance of ERK activation in As2O3-induced differentiation and promyelocytic leukemia nuclear bodies formation in neuroblastoma cells.

PubMed

Petit, A; Delaune, A; Falluel-Morel, A; Goullé, J-P; Vannier, J-P; Dubus, I; Vasse, M

2013-11-01

Neuroblastoma malignant cell growth is dependent on their undifferentiated status. Arsenic trioxide (As2O3) induces neuroblastoma cell differentiation in vitro, but its mechanisms still remains unknown. We used three human neuroblastoma cell lines (SH-SY5Y, IGR-N-91, LAN-1) that differ from their MYCN and p53 status to explore the intracellular events activated by As2O3 and involved in neurite outgrowth, a morphological marker of differentiation. As2O3 (2μM) induced neurite outgrowth in all cell lines, which was dependent on ERK activation but independent on MYCN status. This process was induced either by a sustained (3 days) or a transient (2h) incubation with As2O3, indicating that very early events trigger the induction of differentiation. In parallel, As2O3 induced a rapid assembly of promyelocytic leukemia nuclear bodies (PML-NB) in an ERK-dependent manner. In conclusion, mechanisms leading to neuroblastoma cell differentiation in response to As2O3 appear to involve the ERK pathway activation and PML-NB formation, which are observed in response to other differentiating molecules such as retinoic acid derivates. This open new perspectives based on the use of treatment combinations to potentiate the differentiating effects of each drug alone and reduce their adverse side effects. Copyright © 2013 Elsevier Ltd. All rights reserved.

Fetal bovine serum enables cardiac differentiation of human embryonic stem cells.

PubMed

Bettiol, Esther; Sartiani, Laura; Chicha, Laurie; Krause, Karl Heinz; Cerbai, Elisabetta; Jaconi, Marisa E

2007-10-01

During development, cardiac commitment within the mesoderm requires endoderm-secreted factors. Differentiation of embryonic stem cells into the three germ layers in vitro recapitulates developmental processes and can be influenced by supplements added to culture medium. Hence, we investigated the effect of fetal bovine serum (FBS) and KnockOut serum replacement (SR) on germ layers specification and cardiac differentiation of H1 human embryonic stem cells (hESC) within embryoid bodies (EB). At the time of EB formation, FBS triggered an increased apoptosis. As assessed by quantitative PCR on 4-, 10-, and 20-day-old EB, FBS promoted a faster down-regulation of pluripotency marker Oct4 and an increased expression of endodermal (Sox17, alpha-fetoprotein, AFP) and mesodermal genes (Brachyury, CSX). While neuronal and hematopoietic differentiation occurred in both supplements, spontaneously beating cardiomyocytes were only observed in FBS. Action potential (AP) morphology of hESC-derived cardiomyocytes indicated that ventricular cells were present only after 2 months of culture. However, quantification of myosin light chain 2 ventricular (mlc2v)-positive areas revealed that mlc2v-expressing cardiomyocytes could be detected already after 2 weeks of differentiation, but not in all beating clusters. In conclusion, FBS enabled cardiac differentiation of hESC, likely in an endodermal-dependent pathway. Among cardiac cells, ventricular cardiomyocytes differentiated over time, but not as the predominant cardiac cell subtype.

Temporal Transcriptional Profiling of Somatic and Germ Cells Reveals Biased Lineage Priming of Sexual Fate in the Fetal Mouse Gonad

PubMed Central

Jameson, Samantha A.; Natarajan, Anirudh; Cool, Jonah; DeFalco, Tony; Maatouk, Danielle M.; Mork, Lindsey; Munger, Steven C.; Capel, Blanche

2012-01-01

The divergence of distinct cell populations from multipotent progenitors is poorly understood, particularly in vivo. The gonad is an ideal place to study this process, because it originates as a bipotential primordium where multiple distinct lineages acquire sex-specific fates as the organ differentiates as a testis or an ovary. To gain a more detailed understanding of the process of gonadal differentiation at the level of the individual cell populations, we conducted microarrays on sorted cells from XX and XY mouse gonads at three time points spanning the period when the gonadal cells transition from sexually undifferentiated progenitors to their respective sex-specific fates. We analyzed supporting cells, interstitial/stromal cells, germ cells, and endothelial cells. This work identified genes specifically depleted and enriched in each lineage as it underwent sex-specific differentiation. We determined that the sexually undifferentiated germ cell and supporting cell progenitors showed lineage priming. We found that germ cell progenitors were primed with a bias toward the male fate. In contrast, supporting cells were primed with a female bias, indicative of the robust repression program involved in the commitment to XY supporting cell fate. This study provides a molecular explanation reconciling the female default and balanced models of sex determination and represents a rich resource for the field. More importantly, it yields new insights into the mechanisms by which different cell types in a single organ adopt their respective fates. PMID:22438826

TGFβ pathway limits dedifferentiation following WNT and MAPK pathway activation to suppress intestinal tumourigenesis

PubMed Central

Cammareri, Patrizia; Vincent, David F; Hodder, Michael C; Ridgway, Rachel A; Murgia, Claudio; Nobis, Max; Campbell, Andrew D; Varga, Julia; Huels, David J; Subramani, Chithra; Prescott, Katie L H; Nixon, Colin; Hedley, Ann; Barry, Simon T; Greten, Florian R; Inman, Gareth J; Sansom, Owen J

2017-01-01

Recent studies have suggested increased plasticity of differentiated cells within the intestine to act both as intestinal stem cells (ISCs) and tumour-initiating cells. However, little is known of the processes that regulate this plasticity. Our previous work has shown that activating mutations of Kras or the NF-κB pathway can drive dedifferentiation of intestinal cells lacking Apc. To investigate this process further, we profiled both cells undergoing dedifferentiation in vitro and tumours generated from these cells in vivo by gene expression analysis. Remarkably, no clear differences were observed in the tumours; however, during dedifferentiation in vitro we found a marked upregulation of TGFβ signalling, a pathway commonly mutated in colorectal cancer (CRC). Genetic inactivation of TGFβ type 1 receptor (Tgfbr1/Alk5) enhanced the ability of KrasG12D/+ mutation to drive dedifferentiation and markedly accelerated tumourigenesis. Mechanistically this is associated with a marked activation of MAPK signalling. Tumourigenesis from differentiated compartments is potently inhibited by MEK inhibition. Taken together, we show that tumours arising in differentiated compartments will be exposed to different suppressive signals, for example, TGFβ and blockade of these makes tumourigenesis more efficient from this compartment. PMID:28622298

Phenotypic stability and plasticity in GMP-derived cells as determined by their underlying regulatory network.

PubMed

Ramírez, Carlos; Mendoza, Luis

2018-04-01

Blood cell formation has been recognized as a suitable system to study celular differentiation mainly because of its experimental accessibility, and because it shows characteristics such as hierarchical and gradual bifurcated patterns of commitment, which are present in several developmental processes. Although hematopoiesis has been extensively studied and there is a wealth of molecular and cellular data about it, it is not clear how the underlying molecular regulatory networks define or restrict cellular differentiation processes. Here, we infer the molecular regulatory network that controls the differentiation of a blood cell subpopulation derived from the granulocyte-monocyte precursor (GMP), comprising monocytes, neutrophils, eosinophils, basophils and mast cells. We integrate published qualitative experimental data into a model to describe temporal expression patterns observed in GMP-derived cells. The model is implemented as a Boolean network, and its dynamical behavior is studied. Steady states of the network can be clearly identified with the expression profiles of monocytes, mast cells, neutrophils, basophils, and eosinophils, under wild-type and mutant backgrounds. All scripts are publicly available at https://github.com/caramirezal/RegulatoryNetworkGMPModel. lmendoza@biomedicas.unam.mx. Supplementary data are available at Bioinformatics online.

Gene expression profiling of bone marrow mesenchymal stem cells from Osteogenesis Imperfecta patients during osteoblast differentiation.

PubMed

Kaneto, Carla Martins; Pereira Lima, Patrícia S; Prata, Karen Lima; Dos Santos, Jane Lima; de Pina Neto, João Monteiro; Panepucci, Rodrigo Alexandre; Noushmehr, Houtan; Covas, Dimas Tadeu; de Paula, Francisco José Alburquerque; Silva, Wilson Araújo

2017-06-01

Mesenchymal stem cells (MSCs) are precursors present in adult bone marrow that are able to differentiate into osteoblasts, adipocytes and chondroblasts that have gained great importance as a source for cell therapy. Recently, a number of studies involving the analysis of gene expression of undifferentiated MSCs and of MSCs in the differentiation into multiple lineage processes were observed but there is no information concerning the gene expression of MSCs from Osteogenesis Imperfecta (OI) patients. Osteogenesis Imperfecta is characterized as a genetic disorder in which a generalized osteopenia leads to excessive bone fragility and severe bone deformities. The aim of this study was to analyze gene expression profile during osteogenic differentiation from BMMSCs (Bone Marrow Mesenchymal Stem Cells) obtained from patients with Osteogenesis Imperfecta and from control subjects. Bone marrow samples were collected from three normal subjects and five patients with OI. Mononuclear cells were isolated for obtaining mesenchymal cells that had been expanded until osteogenic differentiation was induced. RNA was harvested at seven time points during the osteogenic differentiation period (D0, D+1, D+2, D+7, D+12, D+17 and D+21). Gene expression analysis was performed by the microarray technique and identified several differentially expressed genes. Some important genes for osteoblast differentiation had lower expression in OI patients, suggesting a smaller commitment of these patient's MSCs with the osteogenic lineage. Other genes also had their differential expression confirmed by RT-qPCR. An increase in the expression of genes related to adipocytes was observed, suggesting an increase of adipogenic differentiation at the expense osteogenic differentiation. Copyright © 2017. Published by Elsevier Masson SAS.

βIII-Gal is involved in galactan reduction during phloem element differentiation in chickpea stems.

PubMed

Martín, Ignacio; Hernández-Nistal, Josefina; Albornos, Lucía; Labrador, Emilia; Dopico, Berta

2013-06-01

βIII-Gal, a member of the chickpea β-galactosidase family, is the enzyme responsible for the cell wall autolytic process. This enzyme, whose activity increases during epicotyl growth, displays significant hydrolytic activity against cell wall pectins, and its natural substrate has been determined as an arabinogalactan from the pectic fraction of the cell wall. In the present work, the localization of βIII-Gal in different seedling and plant organs was analyzed by using specific anti-βIII-Gal antibodies. Our results revealed that besides its possible role in cell wall loosening and in early events during primary xylem and phloem fiber differentiation βIII-Gal acts on the development of sieve elements. Localization of the enzyme in this tissue, both in epicotyls and radicles from seedlings and in the different stem internodes, is consistent with the reduction in galactan during the maturation of phloem elements, as can be observed with LM5 antibodies. Thus, βIII-Gal could act on its natural substrate, the neutral side chains of rhamnogalacturonan I, contributing to cell wall reinforcement allowing phloem elements to differentiate, and conferring the necessary strengthening of the cell wall to fulfill its function. This work completes the immunolocation studies of all known chickpea β-galactosidases. Taken together, our results reflect the broad range of developmental processes covered by different members of this protein family, and confirm their crucial role in cell wall remodeling during tissue differentiation.

Arabidopsis and Tobacco SUPERMAN regulate hormone signalling and mediate cell proliferation and differentiation

PubMed Central

Nibau, Candida; Di Stilio, Verónica S.; Wu, Hen-ming; Cheung, Alice Y.

2011-01-01

Arabidopsis thaliana SUPERMAN (SUP) plays an important role during flower development by maintaining the boundary between stamens and carpels in the inner two whorls. It was proposed that SUP maintains this boundary by regulating cell proliferation in both whorls, as loss-of-function superman mutants produce more stamens at the expense of carpels. However, the cellular mechanism that underlies SUP function remains unknown. Here Arabidopsis or tobacco (Nicotiana tabacum) SUP was overexpressed in tobacco plants to substantiate SUP's role as a regulator of cell proliferation and boundary definition and provide evidence that its biological role may be mediated via hormonal changes. It was found that moderate levels of SUP stimulated cell growth and proliferation, whereas high levels were inhibitory. SUP stimulated auxin- and cytokinin-regulated processes, and cells overexpressing SUP displayed reduced hormone dependency for proliferation and regeneration into plants. SUP also induced proliferation of female traits in the second and third flower whorls and promoted differentiation of petaloid properties in sepals, further supporting a role for SUP as a boundary regulator. Moreover, cytokinin suppressed stamen development and promoted differentiation of carpeloid tissues, suggesting that SUP may regulate male and female development via its effect on cytokinin signalling. Taken together, these observations suggest a model whereby the effect of SUP on cell growth and proliferation involves the modulation of auxin- and cytokinin-regulated processes. Furthermore, differential SUP expression or different sensitivities of different cell types to SUP may determine whether SUP stimulates or suppresses their proliferation. PMID:20980362

EVI2B is a C/EBPα target gene required for granulocytic differentiation and functionality of hematopoietic progenitors.

PubMed

Zjablovskaja, Polina; Kardosova, Miroslava; Danek, Petr; Angelisova, Pavla; Benoukraf, Touati; Wurm, Alexander A; Kalina, Tomas; Sian, Stephanie; Balastik, Martin; Delwel, Ruud; Brdicka, Tomas; Tenen, Daniel G; Behre, Gerhard; Fiore, Fréderic; Malissen, Bernard; Horejsi, Vaclav; Alberich-Jorda, Meritxell

2017-04-01

Development of hematopoietic populations through the process of differentiation is critical for proper hematopoiesis. The transcription factor CCAAT/enhancer binding protein alpha (C/EBPα) is a master regulator of myeloid differentiation, and the identification of C/EBPα target genes is key to understand this process. Here we identified the Ecotropic Viral Integration Site 2B (EVI2B) gene as a direct target of C/EBPα. We showed that the product of the gene, the transmembrane glycoprotein EVI2B (CD361), is abundantly expressed on the surface of primary hematopoietic cells, the highest levels of expression being reached in mature granulocytes. Using shRNA-mediated downregulation of EVI2B in human and murine cell lines and in primary hematopoietic stem and progenitor cells, we demonstrated impaired myeloid lineage development and altered progenitor functions in EVI2B-silenced cells. We showed that the compromised progenitor functionality in Evi2b-depleted cells can be in part explained by deregulation of cell proliferation and apoptosis. In addition, we generated an Evi2b knockout murine model and demonstrated altered properties of hematopoietic progenitors, as well as impaired G-CSF dependent myeloid colony formation in the knockout cells. Remarkably, we found that EVI2B is significantly downregulated in human acute myeloid leukemia samples characterized by defects in CEBPA. Altogether, our data demonstrate that EVI2B is a downstream target of C/EBPα, which regulates myeloid differentiation and functionality of hematopoietic progenitors.

Dietary Micronutrients Promote Neuronal Differentiation by Modulating the Mitochondrial-Nuclear Dialogue.

PubMed

Xie, Kui; Sheppard, Allan

2018-07-01

The metabolic requirements of differentiated neurons are significantly different from that of neuronal precursor and neural stem cells. While a re-programming of metabolism is tightly coupled to the neuronal differentiation process, whether shifts in mitochondrial mass, glycolysis, and oxidative phosphorylation are required (or merely consequential) in differentiation is not yet certain. In addition to providing more energy, enhanced metabolism facilitates differentiation by supporting increased neurotransmitter signaling and underpinning epigenetic regulation of gene expression. Both epidemiological and animal studies demonstrate that micronutrients (MNs) significantly influence many aspects of neonatal brain development, particularly neural migration and survival, neurite outgrowth, and process maturation. Here we review recent insights into the importance of metabolic reprogramming in neuronal differentiation, before considering evidence that micronutrient signaling may be key to regulating these processes. © 2018 WILEY Periodicals, Inc.

Improved human endometrial stem cells differentiation into functional hepatocyte-like cells on a glycosaminoglycan/collagen-grafted polyethersulfone nanofibrous scaffold.

PubMed

Khademi, Farzaneh; Ai, Jafar; Soleimani, Masoud; Verdi, Javad; Mohammad Tavangar, Seyed; Sadroddiny, Esmaeil; Massumi, Mohammad; Mahmoud Hashemi, Seyed

2017-11-01

Liver tissue engineering (TE) is rapidly emerging as an effective technique which combines engineering and biological processes to compensate for the shortage of damaged or destroyed liver tissues. We examined the viability, differentiation, and integration of hepatocyte-like cells on an electrospun polyethersulfone (PES) scaffold, derived from human endometrial stem cells (hEnSCs). Natural polymers were separately grafted on plasma-treated PES nanofibers, that is, collagen, heparan sulfate (HS) and collagen-HS. Galactosilated PES (PES-Gal) nanofibrous were created. The engineering and cell growth parameters were considered and compared with each sample. The cellular studies revealed increased cell survival, attachment, and normal morphology on the bioactive natural polymer-grafted scaffolds after 30 days of hepatic differentiation. The chemical and molecular assays displayed hepatocyte differentiation. These cells were also functional, showing glycogen storage, α-fetoprotein, and albumin secretion. The HS nanoparticle-grafted PES nanofibers demonstrated a high rate of cell proliferation, differentiation, and integration. Based on the observations mentioned above, engineered tissue is a good option in the future, for the commercial production of three-dimensional liver tissues for clinical purposes. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2516-2529, 2017. © 2016 Wiley Periodicals, Inc.

Angiotensin II promotes differentiation of mouse c-kit-positive cardiac stem cells into pacemaker-like cells

PubMed Central

XUE, CHENG; ZHANG, JUN; LV, ZHAN; LIU, HUI; HUANG, CONGXIN; YANG, JING; WANG, TEN

2015-01-01

Cardiac stem cells (CSCs) can differentiate into cardiac muscle-like cells; however, it remains unknown whether CSCs may possess the ability to differentiate into pacemaker cells. The aim of the present study was to determine whether angiotensin II (Ang II) could promote the specialization of CSCs into pacemaker-like cells. Mouse CSCs were treated with Ang II from day 3–5, after cell sorting. The differentiation potential of the cells was then analyzed by morphological analysis, flow cytometry, reverse transcription-polymerase chain reaction, immunohistochemistry and patch clamp analysis. Treatment with Ang II resulted in an increased number of cardiac muscle-like cells (32.7±4.8% vs. 21.5±4.8%; P<0.05), and inhibition of smooth muscle-like cells (6.2±7.3% vs. 20.5±5.1%; P<0.05). Following treatment with Ang II, increased levels of the cardiac progenitor-specific markers GATA4 and Nkx2.5 were observed in the cells. Furthermore, the transcript levels of pacemaker function-related genes, including hyperpolarization-activated cyclic nucleotide-gated (HCN)2, HCN4, T-box (Tbx)2 and Tbx3, were significantly upregulated. Immunofluorescence analysis confirmed the increased number of pacemaker-like cells. The pacemaker current (If) was recorded in the cells derived from CSCs, treated with Ang II. In conclusion, treatment of CSCs with Ang II during the differentiation process modified cardiac-specific gene expression and resulted in the enhanced formation of pacemaker-like cells. PMID:25572000

Connections Between Metabolism and Epigenetics in Programming Cellular Differentiation.

PubMed

Chisolm, Danielle A; Weinmann, Amy S

2018-04-26

Researchers are intensifying efforts to understand the mechanisms by which changes in metabolic states influence differentiation programs. An emerging objective is to define how fluctuations in metabolites influence the epigenetic states that contribute to differentiation programs. This is because metabolites such as S-adenosylmethionine, acetyl-CoA, α-ketoglutarate, 2-hydroxyglutarate, and butyrate are donors, substrates, cofactors, and antagonists for the activities of epigenetic-modifying complexes and for epigenetic modifications. We discuss this topic from the perspective of specialized CD4 + T cells as well as effector and memory T cell differentiation programs. We also highlight findings from embryonic stem cells that give mechanistic insight into how nutrients processed through pathways such as glycolysis, glutaminolysis, and one-carbon metabolism regulate metabolite levels to influence epigenetic events and discuss similar mechanistic principles in T cells. Finally, we highlight how dysregulated environments, such as the tumor microenvironment, might alter programming events.

NBR1 is a new PB1 signalling adapter in Th2 differentiation and allergic airway inflammation in vivo

PubMed Central

Yang, Jun-Qi; Liu, Hongzhu; Diaz-Meco, Maria T; Moscat, Jorge

2010-01-01

Allergic airway inflammation is a disease in which T helper 2 (Th2) cells have a critical function. The molecular mechanisms controlling Th2 differentiation and function are of paramount importance in biology and immunology. Recently, a network of PB1-containing adapters and kinases has been shown to be essential in this process owing to its function in regulating cell polarity and the activation of critical transcription factors. Here, we show in vivo data showing that T-cell-specific NBR1-deficient mice show impaired lung inflammation and have defective Th2 differentiation ex vivo with alterations in T-cell polarity and the selective inhibition of Gata3 and nuclear factor of activated T c1 activation. These results establish NBR1 as a novel PB1 adapter in Th2 differentiation and asthma. PMID:20808283

Cytoplasmic sequestration of cyclin D1 associated with cell cycle withdrawal of neuroblastoma cells

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

Sumrejkanchanakij, Piyamas; Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330; Eto, Kazuhiro

2006-02-03

The regulation of D-type cyclin-dependent kinase activity is critical for neuronal differentiation and apoptosis. We recently showed that cyclin D1 is sequestered in the cytoplasm and that its nuclear localization induces apoptosis in postmitotic primary neurons. Here, we further investigated the role of the subcellular localization of cyclin D1 in cell cycle withdrawal during the differentiation of N1E-115 neuroblastoma cells. We show that cyclin D1 became predominantly cytoplasmic after differentiation. Targeting cyclin D1 expression to the nucleus induced phosphorylation of Rb and cdk2 kinase activity. Furthermore, cyclin D1 nuclear localization promoted differentiated N1E-115 cells to reenter the cell cycle, amore » process that was inhibited by p16{sup INK4a}, a specific inhibitor of D-type cyclin activity. These results indicate that cytoplasmic sequestration of cyclin D1 plays a role in neuronal cell cycle withdrawal, and suggests that the abrogation of machinery involved in monitoring aberrant nuclear cyclin D1 activity contributes to neuronal tumorigenesis.« less

Cytoplasmic sequestration of cyclin D1 associated with cell cycle withdrawal of neuroblastoma cells.

PubMed

Sumrejkanchanakij, Piyamas; Eto, Kazuhiro; Ikeda, Masa-Aki

2006-02-03

The regulation of D-type cyclin-dependent kinase activity is critical for neuronal differentiation and apoptosis. We recently showed that cyclin D1 is sequestered in the cytoplasm and that its nuclear localization induces apoptosis in postmitotic primary neurons. Here, we further investigated the role of the subcellular localization of cyclin D1 in cell cycle withdrawal during the differentiation of N1E-115 neuroblastoma cells. We show that cyclin D1 became predominantly cytoplasmic after differentiation. Targeting cyclin D1 expression to the nucleus induced phosphorylation of Rb and cdk2 kinase activity. Furthermore, cyclin D1 nuclear localization promoted differentiated N1E-115 cells to reenter the cell cycle, a process that was inhibited by p16(INK4a), a specific inhibitor of D-type cyclin activity. These results indicate that cytoplasmic sequestration of cyclin D1 plays a role in neuronal cell cycle withdrawal, and suggests that the abrogation of machinery involved in monitoring aberrant nuclear cyclin D1 activity contributes to neuronal tumorigenesis.

Vinpocetine Attenuates the Osteoblastic Differentiation of Vascular Smooth Muscle Cells

PubMed Central

Chen, Xiu-Juan; Wang, Na; Yi, Peng-Fei; Song, Min; Zhang, Bo; Wang, Yu-Zhong; Liang, Qiu-Hua

2016-01-01

Vascular calcification is an active process of osteoblastic differentiation of vascular smooth muscle cells; however, its definite mechanism remains unknown. Vinpocetine, a derivative of the alkaloid vincamine, has been demonstrated to inhibit the high glucose-induced proliferation of vascular smooth muscle cells; however, it remains unknown whether vinpocetine can affect the osteoblastic differentiation of vascular smooth muscle cells. We hereby investigated the effect of vinpocetine on vascular calcification using a beta-glycerophosphate-induced cell model. Our results showed that vinpocetine significantly reduced the osteoblast-like phenotypes of vascular smooth muscle cells including ALP activity, osteocalcin, collagen type I, Runx2 and BMP-2 expression as well as the formation of mineralized nodule. Vinpocetine, binding to translocation protein, induced phosphorylation of extracellular signal-related kinase and Akt and thus inhibited the translocation of nuclear factor-kappa B into the nucleus. Silencing of translocator protein significantly attenuated the inhibitory effect of vinpocetine on osteoblastic differentiation of vascular smooth muscle cells. Taken together, vinpocetine may be a promising candidate for the clinical therapy of vascular calcification. PMID:27589055

Induction of polyploidization in leukemic cell lines and primary bone marrow by Src kinase inhibitor SU6656

PubMed Central

Lannutti, Brian J.; Blake, Noel; Gandhi, Manish J.; Reems, Jo Anna; Drachman, Jonathan G.

2005-01-01

Megakaryocytes (MKs) undergo successive rounds of endomitosis during differentiation, resulting in polyploidy (typically, 16-64N). Previous studies have demonstrated that this occurs through an interruption of normal cell cycle progression during anaphase. However, the molecular mechanism(s) controlling this unique process is undefined. In the present report, we examine the effect of an Src kinase inhibitor, SU6656, on thrombopoietin (TPO)-induced growth and differentiation. Remarkably, when SU6656 (2.5 μM) was added to a megakaryocytic cell line, UT-7/TPO, the cells ceased cell division but continued to accumulate DNA by endomitosis. During this interval, CD41 and CD61 expression on the cell surface increased. Similar effects on polyploidization and MK differentiation were seen with expanded primary MKs, bone marrow from 2 patients with myelodysplastic syndrome, and other cell lines with MK potential. Our data suggest that SU6656 might be useful as a differentiation-inducing agent for MKs and is an important tool for understanding the molecular basis of MK endomitosis. PMID:15677565

Glucose metabolism regulates T cell activation, differentiation, and functions.

PubMed

Palmer, Clovis S; Ostrowski, Matias; Balderson, Brad; Christian, Nicole; Crowe, Suzanne M

2015-01-01

The adaptive immune system is equipped to eliminate both tumors and pathogenic microorganisms. It requires a series of complex and coordinated signals to drive the activation, proliferation, and differentiation of appropriate T cell subsets. It is now established that changes in cellular activation are coupled to profound changes in cellular metabolism. In addition, emerging evidence now suggest that specific metabolic alterations associated with distinct T cell subsets may be ancillary to their differentiation and influential in their immune functions. The "Warburg effect" originally used to describe a phenomenon in which most cancer cells relied on aerobic glycolysis for their growth is a key process that sustain T cell activation and differentiation. Here, we review how different aspects of metabolism in T cells influence their functions, focusing on the emerging role of key regulators of glucose metabolism such as HIF-1α. A thorough understanding of the role of metabolism in T cell function could provide insights into mechanisms involved in inflammatory-mediated conditions, with the potential for developing novel therapeutic approaches to treat these diseases.

Identification and Characterization of FGF2-Dependent mRNA: microRNA Networks During Lens Fiber Cell Differentiation

PubMed Central

Wolf, Louise; Gao, Chun S.; Gueta, Karen; Xie, Qing; Chevallier, Tiphaine; Podduturi, Nikhil R.; Sun, Jian; Conte, Ivan; Zelenka, Peggy S.; Ashery-Padan, Ruth; Zavadil, Jiri; Cvekl, Ales

2013-01-01

MicroRNAs (miRNAs) and fibroblast growth factor (FGF) signaling regulate a wide range of cellular functions, including cell specification, proliferation, migration, differentiation, and survival. In lens, both these systems control lens fiber cell differentiation; however, a possible link between these processes remains to be examined. Herein, the functional requirement for miRNAs in differentiating lens fiber cells was demonstrated via conditional inactivation of Dicer1 in mouse (Mus musculus) lens. To dissect the miRNA-dependent pathways during lens differentiation, we used a rat (Rattus norvegicus) lens epithelial explant system, induced by FGF2 to differentiate, followed by mRNA and miRNA expression profiling. Transcriptome and miRNome analysis identified extensive FGF2-regulated cellular responses that were both independent and dependent on miRNAs. We identified 131 FGF2-regulated miRNAs. Seventy-six of these miRNAs had at least two in silico predicted and inversely regulated target mRNAs. Genes modulated by the greatest number of FGF-regulated miRNAs include DNA-binding transcription factors Nfib, Nfat5/OREBP, c-Maf, Ets1, and N-Myc. Activated FGF signaling influenced bone morphogenetic factor/transforming growth factor-β, Notch, and Wnt signaling cascades implicated earlier in lens differentiation. Specific miRNA:mRNA interaction networks were predicted for c-Maf, N-Myc, and Nfib (DNA-binding transcription factors); Cnot6, Cpsf6, Dicer1, and Tnrc6b (RNA to miRNA processing); and Ash1l, Med1/PBP, and Kdm5b/Jarid1b/Plu1 (chromatin remodeling). Three miRNAs, including miR-143, miR-155, and miR-301a, down-regulated expression of c-Maf in the 3′-UTR luciferase reporter assays. These present studies demonstrate for the first time global impact of activated FGF signaling in lens cell culture system and predicted novel gene regulatory networks connected by multiple miRNAs that regulate lens differentiation. PMID:24142921

Induction of Adipocyte Differentiation by Polybrominated Diphenyl Ethers (PBDEs) in 3T3-L1 Cells

PubMed Central

Tung, Emily W. Y.; Boudreau, Adèle; Wade, Michael G.; Atlas, Ella

2014-01-01

Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame retardants that were extensively used in commercial products. PBDEs are ubiquitous environmental contaminants that are both lipophilic and bioaccumulative. Effects of PBDEs on adipogenesis were studied in the 3T3-L1 preadipocyte cell model in the presence and absence of a known adipogenic agent, dexamethasone (DEX). A PBDE mixture designed to mimic body burden of North Americans was tested, in addition to the technical mixture DE-71 and the individual congener BDE-47. The mixture, DE-71, and BDE-47 all induced adipocyte differentiation as assessed by markers for terminal differentiation [fatty acid binding protein 4 (aP2) and perilipin] and lipid accumulation. Characterization of the differentiation process in response to PBDEs indicated that adipogenesis induced by a minimally effective dose of DEX was enhanced by these PBDEs. Moreover, C/EBPα, PPARγ, and LXRα were induced late in the differentiation process. Taken together, these data indicate that adipocyte differentiation is induced by PBDEs; they act in the absence of glucocorticoid and enhance glucocorticoid-mediated adipogenesis. PMID:24722056

Directed Neural Differentiation of Mouse Embryonic Stem Cells Is a Sensitive System for the Identification of Novel Hox Gene Effectors

PubMed Central

Bami, Myrto; Episkopou, Vasso; Gavalas, Anthony; Gouti, Mina

2011-01-01

The evolutionarily conserved Hox family of homeodomain transcription factors plays fundamental roles in regulating cell specification along the anterior posterior axis during development of all bilaterian animals by controlling cell fate choices in a highly localized, extracellular signal and cell context dependent manner. Some studies have established downstream target genes in specific systems but their identification is insufficient to explain either the ability of Hox genes to direct homeotic transformations or the breadth of their patterning potential. To begin delineating Hox gene function in neural development we used a mouse ES cell based system that combines efficient neural differentiation with inducible Hoxb1 expression. Gene expression profiling suggested that Hoxb1 acted as both activator and repressor in the short term but predominantly as a repressor in the long run. Activated and repressed genes segregated in distinct processes suggesting that, in the context examined, Hoxb1 blocked differentiation while activating genes related to early developmental processes, wnt and cell surface receptor linked signal transduction and cell-to-cell communication. To further elucidate aspects of Hoxb1 function we used loss and gain of function approaches in the mouse and chick embryos. We show that Hoxb1 acts as an activator to establish the full expression domain of CRABPI and II in rhombomere 4 and as a repressor to restrict expression of Lhx5 and Lhx9. Thus the Hoxb1 patterning activity includes the regulation of the cellular response to retinoic acid and the delay of the expression of genes that commit cells to neural differentiation. The results of this study show that ES neural differentiation and inducible Hox gene expression can be used as a sensitive model system to systematically identify Hox novel target genes, delineate their interactions with signaling pathways in dictating cell fate and define the extent of functional overlap among different Hox genes. PMID:21637844

2-(trimethylammonium)ethyl (R)-3-methoxy-3-oxo-2-stearamidopropyl phosphate promotes megakaryocytic differentiation of myeloid leukaemia cells and primary human CD34⁺ haematopoietic stem cells.

PubMed

Limb, Jin-Kyung; Song, Doona; Jeon, Mijeong; Han, So-Yeop; Han, Gyoonhee; Jhon, Gil-Ja; Bae, Yun Soo; Kim, Jaesang

2015-04-01

In this study we showed that 2-(trimethylammonium)ethyl (R)-3-methoxy-3-oxo-2-stearamidopropyl phosphate [(R)-TEMOSPho], a derivative of an organic chemical identified from a natural product library, promotes highly efficient differentiation of megakaryocytes. Specifically, (R)-TEMOSPho induces cell cycle arrest, cell size increase and polyploidization from K562 and HEL cells, which are used extensively to model megakaryocytic differentiation. In addition, megakaryocyte-specific cell surface markers showed a dramatic increase in expression in response to (R)-TEMOSPho treatment. Importantly, we demonstrated that such megakaryocytic differentiation can also be induced from primary human CD34(+) haematopoietic stem cells. Activation of the PI3K-AKT pathway and, to a lesser extent, the MEK-ERK pathway appears to be required for this process, as blocking with specific inhibitors interferes with the differentiation of K562 cells. A subset of (R)-TEMOSPho-treated K562 cells undergoes spontaneous apoptosis and produces platelets that are apparently functional, as they bind to fibrinogen, express P-selectin and aggregate in response to SFLLRN and AYPGFK, the activating peptides for the PAR1 and PAR4 receptors, respectively. Taken together, these results indicate that (R)-TEMOSPho will be useful for dissecting the molecular mechanisms of megakaryocytic differentiation, and that this class of compounds represents potential therapeutic reagents for thrombocytopenia. Copyright © 2012 John Wiley & Sons, Ltd.

Epithelialization and stromalization of porcine follicular granulosa cells during real-time proliferation - a primary cell culture approach.

PubMed

Ciesiółka, S; Bryja, A; Budna, J; Kranc, W; Chachuła, A; Bukowska, D; Piotrowska, H; Porowski, L; Antosik, P; Bruska, M; Brüssow, K P; Nowicki, M; Zabel, M; Kempisty, B

2016-01-01

The process of oocyte growth and development takes place during long stages of folliculogenesis and oogenesis. This is accompanied by biochemical and morphological changes, occurring from the preantral to antral stages during ovarian follicle differentiation. It is well known that the process of follicle growth is associated with morphological modifications of theca (TCs) and granulosa cells (GCs). However, the relationship between proliferation and/or differentiation of porcine GCs during long-term in vitro culture requires further investigation. Moreover, the expression of cytokeratins and vimentin in porcine GCs, in relation to real-time cell proliferation, has yet to be explored. Utilizing confocal microscopy, we analyzed cytokeratin 18 (CK18), cytokeratin 8 + 18 + 19 (panCK), and vimentin (Vim) expression, as well as their protein distribution, within GCs isolated from slaughtered ovarian follicles. The cells were cultured for 168 h with protein expression and cell proliferation index analyzed at 24-h intervals. We found the highest expression of CK18, panCK, and Vim occurred at 120 h of in vitro culture (IVC) as compared with other experimental time intervals. All of the investigated proteins displayed cytoplasmic distribution. Analysis of real-time cell proliferation revealed an increased cell index after the first 24 h of IVC. Additionally, during each period between 24-168 h of IVC, a significant difference in the proliferation profile, expressed as the cell index, was also observed. We concluded that higher expression of vimentin at 120 h of in vitro proliferation might explain the culmination of the stromalization process associated with growth and domination of stromal cells in GC culture. Cytokeratin expression within GC cytoplasm confirms the presence of epithelial cells as well as epithelial-related GC development during IVC. Moreover, expression of both cytokeratins and vimentin during short-term culture suggests that the process of GC proliferation is also highly associated with porcine ovarian follicular granulosa cell differentiation in vitro.

Proangiogenic hematopoietic cells of monocytic origin: roles in vascular regeneration and pathogenic processes of systemic sclerosis.

PubMed

Yamaguchi, Yukie; Kuwana, Masataka

2013-02-01

New blood vessel formation is critical, not only for organ development and tissue regeneration, but also for various pathologic processes, such as tumor development and vasculopathy. The maintenance of the postnatal vascular system requires constant remodeling, which occurs through angiogenesis, vasculogenesis, and arteriogenesis. Vasculogenesis is mediated by the de novo differentiation of mature endothelial cells from endothelial progenitor cells (EPCs). Early studies provided evidence that bone marrow-derived CD14⁺ monocytes can serve as a subset of EPCs because of their expression of endothelial markers and ability to promote neovascularization in vitro and in vivo. However, the current consensus is that monocytic cells do not give rise to endothelial cells in vivo, but function as support cells, by promoting vascular formation and repair through their immediate recruitment to the site of vascular injury, secretion of proangiogenic factors, and differentiation into mural cells. These monocytes that function in a supporting role in vascular repair are now termed monocytic pro-angiogenic hematopoietic cells (PHCs). Systemic sclerosis (SSc) is a multisystem connective tissue disease characterized by excessive fibrosis and microvasculopathy, along with poor vascular formation and repair. We recently showed that in patients with SSc, circulating monocytic PHCs increase dramatically and have enhanced angiogenic potency. These effects may be induced in response to defective vascular repair machinery. Since CD14⁺ monocytes can also differentiate into fibroblast-like cells that produce extracellular matrix proteins, here we propose a new hypothesis that aberrant monocytic PHCs, once mobilized into circulation, may also contribute to the fibrotic process of SSc.

Metabolic effects of the HIV protease inhibitor--saquinavir in differentiating human preadipocytes.

PubMed

Bociąga-Jasik, Monika; Polus, Anna; Góralska, Joanna; Czech, Urszula; Gruca, Anna; Śliwa, Agnieszka; Garlicki, Aleksander; Mach, Tomasz; Dembińska-Kieć, Aldona

2013-01-01

The iatrogenic, HIV-related lipodystrophy is associated with development of the significant metabolic and cardiovascular complications. The underlying mechanisms of antiretroviral (ARV) drugs are not completely explored. The aim of the study was to characterize effects of the protease inhibitor (PI)--saquinavir (SQV) on metabolic functions, and gene expression during differentiation in cells (Chub-S7) culture. SQV in concentrations observed during antiretroviral therapy (ART) significantly decreased mitochondrial membrane potential (MMP), oxygen consumption and ATP generation. The effects were greater in already differentiated cells. This was accompanied by characteristic changes in the expression of the genes involved in endoplasmic reticulum (ER) stress, and differentiation (lipid droplet formation) process such as: WNT10a, C/EBPa, AFT4, CIDEC, ADIPOQ, LPIN1. The results indicate that SQV affects not only metabolic (mitochondrial) activity of adipocytes, but affects the expression of genes related to differentiation and to a lesser extent to cell apoptosis.

Differentiation as symbiosis.

PubMed

Chigira, M; Watanabe, H

1994-07-01

Preservation of the identity of DNA is the ultimate goal of multicellular organisms. An abnormal DNA sequence in cells within an individual means its parasitic nature in cell society as shown in tumors. Somatic gene arrangement and gene mutation in development may be considered as de novo formation of parasites. It is likely that the developmental process with genetic alterations means symbiosis between altered cells and germ line cells preserving genetic information without alterations, when somatic alteration of DNA sequence is a major mechanism of differentiation. According to the selfish gene theory of Dawkins, germ line cells permit symbiosis when somatic cell society derives clear profit for the replication of original DNA copies.

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

He, Yonghan; Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3; State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223

Highlights: •Radicicol suppressed intracellular fat accumulation in 3T3-L1 adipocytes. •Radicicol inhibited the expression of FAS and FABP4. •Radicicol blocked cell cycle at the G1-S phase during cell differentiation. •Radicicol inhibited the PDK1/Akt pathway in adipocyte differentiation. -- Abstract: Heat shock protein 90 (Hsp90) is involved in various cellular processes, such as cell proliferation, differentiation and apoptosis. As adipocyte differentiation plays a critical role in obesity development, the present study investigated the effect of an Hsp90 inhibitor radicicol on the differentiation of 3T3-L1 preadipocytes and potential mechanisms. The cells were treated with different concentrations of radicicol during the first 8 daysmore » of cell differentiation. Adipogenesis, the expression of adipogenic transcriptional factors, differentiation makers and cell cycle were determined. It was found that radicicol dose-dependently decreased intracellular fat accumulation through down-regulating the expression of peroxisome proliferator-activated receptor γ (PPAR{sub γ}) and CCAAT element binding protein α (C/EBP{sub α}), fatty acid synthase (FAS) and fatty acid-binding protein 4 (FABP4). Flow cytometry analysis revealed that radicicol blocked cell cycle at G1-S phase. Radicicol redcued the phosphorylation of Akt while showing no effect on β-catenin expression. Radicicol decreased the phosphorylation of phosphoinositide-dependent kinase 1 (PDK1). The results suggest that radicicol inhibited 3T3-L1 preadipocyte differentiation through affecting the PDK1/Akt pathway and subsequent inhibition of mitotic clonal expansion and the expression/activity of adipogenic transcriptional factors and their downstream adipogenic proteins.« less

The importance of connexin hemichannels during chondroprogenitor cell differentiation in hydrogel versus microtissue culture models.

PubMed

Schrobback, Karsten; Klein, Travis Jacob; Woodfield, Tim B F

2015-06-01

Appropriate selection of scaffold architecture is a key challenge in cartilage tissue engineering. Gap junction-mediated intercellular contacts play important roles in precartilage condensation of mesenchymal cells. However, scaffold architecture could potentially restrict cell-cell communication and differentiation. This is particularly important when choosing the appropriate culture platform as well as scaffold-based strategy for clinical translation, that is, hydrogel or microtissues, for investigating differentiation of chondroprogenitor cells in cartilage tissue engineering. We, therefore, studied the influence of gap junction-mediated cell-cell communication on chondrogenesis of bone marrow-derived mesenchymal stromal cells (BM-MSCs) and articular chondrocytes. Expanded human chondrocytes and BM-MSCs were either (re-) differentiated in micromass cell pellets or encapsulated as isolated cells in alginate hydrogels. Samples were treated with and without the gap junction inhibitor 18-α glycyrrhetinic acid (18αGCA). DNA and glycosaminoglycan (GAG) content and gene expression levels (collagen I/II/X, aggrecan, and connexin 43) were quantified at various time points. Protein localization was determined using immunofluorescence, and adenosine-5'-triphosphate (ATP) was measured in conditioned media. While GAG/DNA was higher in alginate compared with pellets for chondrocytes, there were no differences in chondrogenic gene expression between culture models. Gap junction blocking reduced collagen II and extracellular ATP in all chondrocyte cultures and in BM-MSC hydrogels. However, differentiation capacity was not abolished completely by 18αGCA. Connexin 43 levels were high throughout chondrocyte cultures and peaked only later during BM-MSC differentiation, consistent with the delayed response of BM-MSCs to 18αGCA. Alginate hydrogels and microtissues are equally suited culture platforms for the chondrogenic (re-)differentiation of expanded human articular chondrocytes and BM-MSCs. Therefore, reducing direct cell-cell contacts does not affect in vitro chondrogenesis. However, blocking gap junctions compromises cell differentiation, pointing to a prominent role for hemichannel function in this process. Therefore, scaffold design strategies that promote an increasing distance between single chondroprogenitor cells do not restrict their differentiation potential in tissue-engineered constructs.