Derivation of Pluripotent Stem Cells with In Vivo Embryonic and Extraembryonic Potency.

PubMed

Yang, Yang; Liu, Bei; Xu, Jun; Wang, Jinlin; Wu, Jun; Shi, Cheng; Xu, Yaxing; Dong, Jiebin; Wang, Chengyan; Lai, Weifeng; Zhu, Jialiang; Xiong, Liang; Zhu, Dicong; Li, Xiang; Yang, Weifeng; Yamauchi, Takayoshi; Sugawara, Atsushi; Li, Zhongwei; Sun, Fangyuan; Li, Xiangyun; Li, Chen; He, Aibin; Du, Yaqin; Wang, Ting; Zhao, Chaoran; Li, Haibo; Chi, Xiaochun; Zhang, Hongquan; Liu, Yifang; Li, Cheng; Duo, Shuguang; Yin, Ming; Shen, Huan; Belmonte, Juan Carlos Izpisua; Deng, Hongkui

2017-04-06

Of all known cultured stem cell types, pluripotent stem cells (PSCs) sit atop the landscape of developmental potency and are characterized by their ability to generate all cell types of an adult organism. However, PSCs show limited contribution to the extraembryonic placental tissues in vivo. Here, we show that a chemical cocktail enables the derivation of stem cells with unique functional and molecular features from mice and humans, designated as extended pluripotent stem (EPS) cells, which are capable of chimerizing both embryonic and extraembryonic tissues. Notably, a single mouse EPS cell shows widespread chimeric contribution to both embryonic and extraembryonic lineages in vivo and permits generating single-EPS-cell-derived mice by tetraploid complementation. Furthermore, human EPS cells exhibit interspecies chimeric competency in mouse conceptuses. Our findings constitute a first step toward capturing pluripotent stem cells with extraembryonic developmental potentials in culture and open new avenues for basic and translational research. VIDEO ABSTRACT. Copyright © 2017 Elsevier Inc. All rights reserved.

A single-cell resolution map of mouse hematopoietic stem and progenitor cell differentiation.

PubMed

Nestorowa, Sonia; Hamey, Fiona K; Pijuan Sala, Blanca; Diamanti, Evangelia; Shepherd, Mairi; Laurenti, Elisa; Wilson, Nicola K; Kent, David G; Göttgens, Berthold

2016-08-25

Maintenance of the blood system requires balanced cell fate decisions by hematopoietic stem and progenitor cells (HSPCs). Because cell fate choices are executed at the individual cell level, new single-cell profiling technologies offer exciting possibilities for mapping the dynamic molecular changes underlying HSPC differentiation. Here, we have used single-cell RNA sequencing to profile more than 1600 single HSPCs, and deep sequencing has enabled detection of an average of 6558 protein-coding genes per cell. Index sorting, in combination with broad sorting gates, allowed us to retrospectively assign cells to 12 commonly sorted HSPC phenotypes while also capturing intermediate cells typically excluded by conventional gating. We further show that independently generated single-cell data sets can be projected onto the single-cell resolution expression map to directly compare data from multiple groups and to build and refine new hypotheses. Reconstruction of differentiation trajectories reveals dynamic expression changes associated with early lymphoid, erythroid, and granulocyte-macrophage differentiation. The latter two trajectories were characterized by common upregulation of cell cycle and oxidative phosphorylation transcriptional programs. By using external spike-in controls, we estimate absolute messenger RNA (mRNA) levels per cell, showing for the first time that despite a general reduction in total mRNA, a subset of genes shows higher expression levels in immature stem cells consistent with active maintenance of the stem-cell state. Finally, we report the development of an intuitive Web interface as a new community resource to permit visualization of gene expression in HSPCs at single-cell resolution for any gene of choice. © 2016 by The American Society of Hematology.

Single-Cell Transcriptomics and Fate Mapping of Ependymal Cells Reveals an Absence of Neural Stem Cell Function.

PubMed

Shah, Prajay T; Stratton, Jo A; Stykel, Morgan Gail; Abbasi, Sepideh; Sharma, Sandeep; Mayr, Kyle A; Koblinger, Kathrin; Whelan, Patrick J; Biernaskie, Jeff

2018-05-03

Ependymal cells are multi-ciliated cells that form the brain's ventricular epithelium and a niche for neural stem cells (NSCs) in the ventricular-subventricular zone (V-SVZ). In addition, ependymal cells are suggested to be latent NSCs with a capacity to acquire neurogenic function. This remains highly controversial due to a lack of prospective in vivo labeling techniques that can effectively distinguish ependymal cells from neighboring V-SVZ NSCs. We describe a transgenic system that allows for targeted labeling of ependymal cells within the V-SVZ. Single-cell RNA-seq revealed that ependymal cells are enriched for cilia-related genes and share several stem-cell-associated genes with neural stem or progenitors. Under in vivo and in vitro neural-stem- or progenitor-stimulating environments, ependymal cells failed to demonstrate any suggestion of latent neural-stem-cell function. These findings suggest remarkable stability of ependymal cell function and provide fundamental insights into the molecular signature of the V-SVZ niche. Copyright © 2018 Elsevier Inc. All rights reserved.

Understanding development and stem cells using single cell-based analyses of gene expression

PubMed Central

Kumar, Pavithra; Tan, Yuqi

2017-01-01

In recent years, genome-wide profiling approaches have begun to uncover the molecular programs that drive developmental processes. In particular, technical advances that enable genome-wide profiling of thousands of individual cells have provided the tantalizing prospect of cataloging cell type diversity and developmental dynamics in a quantitative and comprehensive manner. Here, we review how single-cell RNA sequencing has provided key insights into mammalian developmental and stem cell biology, emphasizing the analytical approaches that are specific to studying gene expression in single cells. PMID:28049689

Eight types of stem cells in the life cycle of the moss Physcomitrella patens.

PubMed

Kofuji, Rumiko; Hasebe, Mitsuyasu

2014-02-01

Stem cells self-renew and produce cells that differentiate to become the source of the plant body. The moss Physcomitrella patens forms eight types of stem cells during its life cycle and serves as a useful model in which to explore the evolution of such cells. The common ancestor of land plants is inferred to have been haplontic and to have formed stem cells only in the gametophyte generation. A single stem cell would have been maintained in the ancestral gametophyte meristem, as occurs in extant basal land plants. During land plant evolution, stem cells diverged in the gametophyte generation to form different types of body parts, including the protonema and rhizoid filaments, leafy-shoot and thalloid gametophores, and gametangia formed in moss. A simplex meristem with a single stem cell was acquired in the sporophyte generation early in land plant evolution. Subsequently, sporophyte stem cells became multiple in the meristem and were elaborated further in seed plant lineages, although the evolutionary origin of niche cells, which maintain stem cells is unknown. Comparisons of gene regulatory networks are expected to give insights into the general mechanisms of stem cell formation and maintenance in land plants and provide information about their evolution. P. patens develops at least seven types of simplex meristem in the gametophyte and at least one type in the sporophyte generation and is a good material for regulatory network comparisons. In this review, we summarize recently revealed molecular mechanisms of stem cell initiation and maintenance in the moss. Copyright © 2013 Elsevier Ltd. All rights reserved.

Stem cell plasticity.

PubMed

Lakshmipathy, Uma; Verfaillie, Catherine

2005-01-01

The central dogma in stem cell biology has been that cells isolated from a particular tissue can renew and differentiate into lineages of the tissue it resides in. Several studies have challenged this idea by demonstrating that tissue specific cell have considerable plasticity and can cross-lineage restriction boundary and give rise to cell types of other lineages. However, the lack of a clear definition for plasticity has led to confusion with several reports failing to demonstrate that a single cell can indeed differentiate into multiple lineages at significant levels. Further, differences between results obtained in different labs has cast doubt on some results and several studies still await independent confirmation. In this review, we critically evaluate studies that report stem cell plasticity using three rigid criteria to define stem cell plasticity; differentiation of a single cell into multiple cell lineages, functionality of differentiated cells in vitro and in vivo, robust and persistent engraft of transplanted cells.

A cGMP-applicable expansion method for aggregates of human neural stem and progenitor cells derived from pluripotent stem cells or fetal brain tissue.

PubMed

Shelley, Brandon C; Gowing, Geneviève; Svendsen, Clive N

2014-06-15

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as "chopping" that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

A Bilayer Construct Controls Adipose-Derived Stem Cell Differentiation into Endothelial Cells and Pericytes without Growth Factor Stimulation

DTIC Science & Technology

2011-01-01

A Bilayer Construct Controls Adipose-Derived Stem Cell Differentiation into Endothelial Cells and Pericytes Without Growth Factor Stimulation...Ph.D.3 This work describes the differentiation of adipose-derived mesenchymal stem cells (ASC) in a composite hy- drogel for use as a vascularized...tissue from a single population of ASC. This work underscores the importance of the extracellular matrix in controlling stem cell phenotype. It is our

Feasibility of mesenchymal stem cell culture expansion for a phase I clinical trial in multiple sclerosis.

PubMed

Planchon, Sarah M; Lingas, Karen T; Reese Koç, Jane; Hooper, Brittney M; Maitra, Basabi; Fox, Robert M; Imrey, Peter B; Drake, Kylie M; Aldred, Micheala A; Lazarus, Hillard M; Cohen, Jeffrey A

2018-01-01

Multiple sclerosis is an inflammatory, neurodegenerative disease of the central nervous system for which therapeutic mesenchymal stem cell transplantation is under study. Published experience of culture-expanding multiple sclerosis patients' mesenchymal stem cells for clinical trials is limited. To determine the feasibility of culture-expanding multiple sclerosis patients' mesenchymal stem cells for clinical use. In a phase I trial, autologous, bone marrow-derived mesenchymal stem cells were isolated from 25 trial participants with multiple sclerosis and eight matched controls, and culture-expanded to a target single dose of 1-2 × 10 6 cells/kg. Viability, cell product identity and sterility were assessed prior to infusion. Cytogenetic stability was assessed by single nucleotide polymorphism analysis of mesenchymal stem cells from 18 multiple sclerosis patients and five controls. One patient failed screening. Mesenchymal stem cell culture expansion was successful for 24 of 25 multiple sclerosis patients and six of eight controls. The target dose was achieved in 16-62 days, requiring two to three cell passages. Growth rate and culture success did not correlate with demographic or multiple sclerosis disease characteristics. Cytogenetic studies identified changes on one chromosome of one control (4.3%) after extended time in culture. Culture expansion of mesenchymal stem cells from multiple sclerosis patients as donors is feasible. However, culture time should be minimized for cell products designated for therapeutic administration.

Enhancing the reliability and throughput of neurosphere culture on hydrogel microwell arrays.

PubMed

Cordey, Myriam; Limacher, Monika; Kobel, Stefan; Taylor, Verdon; Lutolf, Matthias P

2008-10-01

The neurosphere assay is the standard retrospective assay to test the self-renewal capability and multipotency of neural stem cells (NSCs) in vitro. However, it has recently become clear that not all neurospheres are derived from a NSC and that on conventional cell culture substrates, neurosphere motility may cause frequent neurosphere "merging" [Nat Methods 2006;3:801-806; Stem Cells 2007;25:871-874]. Combining biomimetic hydrogel matrix technology with microengineering, we developed a microwell array platform on which NSC fate and neurosphere formation can be unequivocally attributed to a single founding cell. Using time-lapse microscopy and retrospective immunostaining, the fate of several hundred single NSCs was quantified. Compared with conventional neurosphere culture methods on plastic dishes, we detected a more than 100% increase in single NSC viability on soft hydrogels. Effective confinement of single proliferating cells to microwells led to neurosphere formation of vastly different sizes, a high percentage of which showed stem cell phenotypes after one week in culture. The reliability and increased throughput of this platform should help to better elucidate the function of sphere-forming stem/progenitor cells independent of their proliferation dynamics. Disclosure of potential conflicts of interest is found at the end of this article.

Understanding development and stem cells using single cell-based analyses of gene expression.

PubMed

Kumar, Pavithra; Tan, Yuqi; Cahan, Patrick

2017-01-01

In recent years, genome-wide profiling approaches have begun to uncover the molecular programs that drive developmental processes. In particular, technical advances that enable genome-wide profiling of thousands of individual cells have provided the tantalizing prospect of cataloging cell type diversity and developmental dynamics in a quantitative and comprehensive manner. Here, we review how single-cell RNA sequencing has provided key insights into mammalian developmental and stem cell biology, emphasizing the analytical approaches that are specific to studying gene expression in single cells. © 2017. Published by The Company of Biologists Ltd.

A Simplified and Systematic Method to Isolate, Culture, and Characterize Multiple Types of Human Dental Stem Cells from a Single Tooth.

PubMed

Bakkar, Mohammed; Liu, Younan; Fang, Dongdong; Stegen, Camille; Su, Xinyun; Ramamoorthi, Murali; Lin, Li-Chieh; Kawasaki, Takako; Makhoul, Nicholas; Pham, Huan; Sumita, Yoshinori; Tran, Simon D

2017-01-01

This chapter describes a simplified method that allows the systematic isolation of multiple types of dental stem cells such as dental pulp stem cells (DPSC), periodontal ligament stem cells (PDLSC), and stem cells of the apical papilla (SCAP) from a single tooth. Of specific interest is the modified laboratory approach to harvest/retrieve the dental pulp tissue by minimizing trauma to DPSC by continuous irrigation, reduction of frictional heat from the bur rotation, and reduction of the bur contact time with the dentin. Also, the use of a chisel and a mallet will maximize the number of live DPSC for culture. Steps demonstrating the potential for multiple cell differentiation lineages of each type of dental stem cell into either osteocytes, adipocytes, or chondrocytes are described. Flow cytometry, with a detailed strategy for cell gating and analysis, is described to verify characteristic markers of human mesenchymal multipotent stromal cells (MSC) from DPSC, PDLSC, or SCAP for subsequent experiments in cell therapy and in tissue engineering. Overall, this method can be adapted to any laboratory with a general setup for cell culture experiments.

[Single nucleotide polymorphism and its application in allogeneic hematopoietic stem cell transplantation--review].

PubMed

Li, Su-Xia

2004-12-01

Single nucleotide polymorphism (SNP) is the third genetic marker after restriction fragment length polymorphism (RFLP) and short tandem repeat. It represents the most density genetic variability in the human genome and has been widely used in gene location, cloning, and research of heredity variation, as well as parenthood identification in forensic medicine. As steady heredity polymorphism, single nucleotide polymorphism is becoming the focus of attention in monitoring chimerism and minimal residual disease in the patients after allogeneic hematopoietic stem cell transplantation. The article reviews SNP heredity characterization, analysis techniques and its applications in allogeneic stem cell transplantation and other fields.

Investigation of adhesion and mechanical properties of human glioma cells by single cell force spectroscopy and atomic force microscopy.

PubMed

Andolfi, Laura; Bourkoula, Eugenia; Migliorini, Elisa; Palma, Anita; Pucer, Anja; Skrap, Miran; Scoles, Giacinto; Beltrami, Antonio Paolo; Cesselli, Daniela; Lazzarino, Marco

2014-01-01

Active cell migration and invasion is a peculiar feature of glioma that makes this tumor able to rapidly infiltrate into the surrounding brain tissue. In our recent work, we identified a novel class of glioma-associated-stem cells (defined as GASC for high-grade glioma--HG--and Gasc for low-grade glioma--LG) that, although not tumorigenic, act supporting the biological aggressiveness of glioma-initiating stem cells (defined as GSC for HG and Gsc for LG) favoring also their motility. Migrating cancer cells undergo considerable molecular and cellular changes by remodeling their cytoskeleton and cell interactions with surrounding environment. To get a better understanding about the role of the glioma-associated-stem cells in tumor progression, cell deformability and interactions between glioma-initiating stem cells and glioma-associated-stem cells were investigated. Adhesion of HG/LG-cancer cells on HG/LG-glioma-associated stem cells was studied by time-lapse microscopy, while cell deformability and cell-cell adhesion strengths were quantified by indentation measurements by atomic force microscopy and single cell force spectroscopy. Our results demonstrate that for both HG and LG glioma, cancer-initiating-stem cells are softer than glioma-associated-stem cells, in agreement with their neoplastic features. The adhesion strength of GSC on GASC appears to be significantly lower than that observed for Gsc on Gasc. Whereas, GSC spread and firmly adhere on Gasc with an adhesion strength increased as compared to that obtained on GASC. These findings highlight that the grade of glioma-associated-stem cells plays an important role in modulating cancer cell adhesion, which could affect glioma cell migration, invasion and thus cancer aggressiveness. Moreover this work provides evidence about the importance of investigating cell adhesion and elasticity for new developments in disease diagnostics and therapeutics.

Isolation and gene expression analysis of single potential human spermatogonial stem cells.

PubMed

von Kopylow, K; Schulze, W; Salzbrunn, A; Spiess, A-N

2016-04-01

It is possible to isolate pure populations of single potential human spermatogonial stem cells without somatic contamination for down-stream applications, for example cell culture and gene expression analysis. We isolated pure populations of single potential human spermatogonial stem cells (hSSC) without contaminating somatic cells and analyzed gene expression of these cells via single-cell real-time RT-PCR. The isolation of a pure hSSC fraction could enable clinical applications such as fertility preservation for prepubertal boys and in vitro-spermatogenesis. By utilizing largely nonspecific markers for the isolation of spermatogonia (SPG) and hSSC, previously published cell selection methods are not able to deliver pure target cell populations without contamination by testicular somatic cells. However, uniform cell populations free of somatic cells are necessary to guarantee defined growth conditions in cell culture experiments and to prevent unintended stem cell differentiation. Fibroblast growth factor receptor 3 (FGFR3) is a cell surface protein of human undifferentiated A-type SPG and a promising candidate marker for hSSC. It is exclusively expressed in small, non-proliferating subgroups of this spermatogonial cell type together with the pluripotency-associated protein and spermatogonial nuclear marker undifferentiated embryonic cell transcription factor 1 (UTF1). We specifically selected the FGFR3-positive spermatogonial subpopulation from two 30 mg biopsies per patient from a total of 37 patients with full spermatogenesis and three patients with meiotic arrest. We then employed cell selection with magnetic beads in combination with a fluorescence-activated cell sorter antibody directed against human FGFR3 to tag and visually identify human FGFR3-positive spermatogonia. Positively selected and bead-labeled cells were subsequently picked with a micromanipulator. Analysis of the isolated cells was carried out by single-cell real-time RT-PCR, real-time RT-PCR, immunocytochemistry and live/dead staining. Single-cell real-time RT-PCR and real-time RT-PCR of pooled cells indicate that bead-labeled single cells express FGFR3 with high heterogeneity at the mRNA level, while bead-unlabeled cells lack FGFR3 mRNA. Furthermore, isolated cells exhibit strong immunocytochemical staining for the stem cell factor UTF1 and are viable. The cell population isolated in this study has to be tested for their potential stem cell characteristics via xenotransplantation. Due to the small amount of the isolated cells, propagation by cell culture will be essential. Other potential hSSC without FGFR3 surface expression will not be captured with the provided experimental design. The technical approach as developed in this work could encourage the scientific community to test other established or novel hSSC markers on single SPG that present with potential stem cell-like features. The project was funded by the DFG Research Unit FOR1041 Germ cell potential (SCH 587/3-2) and DFG grants to K.v.K. (KO 4769/2-1) and A.-N.S. (SP 721/4-1). The authors declare no competing interests. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Stable chromosomal aberrations in haemopoietic stem cells in the blood of radiation accident victims.

PubMed

Kreja, L; Greulich, K M; Fliedner, T M; Heinze, B

1999-10-01

The detection of long-term persistent chromosome aberrations in circulating haemopoietic stem cells after accidental radiation exposure. Peripheral blood samples from highly exposed persons were collected 7-25 years after the radiation accidents in Moscow (1971), Kazan (1975) and Chernobyl (1996). Haemopoietic blood stem cells were analysed when investigating individual colonies derived from haemopoietic progenitor cells: burst-forming units-erythroid (BFU-E), granulocyte-macrophage-colony-forming cells (GM-CFC) and multipotent granulocyte-erythrocyte-macrophage- megakaryocyte-colony-forming cells (GEMM-CFC). Colony formation was obtained in methylcellulose cultures. Chromosome preparations in single colonies were performed using a microtechnique. Nine patients were investigated at 1 to 4 follow-up time points after radiation exposure. Three hundred and thirty-four single colonies were analyzed resulting in 1375 mitoses. It was found that colonies showed chromosome aberrations (ChA) up to 25 years after radiation exposure by classical cytogenetics and by fluorescence in situ hybridization (FISH). Stable aberrations were detected in 21% of colonies. They were clonal in 19% of colonies, i.e. the same abnormality was found in all cells derived from a single colony. In 2% of colonies ChA were stable but non-clonal; unstable ChA were not observed. The results indicate that blood-derived haemopoietic stem cells may serve as a biological indicator to detect radiation-induced ChA. Since they are considered to be in dynamic and functional exchange with stem cells in the medullary sites of blood cell formation such as bone marrow, the use of blood stem cells as a marker of radiation effects should be explored to assess the repair status of the stem cell pool as such.

Differential Radiosensitizing Effect of Valproic Acid in Differentiation Versus Self-Renewal Promoting Culture Conditions

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

Debeb, Bisrat G.; Xu Wei; Mok, Henry

2010-03-01

Purpose: It has been shown that valproic acid (VA) enhances the proliferation and self-renewal of normal hematopoietic stem cells and that breast cancer stem/progenitor cells can be resistant to radiation. From these data, we hypothesized that VA would fail to radiosensitize breast cancer stem/progenitor cells grown to three-dimensional (3D) mammospheres. Methods and Materials: We used the MCF7 breast cancer cell line grown under stem cell-promoting culture conditions (3D mammosphere) and standard nonstem cell monolayer culture conditions (two-dimensional) to examine the effect of pretreatment with VA on radiation sensitivity in clonogenic survival assays and on the expression of embryonic stem cellmore » transcription factors. Results: 3D-cultured MCF-7 cells expressed higher levels of Oct4, Nanog, and Sox2. The 3D passage enriched self-renewal and increased radioresistance in the 3D mammosphere formation assays. VA radiosensitized adherent cells but radioprotected 3D cells in single-fraction clonogenic assays. Moreover, fractionated radiation sensitized VA-treated adherent MCF7 cells but did not have a significant effect on VA-treated single cells grown to mammospheres. Conclusion: We have concluded that VA might preferentially radiosensitize differentiated cells compared with those expressing stem cell surrogates and that stem cell-promoting culture is a useful tool for in vitro evaluation of novel cancer therapeutic agents and radiosensitizers.« less

Ovary and fimbrial stem cells: biology, niche and cancer origins.

PubMed

Ng, Annie; Barker, Nick

2015-10-01

The mammalian ovary is covered by a single-layered epithelium that undergoes rupture and remodelling following each ovulation. Although resident stem cells are presumed to be crucial for this cyclic regeneration, their identity and mode of action have been elusive. Surrogate stemness assays and in vivo fate-mapping studies using recently discovered stem cell markers have identified stem cell pools in the ovary and fimbria that ensure epithelial homeostasis. Recent findings provide insights into intrinsic mechanisms and local extrinsic cues that govern the function of ovarian and fimbrial stem cells. These discoveries have advanced our understanding of stem cell biology in the ovary and fimbria, and lay the foundations for evaluating the contribution of resident stem cells to the initiation and progression of human epithelial ovarian cancer.

Somatic stem cell heterogeneity: diversity in the blood, skin and intestinal stem cell compartments

PubMed Central

Goodell, Margaret A.; Nguyen, Hoang; Shroyer, Noah

2017-01-01

Somatic stem cells replenish many tissues throughout life to repair damage and to maintain tissue homeostasis. Stem cell function is frequently described as following a hierarchical model in which a single master cell undergoes self-renewal and differentiation into multiple cell types and is responsible for most regenerative activity. However, recent data from studies on blood, skin and intestinal epithelium all point to the concomitant action of multiple types of stem cells with distinct everyday roles. Under stress conditions such as acute injury, the surprising developmental flexibility of these stem cells enables them to adapt to diverse roles and to acquire different regeneration capabilities. This paradigm shift raises many new questions about the developmental origins, inter-relationships and molecular regulation of these multiple stem cell types. PMID:25907613

Fusion with stem cell makes the hepatocellular carcinoma cells similar to liver tumor-initiating cells.

PubMed

Wang, Ran; Chen, Shuxun; Li, Changxian; Ng, Kevin Tak Pan; Kong, Chi-wing; Cheng, Jinping; Cheng, Shuk Han; Li, Ronald A; Lo, Chung Mau; Man, Kwan; Sun, Dong

2016-02-04

Cell fusion is a fast and highly efficient technique for cells to acquire new properties. The fusion of somatic cells with stem cells can reprogram somatic cells to a pluripotent state. Our research on the fusion of stem cells and cancer cells demonstrates that the fused cells can exhibit stemness and cancer cell-like characteristics. Thus, tumor-initiating cell-like cells are generated. We employed laser-induced single-cell fusion technique to fuse the hepatocellular carcinoma cells and human embryonic stem cells (hESC). Real-time RT-PCR, flow cytometry and in vivo tumorigenicity assay were adopted to identify the gene expression difference. We successfully produced a fused cell line that coalesces the gene expression information of hepatocellular carcinoma cells and stem cells. Experimental results showed that the fused cells expressed cancer and stemness markers as well as exhibited increased resistance to drug treatment and enhanced tumorigenesis. Fusion with stem cells transforms liver cancer cells into tumor initiating-like cells. Results indicate that fusion between cancer cell and stem cell may generate tumor initiating-like cells.

High-performance imaging of stem cells using single-photon emissions

NASA Astrophysics Data System (ADS)

Wagenaar, Douglas J.; Moats, Rex A.; Hartsough, Neal E.; Meier, Dirk; Hugg, James W.; Yang, Tang; Gazit, Dan; Pelled, Gadi; Patt, Bradley E.

2011-10-01

Radiolabeled cells have been imaged for decades in the field of autoradiography. Recent advances in detector and microelectronics technologies have enabled the new field of "digital autoradiography" which remains limited to ex vivo specimens of thin tissue slices. The 3D field-of-view (FOV) of single cell imaging can be extended to millimeters if the low energy (10-30 keV) photon emissions of radionuclides are used for single-photon nuclear imaging. This new microscope uses a coded aperture foil made of highly attenuating elements such as gold or platinum to form the image as a kind of "lens". The detectors used for single-photon emission microscopy are typically silicon detectors with a pixel pitch less than 60 μm. The goal of this work is to image radiolabeled mesenchymal stem cells in vivo in an animal model of tendon repair processes. Single-photon nuclear imaging is an attractive modality for translational medicine since the labeled cells can be imaged simultaneously with the reparative processes by using the dual-isotope imaging technique. The details our microscope's two-layer gold aperture and the operation of the energy-dispersive, pixellated silicon detector are presented along with the first demonstration of energy discrimination with a 57Co source. Cell labeling techniques have been augmented by genetic engineering with the sodium-iodide symporter, a type of reporter gene imaging method that enables in vivo uptake of free 99mTc or an iodine isotope at a time point days or weeks after the insertion of the genetically modified stem cells into the animal model. This microscopy work in animal research may expand to the imaging of reporter-enabled stem cells simultaneously with the expected biological repair process in human clinical trials of stem cell therapies.

Transcriptional Networks in Single Perivascular Cells Sorted from Human Adipose Tissue Reveal a Hierarchy of Mesenchymal Stem Cells.

PubMed

Hardy, W Reef; Moldovan, Nicanor I; Moldovan, Leni; Livak, Kenneth J; Datta, Krishna; Goswami, Chirayu; Corselli, Mirko; Traktuev, Dmitry O; Murray, Iain R; Péault, Bruno; March, Keith

2017-05-01

Adipose tissue is a rich source of multipotent mesenchymal stem-like cells, located in the perivascular niche. Based on their surface markers, these have been assigned to two main categories: CD31 - /CD45 - /CD34 + /CD146 - cells (adventitial stromal/stem cells [ASCs]) and CD31 - /CD45 - /CD34 - /CD146 + cells (pericytes [PCs]). These populations display heterogeneity of unknown significance. We hypothesized that aldehyde dehydrogenase (ALDH) activity, a functional marker of primitivity, could help to better define ASC and PC subclasses. To this end, the stromal vascular fraction from a human lipoaspirate was simultaneously stained with fluorescent antibodies to CD31, CD45, CD34, and CD146 antigens and the ALDH substrate Aldefluor, then sorted by fluorescence-activated cell sorting. Individual ASCs (n = 67) and PCs (n = 73) selected from the extremities of the ALDH-staining spectrum were transcriptionally profiled by Fluidigm single-cell quantitative polymerase chain reaction for a predefined set (n = 429) of marker genes. To these single-cell data, we applied differential expression and principal component and clustering analysis, as well as an original gene coexpression network reconstruction algorithm. Despite the stochasticity at the single-cell level, covariation of gene expression analysis yielded multiple network connectivity parameters suggesting that these perivascular progenitor cell subclasses possess the following order of maturity: (a) ALDH br ASC (most primitive); (b) ALDH dim ASC; (c) ALDH br PC; (d) ALDH dim PC (least primitive). This order was independently supported by specific combinations of class-specific expressed genes and further confirmed by the analysis of associated signaling pathways. In conclusion, single-cell transcriptional analysis of four populations isolated from fat by surface markers and enzyme activity suggests a developmental hierarchy among perivascular mesenchymal stem cells supported by markers and coexpression networks. Stem Cells 2017;35:1273-1289. © 2017 AlphaMed Press.

Dissecting Transcriptional Heterogeneity in Pluripotency: Single Cell Analysis of Mouse Embryonic Stem Cells.

PubMed

Guedes, Ana M V; Henrique, Domingos; Abranches, Elsa

2016-01-01

Mouse Embryonic Stem cells (mESCs) show heterogeneous and dynamic expression of important pluripotency regulatory factors. Single-cell analysis has revealed the existence of cell-to-cell variability in the expression of individual genes in mESCs. Understanding how these heterogeneities are regulated and what their functional consequences are is crucial to obtain a more comprehensive view of the pluripotent state.In this chapter we describe how to analyze transcriptional heterogeneity by monitoring gene expression of Nanog, Oct4, and Sox2, using single-molecule RNA FISH in single mESCs grown in different cell culture medium. We describe in detail all the steps involved in the protocol, from RNA detection to image acquisition and processing, as well as exploratory data analysis.

The human urothelium consists of multiple clonal units, each maintained by a stem cell.

PubMed

Gaisa, Nadine T; Graham, Trevor A; McDonald, Stuart A C; Cañadillas-Lopez, Sagrario; Poulsom, Richard; Heidenreich, Axel; Jakse, Gerhard; Tadrous, Paul J; Knuechel, Ruth; Wright, Nicholas A

2011-10-01

Little is known about the clonal architecture of human urothelium. It is likely that urothelial stem cells reside within the basal epithelial layer, yet lineage tracing from a single stem cell as a means to show the presence of a urothelial stem cell has never been performed. Here, we identify clonally related cell areas within human bladder mucosa in order to visualize epithelial fields maintained by a single founder/stem cell. Sixteen frozen cystectomy specimens were serially sectioned. Patches of cells deficient for the mitochondrially encoded enzyme cytochrome c oxidase (CCO) were identified using dual-colour enzyme histochemistry. To show that these patches represent clonal proliferations, small CCO-proficient and -deficient areas were individually laser-capture microdissected and the entire mitochondrial genome (mtDNA) in each area was PCR amplified and sequenced to identify mtDNA mutations. Immunohistochemistry was performed for the different cell layers of the urothelium and adjacent mesenchyme. CCO-deficient patches could be observed in normal urothelium of all cystectomy specimens. The two-dimensional length of these negative patches varied from 2-3 cells (about 30 µm) to 4.7 mm. Each cell area within a CCO-deficient patch contained an identical somatic mtDNA mutation, indicating that the patch was a clonal unit. Patches contained all the mature cell differentiation stages present in the urothelium, suggesting the presence of a stem cell. Our results demonstrate that the normal mucosa of human bladder contains stem cell-derived clonal units that actively replenish the urothelium during ageing. The size of the clonal unit attributable to each stem cell was broadly distributed, suggesting replacement of one stem cell clone by another. Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Single-cell cloning and expansion of human induced pluripotent stem cells by a microfluidic culture device.

PubMed

Matsumura, Taku; Tatsumi, Kazuya; Noda, Yuichiro; Nakanishi, Naoyuki; Okonogi, Atsuhito; Hirano, Kunio; Li, Liu; Osumi, Takashi; Tada, Takashi; Kotera, Hidetoshi

2014-10-10

The microenvironment of cells, which includes basement proteins, shear stress, and extracellular stimuli, should be taken into consideration when examining physiological cell behavior. Although microfluidic devices allow cellular responses to be analyzed with ease at the single-cell level, few have been designed to recover cells. We herein demonstrated that a newly developed microfluidic device helped to improve culture conditions and establish a clonality-validated human pluripotent stem cell line after tracing its growth at the single-cell level. The device will be a helpful tool for capturing various cell types in the human body that have not yet been established in vitro. Copyright © 2014 Elsevier Inc. All rights reserved.

Dedifferentiation of Glioma Cells to Glioma Stem-like Cells By Therapeutic Stress-induced HIF Signaling in the Recurrent GBM Model.

PubMed

Lee, Gina; Auffinger, Brenda; Guo, Donna; Hasan, Tanwir; Deheeger, Marc; Tobias, Alex L; Kim, Jeong Yeon; Atashi, Fatemeh; Zhang, Lingjiao; Lesniak, Maciej S; James, C David; Ahmed, Atique U

2016-12-01

Increasing evidence exposes a subpopulation of cancer cells, known as cancer stem cells (CSCs), to be critical for the progression of several human malignancies, including glioblastoma multiforme. CSCs are highly tumorigenic, capable of self-renewal, and resistant to conventional therapies, and thus considered to be one of the key contributors to disease recurrence. To elucidate the poorly understood evolutionary path of tumor recurrence and the role of CSCs in this process, we developed patient-derived xenograft glioblastoma recurrent models induced by anti-glioma chemotherapy, temozolomide. In this model, we observed a significant phenotypic shift towards an undifferentiated population. We confirmed these findings in vitro as sorted CD133-negative populations cultured in differentiation-forcing media were found to acquire CD133 expression following chemotherapy treatment. To investigate this phenotypic switch at the single-cell level, glioma stem cell (GSC)-specific promoter-based reporter systems were engineered to track changes in the GSC population in real time. We observed the active phenotypic and functional switch of single non-stem glioma cells to a stem-like state and that temozolomide therapy significantly increased the rate of single-cell conversions. Importantly, we showed the therapy-induced hypoxia-inducible factors (HIF) 1α and HIF2α play key roles in allowing non-stem glioma cells to acquire stem-like traits, as the expression of both HIFs increase upon temozolomide therapy and knockdown of HIFs expression inhibits the interconversion between non-stem glioma cells and GSCs post-therapy. On the basis of our results, we propose that anti-glioma chemotherapy promotes the accumulation of HIFs in the glioblastoma multiforme cells that induces the formation of therapy-resistant GSCs responsible for recurrence. Mol Cancer Ther; 15(12); 3064-76. ©2016 AACR. ©2016 American Association for Cancer Research.

Beyond the Niche: Tissue-Level Coordination of Stem Cell Dynamics

PubMed Central

O’Brien, Lucy Erin; Bilder, David

2014-01-01

Adult animals rely on populations of stem cells to ensure organ function throughout their lifetime. Stem cells are governed by signals from stem cell niches, and much is known about how single niches promote stemness and direct stem cell behavior. However, most organs contain a multitude of stem cell–niche units, which are often distributed across the entire expanse of the tissue. Beyond the biology of individual stem cell–niche interactions, the next challenge is to uncover the tissue-level processes that orchestrate spatial control of stem-based renewal, repair, and remodeling throughout a whole organ. Here we examine what is known about higher order mechanisms for interniche coordination in epithelial organs, whose simple geometry offers a promising entry point for understanding the regulation of niche number, distribution, and activity. We also consider the potential existence of stem cell territories and how tissue architecture may influence niche coordination. PMID:23937350

Single Stem Cell Imaging and Analysis Reveals Telomere Length Differences in Diseased Human and Mouse Skeletal Muscles.

PubMed

Tichy, Elisia D; Sidibe, David K; Tierney, Matthew T; Stec, Michael J; Sharifi-Sanjani, Maryam; Hosalkar, Harish; Mubarak, Scott; Johnson, F Brad; Sacco, Alessandra; Mourkioti, Foteini

2017-10-10

Muscle stem cells (MuSCs) contribute to muscle regeneration following injury. In many muscle disorders, the repeated cycles of damage and repair lead to stem cell dysfunction. While telomere attrition may contribute to aberrant stem cell functions, methods to accurately measure telomere length in stem cells from skeletal muscles have not been demonstrated. Here, we have optimized and validated such a method, named MuQ-FISH, for analyzing telomere length in MuSCs from either mice or humans. Our analysis showed no differences in telomere length between young and aged MuSCs from uninjured wild-type mice, but MuSCs isolated from young dystrophic mice exhibited significantly shortened telomeres. In corroboration, we demonstrated that telomere attrition is present in human dystrophic MuSCs, which underscores its importance in diseased regenerative failure. The robust technique described herein provides analysis at a single-cell resolution and may be utilized for other cell types, especially rare populations of cells. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Single cell dual adherent-suspension co-culture micro-environment for studying tumor-stromal interactions with functionally selected cancer stem-like cells.

PubMed

Chen, Yu-Chih; Zhang, Zhixiong; Fouladdel, Shamileh; Deol, Yadwinder; Ingram, Patrick N; McDermott, Sean P; Azizi, Ebrahim; Wicha, Max S; Yoon, Euisik

2016-08-07

Considerable evidence suggests that cancer stem-like cells (CSCs) are critical in tumor pathogenesis, but their rarity and transience has led to much controversy about their exact nature. Although CSCs can be functionally identified using dish-based tumorsphere assays, it is difficult to handle and monitor single cells in dish-based approaches; single cell-based microfluidic approaches offer better control and reliable single cell derived sphere formation. However, like normal stem cells, CSCs are heavily regulated by their microenvironment, requiring tumor-stromal interactions for tumorigenic and proliferative behaviors. To enable single cell derived tumorsphere formation within a stromal microenvironment, we present a dual adherent/suspension co-culture device, which combines a suspension environment for single-cell tumorsphere assays and an adherent environment for co-culturing stromal cells in close proximity by selectively patterning polyHEMA in indented microwells. By minimizing dead volume and improving cell capture efficiency, the presented platform allows for the use of small numbers of cells (<100 cells). As a proof of concept, we co-cultured single T47D (breast cancer) cells and primary cancer associated fibroblasts (CAF) on-chip for 14 days to monitor sphere formation and growth. Compared to mono-culture, co-cultured T47D have higher tumorigenic potential (sphere formation rate) and proliferation rates (larger sphere size). Furthermore, 96-multiplexed single-cell transcriptome analyses were performed to compare the gene expression of co-cultured and mono-cultured T47D cells. Phenotypic changes observed in co-culture correlated with expression changes in genes associated with proliferation, apoptotic suppression, tumorigenicity and even epithelial-to-mesechymal transition. Combining the presented platform with single cell transcriptome analysis, we successfully identified functional CSCs and investigated the phenotypic and transcriptome effects induced by tumor-stromal interactions.

High-resolution Myogenic Lineage Mapping by Single-Cell Mass Cytometry

PubMed Central

Porpiglia, Ermelinda; Samusik, Nikolay; Ho, Andrew Tri Van; Cosgrove, Benjamin D.; Mai, Thach; Davis, Kara L.; Jager, Astraea; Nolan, Garry P.; Bendall, Sean C.; Fantl, Wendy J.; Blau, Helen M.

2017-01-01

Muscle regeneration is a dynamic process during which cell state and identity change over time. A major roadblock has been a lack of tools to resolve a myogenic progression in vivo. Here we capitalize on a transformative technology, single-cell mass cytometry (CyTOF), to identify in vivo skeletal muscle stem cell and previously unrecognized progenitor populations that precede differentiation. We discovered two cell surface markers, CD9 and CD104, whose combined expression enabled in vivo identification and prospective isolation of stem and progenitor cells. Data analysis using the X-shift algorithm paired with single-cell force directed layout visualization, defined a molecular signature of the activated stem cell state (CD44+/CD98+/MyoD+) and delineated a myogenic trajectory during recovery from acute muscle injury. Our studies uncover the dynamics of skeletal muscle regeneration in vivo and pave the way for the elucidation of the regulatory networks that underlie cell-state transitions in muscle diseases and aging. PMID:28414312

Single-cell entropy for accurate estimation of differentiation potency from a cell's transcriptome

NASA Astrophysics Data System (ADS)

Teschendorff, Andrew E.; Enver, Tariq

2017-06-01

The ability to quantify differentiation potential of single cells is a task of critical importance. Here we demonstrate, using over 7,000 single-cell RNA-Seq profiles, that differentiation potency of a single cell can be approximated by computing the signalling promiscuity, or entropy, of a cell's transcriptome in the context of an interaction network, without the need for feature selection. We show that signalling entropy provides a more accurate and robust potency estimate than other entropy-based measures, driven in part by a subtle positive correlation between the transcriptome and connectome. Signalling entropy identifies known cell subpopulations of varying potency and drug resistant cancer stem-cell phenotypes, including those derived from circulating tumour cells. It further reveals that expression heterogeneity within single-cell populations is regulated. In summary, signalling entropy allows in silico estimation of the differentiation potency and plasticity of single cells and bulk samples, providing a means to identify normal and cancer stem-cell phenotypes.

Single-cell entropy for accurate estimation of differentiation potency from a cell's transcriptome

PubMed Central

Teschendorff, Andrew E.; Enver, Tariq

2017-01-01

The ability to quantify differentiation potential of single cells is a task of critical importance. Here we demonstrate, using over 7,000 single-cell RNA-Seq profiles, that differentiation potency of a single cell can be approximated by computing the signalling promiscuity, or entropy, of a cell's transcriptome in the context of an interaction network, without the need for feature selection. We show that signalling entropy provides a more accurate and robust potency estimate than other entropy-based measures, driven in part by a subtle positive correlation between the transcriptome and connectome. Signalling entropy identifies known cell subpopulations of varying potency and drug resistant cancer stem-cell phenotypes, including those derived from circulating tumour cells. It further reveals that expression heterogeneity within single-cell populations is regulated. In summary, signalling entropy allows in silico estimation of the differentiation potency and plasticity of single cells and bulk samples, providing a means to identify normal and cancer stem-cell phenotypes. PMID:28569836

Differentiation-Dependent Energy Production and Metabolite Utilization: A Comparative Study on Neural Stem Cells, Neurons, and Astrocytes.

PubMed

Jády, Attila Gy; Nagy, Ádám M; Kőhidi, Tímea; Ferenczi, Szilamér; Tretter, László; Madarász, Emília

2016-07-01

While it is evident that the metabolic machinery of stem cells should be fairly different from that of differentiated neurons, the basic energy production pathways in neural stem cells (NSCs) or in neurons are far from clear. Using the model of in vitro neuron production by NE-4C NSCs, this study focused on the metabolic changes taking place during the in vitro neuronal differentiation. O2 consumption, H(+) production, and metabolic responses to single metabolites were measured in cultures of NSCs and in their neuronal derivatives, as well as in primary neuronal and astroglial cultures. In metabolite-free solutions, NSCs consumed little O2 and displayed a higher level of mitochondrial proton leak than neurons. In stem cells, glycolysis was the main source of energy for the survival of a 2.5-h period of metabolite deprivation. In contrast, stem cell-derived or primary neurons sustained a high-level oxidative phosphorylation during metabolite deprivation, indicating the consumption of own cellular material for energy production. The stem cells increased O2 consumption and mitochondrial ATP production in response to single metabolites (with the exception of glucose), showing rapid adaptation of the metabolic machinery to the available resources. In contrast, single metabolites did not increase the O2 consumption of neurons or astrocytes. In "starving" neurons, neither lactate nor pyruvate was utilized for mitochondrial ATP production. Gene expression studies also suggested that aerobic glycolysis and rapid metabolic adaptation characterize the NE-4C NSCs, while autophagy and alternative glucose utilization play important roles in the metabolism of stem cell-derived neurons.

Single-Crystalline, Nanoporous Gallium Nitride Films With Fine Tuning of Pore Size for Stem Cell Engineering.

PubMed

Han, Lin; Zhou, Jing; Sun, Yubing; Zhang, Yu; Han, Jung; Fu, Jianping; Fan, Rong

2014-11-01

Single-crystalline nanoporous gallium nitride (GaN) thin films were fabricated with the pore size readily tunable in 20-100 nm. Uniform adhesion and spreading of human mesenchymal stem cells (hMSCs) seeded on these thin films peak on the surface with pore size of 30 nm. Substantial cell elongation emerges as pore size increases to ∼80 nm. The osteogenic differentiation of hMSCs occurs preferentially on the films with 30 nm sized nanopores, which is correlated with the optimum condition for cell spreading, which suggests that adhesion, spreading, and stem cell differentiation are interlinked and might be coregulated by nanotopography.

A planarian p53 homolog regulates proliferation and self-renewal in adult stem cell lineages.

PubMed

Pearson, Bret J; Sánchez Alvarado, Alejandro

2010-01-01

The functions of adult stem cells and tumor suppressor genes are known to intersect. However, when and how tumor suppressors function in the lineages produced by adult stem cells is unknown. With a large population of stem cells that can be manipulated and studied in vivo, the freshwater planarian is an ideal system with which to investigate these questions. Here, we focus on the tumor suppressor p53, homologs of which have no known role in stem cell biology in any invertebrate examined thus far. Planaria have a single p53 family member, Smed-p53, which is predominantly expressed in newly made stem cell progeny. When Smed-p53 is targeted by RNAi, the stem cell population increases at the expense of progeny, resulting in hyper-proliferation. However, ultimately the stem cell population fails to self-renew. Our results suggest that prior to the vertebrates, an ancestral p53-like molecule already had functions in stem cell proliferation control and self-renewal.

Characterization of rabbit limbal epithelial side population cells using RNA sequencing and single-cell qRT-PCR.

PubMed

Kameishi, Sumako; Umemoto, Terumasa; Matsuzaki, Yu; Fujita, Masako; Okano, Teruo; Kato, Takashi; Yamato, Masayuki

2016-05-06

Corneal epithelial stem cells reside in the limbus, a transitional zone between the cornea and conjunctiva, and are essential for maintaining homeostasis in the corneal epithelium. Although our previous studies demonstrated that rabbit limbal epithelial side population (SP) cells exhibit stem cell-like phenotypes with Hoechst 33342 staining, the different characteristics and/or populations of these cells remain unclear. Therefore, in this study, we determined the gene expression profiles of limbal epithelial SP cells by RNA sequencing using not only present public databases but also contigs that were created by de novo transcriptome assembly as references for mapping. Our transcriptome data indicated that limbal epithelial SP cells exhibited a stem cell-like phenotype compared with non-SP cells. Importantly, gene ontology analysis following RNA sequencing demonstrated that limbal epithelial SP cells exhibited significantly enhanced expression of mesenchymal/endothelial cell markers rather than epithelial cell markers. Furthermore, single-cell quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) demonstrated that the limbal epithelial SP population consisted of at least two immature cell populations with endothelial- or mesenchymal-like phenotypes. Therefore, our present results may propose the presence of a novel population of corneal epithelial stem cells distinct from conventional epithelial stem cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Regulation of Hemopoietic Stem Cell Turnover and Population Size in Neonatal Mice

DTIC Science & Technology

1975-04-01

Following birth the hematopoietic stem cell population of the liver as measured by the in vivo spleen nodule assay (CFU) declines with a halving time...of about 48 hours. The stem cell population of the spleen grows exponentially with a doubling time of about 17 hours. In vitro incubation with high...single spleen colonies derived from neonatal liver and spleen CFU that both stem cell populations have a high self-renewal capacity. Thus, the decline in

A lineage CLOUD for neoblasts.

PubMed

Tran, Thao Anh; Gentile, Luca

2018-05-10

In planarians, pluripotency can be studied in vivo in the adult animal, making these animals a unique model system where pluripotency-based regeneration (PBR)-and its therapeutic potential-can be investigated. This review focuses on recent findings to build a cloud model of fate restriction likelihood for planarian stem and progenitor cells. Recently, a computational approach based on functional and molecular profiling at the single cell level was proposed for human hematopoietic stem cells. Based on data generated both in vivo and ex vivo, we hypothesized that planarian stem cells could acquire multiple direction lineage biases, following a "badlands" landscape. Instead of a discrete tree-like hierarchy, where the potency of stem/progenitor cells reduces stepwise, we propose a Continuum of LOw-primed UnDifferentiated Planarian Stem/Progenitor Cells (CLOUD-PSPCs). Every subclass of neoblast/progenitor cells is a cloud of likelihood, as the single cell transcriptomics data indicate. The CLOUD-HSPCs concept was substantiated by in vitro data from cell culture; therefore, to confirm the CLOUD-PSPCs model, the planarian community needs to develop new tools, like live cell tracking. Future studies will allow a deeper understanding of PBR in planarian, and the possible implications for regenerative therapies in human. Copyright © 2018 Elsevier Ltd. All rights reserved.

Stem cells are dispensable for lung homeostasis but restore airways after injury.

PubMed

Giangreco, Adam; Arwert, Esther N; Rosewell, Ian R; Snyder, Joshua; Watt, Fiona M; Stripp, Barry R

2009-06-09

Local tissue stem cells have been described in airways of the lung but their contribution to normal epithelial maintenance is currently unknown. We therefore developed aggregation chimera mice and a whole-lung imaging method to determine the relative contributions of progenitor (Clara) and bronchiolar stem cells to epithelial maintenance and repair. In normal and moderately injured airways chimeric patches were small in size and not associated with previously described stem cell niches. This finding suggested that single, randomly distributed progenitor cells maintain normal epithelial homeostasis. In contrast we found that repair following severe lung injury resulted in the generation of rare, large clonal cell patches that were associated with stem cell niches. This study provides evidence that epithelial stem cells are dispensable for normal airway homeostasis. We also demonstrate that stem cell activation and robust clonal cellular expansion occur only during repair from severe lung injury.

The Emerging Role of PEDF in Stem Cell Biology

PubMed Central

Elahy, Mina; Baindur-Hudson, Swati; Dass, Crispin R.

2012-01-01

Encoded by a single gene, PEDF is a 50 kDa glycoprotein that is highly conserved and is widely expressed among many tissues. Most secreted PEDF deposits within the extracellular matrix, with cell-type-specific functions. While traditionally PEDF is known as a strong antiangiogenic factor, more recently, as this paper highlights, PEDF has been linked with stem cell biology, and there is now accumulating evidence demonstrating the effects of PEDF in a variety of stem cells, mainly in supporting stem cell survival and maintaining multipotency. PMID:22675247

Osthole Enhances the Therapeutic Efficiency of Stem Cell Transplantation in Neuroendoscopy Caused Traumatic Brain Injury.

PubMed

Tao, Zhen-Yu; Gao, Peng; Yan, Yu-Hui; Li, Hong-Yan; Song, Jie; Yang, Jing-Xian

2017-01-01

Neuroendoscopy processes can cause severe traumatic brain injury. Existing therapeutic methods, such as neural stem cell transplantation and osthole have not been proven effective. Therefore, there is an emerging need on the development of new techniques for the treatment of brain injuries. In this study we propose to combine the above stem cell based methods and then evaluate the efficiency and accuracy of the new method. Mice were randomly divided into four groups: group 1 (brain injury alone); group 2 (osthole); group 3 (stem cell transplantation); and group 4 (osthole combined with stem cell transplantation). We carried out water maze task to exam spatial memory. Immunocytochemistry was used to test the inflammatory condition of each group, and the differentiation of stem cells. To evaluate the condition of the damaged blood brain barrier restore, we detect the Evans blue (EB) extravasation across the blood brain barrier. The result shows that osthole and stem cell transplantation combined therapeutic method has a potent effect on improving the spatial memory. This combined method was more effective on inhibiting inflammation and preventing neuronal degeneration than the single treated ones. In addition, there was a distinct decline of EB extravasation in the combined treatment groups, which was not observed in single treatment groups. Most importantly, the combined usage of osthole and stem cell transplantation provide a better treatment for the traumatic brain injury caused by neuroendoscopy. The collective evidence indicates osthole combined with neural stem cell transplantation is superior than either method alone for the treatment of traumatic brain injury caused by neuroendoscopy.

Single cell sequencing reveals heterogeneity within ovarian cancer epithelium and cancer associated stromal cells.

PubMed

Winterhoff, Boris J; Maile, Makayla; Mitra, Amit Kumar; Sebe, Attila; Bazzaro, Martina; Geller, Melissa A; Abrahante, Juan E; Klein, Molly; Hellweg, Raffaele; Mullany, Sally A; Beckman, Kenneth; Daniel, Jerry; Starr, Timothy K

2017-03-01

The purpose of this study was to determine the level of heterogeneity in high grade serous ovarian cancer (HGSOC) by analyzing RNA expression in single epithelial and cancer associated stromal cells. In addition, we explored the possibility of identifying subgroups based on pathway activation and pre-defined signatures from cancer stem cells and chemo-resistant cells. A fresh, HGSOC tumor specimen derived from ovary was enzymatically digested and depleted of immune infiltrating cells. RNA sequencing was performed on 92 single cells and 66 of these single cell datasets passed quality control checks. Sequences were analyzed using multiple bioinformatics tools, including clustering, principle components analysis, and geneset enrichment analysis to identify subgroups and activated pathways. Immunohistochemistry for ovarian cancer, stem cell and stromal markers was performed on adjacent tumor sections. Analysis of the gene expression patterns identified two major subsets of cells characterized by epithelial and stromal gene expression patterns. The epithelial group was characterized by proliferative genes including genes associated with oxidative phosphorylation and MYC activity, while the stromal group was characterized by increased expression of extracellular matrix (ECM) genes and genes associated with epithelial-to-mesenchymal transition (EMT). Neither group expressed a signature correlating with published chemo-resistant gene signatures, but many cells, predominantly in the stromal subgroup, expressed markers associated with cancer stem cells. Single cell sequencing provides a means of identifying subpopulations of cancer cells within a single patient. Single cell sequence analysis may prove to be critical for understanding the etiology, progression and drug resistance in ovarian cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

Characterizing human stem cell-derived sensory neurons at the single-cell level reveals their ion channel expression and utility in pain research.

PubMed

Young, Gareth T; Gutteridge, Alex; Fox, Heather DE; Wilbrey, Anna L; Cao, Lishuang; Cho, Lily T; Brown, Adam R; Benn, Caroline L; Kammonen, Laura R; Friedman, Julia H; Bictash, Magda; Whiting, Paul; Bilsland, James G; Stevens, Edward B

2014-08-01

The generation of human sensory neurons by directed differentiation of pluripotent stem cells opens new opportunities for investigating the biology of pain. The inability to generate this cell type has meant that up until now their study has been reliant on the use of rodent models. Here, we use a combination of population and single-cell techniques to perform a detailed molecular, electrophysiological, and pharmacological phenotyping of sensory neurons derived from human embryonic stem cells. We describe the evolution of cell populations over 6 weeks of directed differentiation; a process that results in the generation of a largely homogeneous population of neurons that are both molecularly and functionally comparable to human sensory neurons derived from mature dorsal root ganglia. This work opens the prospect of using pluripotent stem-cell-derived sensory neurons to study human neuronal physiology and as in vitro models for drug discovery in pain and sensory disorders.

Identifying niche-mediated regulatory factors of stem cell phenotypic state: a systems biology approach.

PubMed

Ravichandran, Srikanth; Del Sol, Antonio

2017-02-01

Understanding how the cellular niche controls the stem cell phenotype is often hampered due to the complexity of variegated niche composition, its dynamics, and nonlinear stem cell-niche interactions. Here, we propose a systems biology view that considers stem cell-niche interactions as a many-body problem amenable to simplification by the concept of mean field approximation. This enables approximation of the niche effect on stem cells as a constant field that induces sustained activation/inhibition of specific stem cell signaling pathways in all stem cells within heterogeneous populations exhibiting the same phenotype (niche determinants). This view offers a new basis for the development of single cell-based computational approaches for identifying niche determinants, which has potential applications in regenerative medicine and tissue engineering. © 2017 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

Single Lgr5- or Lgr6-expressing taste stem/progenitor cells generate taste bud cells ex vivo

PubMed Central

Ren, Wenwen; Lewandowski, Brian C.; Watson, Jaime; Aihara, Eitaro; Iwatsuki, Ken; Bachmanov, Alexander A.; Margolskee, Robert F.; Jiang, Peihua

2014-01-01

Leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) and its homologs (e.g., Lgr6) mark adult stem cells in multiple tissues. Recently, we and others have shown that Lgr5 marks adult taste stem/progenitor cells in posterior tongue. However, the regenerative potential of Lgr5-expressing (Lgr5+) cells and the identity of adult taste stem/progenitor cells that regenerate taste tissue in anterior tongue remain elusive. In the present work, we describe a culture system in which single isolated Lgr5+ or Lgr6+ cells from taste tissue can generate continuously expanding 3D structures (“organoids”). Many cells within these taste organoids were cycling and positive for proliferative cell markers, cytokeratin K5 and Sox2, and incorporated 5-bromo-2’-deoxyuridine. Importantly, mature taste receptor cells that express gustducin, carbonic anhydrase 4, taste receptor type 1 member 3, nucleoside triphosphate diphosphohydrolase-2, or cytokeratin K8 were present in the taste organoids. Using calcium imaging assays, we found that cells grown out from taste organoids derived from isolated Lgr5+ cells were functional and responded to tastants in a dose-dependent manner. Genetic lineage tracing showed that Lgr6+ cells gave rise to taste bud cells in taste papillae in both anterior and posterior tongue. RT-PCR data demonstrated that Lgr5 and Lgr6 may mark the same subset of taste stem/progenitor cells both anteriorly and posteriorly. Together, our data demonstrate that functional taste cells can be generated ex vivo from single Lgr5+ or Lgr6+ cells, validating the use of this model for the study of taste cell generation. PMID:25368147

Single Lgr5- or Lgr6-expressing taste stem/progenitor cells generate taste bud cells ex vivo.

PubMed

Ren, Wenwen; Lewandowski, Brian C; Watson, Jaime; Aihara, Eitaro; Iwatsuki, Ken; Bachmanov, Alexander A; Margolskee, Robert F; Jiang, Peihua

2014-11-18

Leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) and its homologs (e.g., Lgr6) mark adult stem cells in multiple tissues. Recently, we and others have shown that Lgr5 marks adult taste stem/progenitor cells in posterior tongue. However, the regenerative potential of Lgr5-expressing (Lgr5(+)) cells and the identity of adult taste stem/progenitor cells that regenerate taste tissue in anterior tongue remain elusive. In the present work, we describe a culture system in which single isolated Lgr5(+) or Lgr6(+) cells from taste tissue can generate continuously expanding 3D structures ("organoids"). Many cells within these taste organoids were cycling and positive for proliferative cell markers, cytokeratin K5 and Sox2, and incorporated 5-bromo-2'-deoxyuridine. Importantly, mature taste receptor cells that express gustducin, carbonic anhydrase 4, taste receptor type 1 member 3, nucleoside triphosphate diphosphohydrolase-2, or cytokeratin K8 were present in the taste organoids. Using calcium imaging assays, we found that cells grown out from taste organoids derived from isolated Lgr5(+) cells were functional and responded to tastants in a dose-dependent manner. Genetic lineage tracing showed that Lgr6(+) cells gave rise to taste bud cells in taste papillae in both anterior and posterior tongue. RT-PCR data demonstrated that Lgr5 and Lgr6 may mark the same subset of taste stem/progenitor cells both anteriorly and posteriorly. Together, our data demonstrate that functional taste cells can be generated ex vivo from single Lgr5(+) or Lgr6(+) cells, validating the use of this model for the study of taste cell generation.

Independent and high-level dual-gene expression in adult stem-progenitor cells from a single lentiviral vector.

PubMed

Tian, J; Andreadis, S T

2009-07-01

Expression of multiple genes from the same target cell is required in several technological and therapeutic applications such as quantitative measurements of promoter activity or in vivo tracking of stem cells. In spite of such need, reaching independent and high-level dual-gene expression cannot be reliably accomplished by current gene transfer vehicles. To address this issue, we designed a lentiviral vector carrying two transcriptional units separated by polyadenylation, terminator and insulator sequences. With this design, the expression level of both genes was as high as that yielded from lentiviral vectors containing only a single transcriptional unit. Similar results were observed with several promoters and cell types including epidermal keratinocytes, bone marrow mesenchymal stem cells and hair follicle stem cells. Notably, we demonstrated quantitative dynamic monitoring of gene expression in primary cells with no need for selection protocols suggesting that this optimized lentivirus may be useful in high-throughput gene expression profiling studies.

Differentiation-Dependent Energy Production and Metabolite Utilization: A Comparative Study on Neural Stem Cells, Neurons, and Astrocytes

PubMed Central

Jády, Attila Gy.; Nagy, Ádám M.; Kőhidi, Tímea; Ferenczi, Szilamér; Tretter, László

2016-01-01

While it is evident that the metabolic machinery of stem cells should be fairly different from that of differentiated neurons, the basic energy production pathways in neural stem cells (NSCs) or in neurons are far from clear. Using the model of in vitro neuron production by NE-4C NSCs, this study focused on the metabolic changes taking place during the in vitro neuronal differentiation. O2 consumption, H+ production, and metabolic responses to single metabolites were measured in cultures of NSCs and in their neuronal derivatives, as well as in primary neuronal and astroglial cultures. In metabolite-free solutions, NSCs consumed little O2 and displayed a higher level of mitochondrial proton leak than neurons. In stem cells, glycolysis was the main source of energy for the survival of a 2.5-h period of metabolite deprivation. In contrast, stem cell-derived or primary neurons sustained a high-level oxidative phosphorylation during metabolite deprivation, indicating the consumption of own cellular material for energy production. The stem cells increased O2 consumption and mitochondrial ATP production in response to single metabolites (with the exception of glucose), showing rapid adaptation of the metabolic machinery to the available resources. In contrast, single metabolites did not increase the O2 consumption of neurons or astrocytes. In “starving” neurons, neither lactate nor pyruvate was utilized for mitochondrial ATP production. Gene expression studies also suggested that aerobic glycolysis and rapid metabolic adaptation characterize the NE-4C NSCs, while autophagy and alternative glucose utilization play important roles in the metabolism of stem cell-derived neurons. PMID:27116891

Droplet barcoding for single cell transcriptomics applied to embryonic stem cells

PubMed Central

Klein, Allon M; Mazutis, Linas; Akartuna, Ilke; Tallapragada, Naren; Veres, Adrian; Li, Victor; Peshkin, Leonid; Weitz, David A; Kirschner, Marc W

2015-01-01

Summary It has long been the dream of biologists to map gene expression at the single cell level. With such data one might track heterogeneous cell sub-populations, and infer regulatory relationships between genes and pathways. Recently, RNA sequencing has achieved single cell resolution. What is limiting is an effective way to routinely isolate and process large numbers of individual cells for quantitative in-depth sequencing. We have developed a high-throughput droplet-microfluidic approach for barcoding the RNA from thousands of individual cells for subsequent analysis by next-generation sequencing. The method shows a surprisingly low noise profile and is readily adaptable to other sequencing-based assays. We analyzed mouse embryonic stem cells, revealing in detail the population structure and the heterogeneous onset of differentiation after LIF withdrawal. The reproducibility of these high-throughput single cell data allowed us to deconstruct cell populations and infer gene expression relationships. PMID:26000487

Imaging Stem Cells Implanted in Infarcted Myocardium

PubMed Central

Zhou, Rong; Acton, Paul D.; Ferrari, Victor A.

2008-01-01

Stem cell–based cellular cardiomyoplasty represents a promising therapy for myocardial infarction. Noninvasive imaging techniques would allow the evaluation of survival, migration, and differentiation status of implanted stem cells in the same subject over time. This review describes methods for cell visualization using several corresponding noninvasive imaging modalities, including magnetic resonance imaging, positron emission tomography, single-photon emission computed tomography, and bioluminescent imaging. Reporter-based cell visualization is compared with direct cell labeling for short- and long-term cell tracking. PMID:17112999

Single-cell analysis of the transcriptome and its application in the characterization of stem cells and early embryos.

PubMed

Liu, Na; Liu, Lin; Pan, Xinghua

2014-07-01

Cellular heterogeneity within a cell population is a common phenomenon in multicellular organisms, tissues, cultured cells, and even FACS-sorted subpopulations. Important information may be masked if the cells are studied as a mass. Transcriptome profiling is a parameter that has been intensively studied, and relatively easier to address than protein composition. To understand the basis and importance of heterogeneity and stochastic aspects of the cell function and its mechanisms, it is essential to examine transcriptomes of a panel of single cells. High-throughput technologies, starting from microarrays and now RNA-seq, provide a full view of the expression of transcriptomes but are limited by the amount of RNA for analysis. Recently, several new approaches for amplification and sequencing the transcriptome of single cells or a limited low number of cells have been developed and applied. In this review, we summarize these major strategies, such as PCR-based methods, IVT-based methods, phi29-DNA polymerase-based methods, and several other methods, including their principles, characteristics, advantages, and limitations, with representative applications in cancer stem cells, early development, and embryonic stem cells. The prospects for development of future technology and application of transcriptome analysis in a single cell are also discussed.

Evaluation of a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay (Keystone Sym)

EPA Science Inventory

Our goal is to establish an in vitro model system to evaluate chemical effects using a single stem cell culture technique that would improve throughput and provide quantitative markers of differentiation and cell number. To this end, we have used an adherent cell differentiation ...

Lung stem cell differentiation in mice directed by endothelial cells via a BMP4-NFATc1-Thrombospondin-1 axis

PubMed Central

Lee, Joo-Hyeon; Bhang, Dong Ha; Beede, Alexander; Huang, Tian Lian; Stripp, Barry R.; Bloch, Kenneth D.; Wagers, Amy J.; Tseng, Yu-Hua; Ryeom, Sandra; Kim, Carla F.

2014-01-01

SUMMARY Lung stem cells are instructed to produce lineage-specific progeny through unknown factors in their microenvironment. We used clonal three-dimensional (3D) co-cultures of endothelial cells and distal lung stem cells, bronchioalveolar stem cells (BASCs), to probe the instructive mechanisms. Single BASCs had bronchiolar and alveolar differentiation potential in lung endothelial cell co-cultures. Gain and loss of function experiments showed BMP4-Bmpr1a signaling triggers calcineurin/NFATc1-dependent expression of Thrombospondin-1 (Tsp1) in lung endothelial cells to drive alveolar lineage-specific BASC differentiation. Tsp1-null mice exhibited defective alveolar injury repair, confirming a crucial role for the BMP4-NFATc1-TSP1 axis in lung epithelial differentiation and regeneration in vivo. Discovery of this pathway points to methods to direct the derivation of specific lung epithelial lineages from multipotent cells. These findings elucidate a pathway that may be a critical target in lung diseases and provide new tools to understand the mechanisms of respiratory diseases at the single cell level. PMID:24485453

Optimality in the Development of Intestinal Crypts

NASA Astrophysics Data System (ADS)

van Oudenaarden, Alexander

2012-02-01

Intestinal crypts in mammals are comprised of long-lived stem cells and shorter-lived progenies, maintained under tight proportions during adult life. Here we ask what are the design principles that govern the dynamics of these proportions during crypt morphogenesis. We use optimal control theory to show that a stem cell proliferation strategy known as a `bang-bang' control minimizes the time to obtain a mature crypt. This strategy consists of a surge of symmetric stem cell divisions, establishing the entire stem cell pool first, followed by a sharp transition to strictly asymmetric stem cell divisions, producing non-stem cells with a delay. We validate these predictions using lineage tracing and single molecule fluorescent in-situ hybridization of intestinal crypts in newborn mice and find that small crypts are entirely composed of Lgr5 stem cells, which become a minority as crypts further grow. Our approach can be used to uncover similar design principles in other developmental systems.

Cryo-imaging of fluorescently labeled single cells in a mouse

NASA Astrophysics Data System (ADS)

Steyer, Grant J.; Roy, Debashish; Salvado, Olivier; Stone, Meredith E.; Wilson, David L.

2009-02-01

We developed a cryo-imaging system to provide single-cell detection of fluorescently labeled cells in mouse, with particular applicability to stem cells and metastatic cancer. The Case cryoimaging system consists of a fluorescence microscope, robotic imaging positioner, customized cryostat, PC-based control system, and visualization/analysis software. The system alternates between sectioning (10-40 μm) and imaging, collecting color brightfield and fluorescent blockface image volumes >60GB. In mouse experiments, we imaged quantum-dot labeled stem cells, GFP-labeled cancer and stem cells, and cell-size fluorescent microspheres. To remove subsurface fluorescence, we used a simplified model of light-tissue interaction whereby the next image was scaled, blurred, and subtracted from the current image. We estimated scaling and blurring parameters by minimizing entropy of subtracted images. Tissue specific attenuation parameters were found [uT : heart (267 +/- 47.6 μm), liver (218 +/- 27.1 μm), brain (161 +/- 27.4 μm)] to be within the range of estimates in the literature. "Next image" processing removed subsurface fluorescence equally well across multiple tissues (brain, kidney, liver, adipose tissue, etc.), and analysis of 200 microsphere images in the brain gave 97+/-2% reduction of subsurface fluorescence. Fluorescent signals were determined to arise from single cells based upon geometric and integrated intensity measurements. Next image processing greatly improved axial resolution, enabled high quality 3D volume renderings, and improved enumeration of single cells with connected component analysis by up to 24%. Analysis of image volumes identified metastatic cancer sites, found homing of stem cells to injury sites, and showed microsphere distribution correlated with blood flow patterns. We developed and evaluated cryo-imaging to provide single-cell detection of fluorescently labeled cells in mouse. Our cryo-imaging system provides extreme (>60GB), micron-scale, fluorescence, and bright field image data. Here we describe our image preprocessing, analysis, and visualization techniques. Processing improves axial resolution, reduces subsurface fluorescence by 97%, and enables single cell detection and counting. High quality 3D volume renderings enable us to evaluate cell distribution patterns. Applications include the myriad of biomedical experiments using fluorescent reporter gene and exogenous fluorophore labeling of cells in applications such as stem cell regenerative medicine, cancer, tissue engineering, etc.

Development of bioengineering system for stem cell proliferation

NASA Astrophysics Data System (ADS)

Park, H. S.; Shah, R.; Shah, C.

2016-08-01

From last decades, intensive research in the field of stem cells proliferation had been promoted due to the unique property of stem cells to self-renew themselves into multiples and has potential to replicate into an organ or tissues and so it's highly demanding though challenging. Bioreactor, a mechanical device, works as a womb for stem cell proliferation by providing nutritious environment for the proper growth of stem cells. Various factors affecting stem cells growth are the bioreactor mechanism, feeding of continuous nutrients, healthy environment, etc., but it always remains a challenge for controlling biological parameters. The present paper unveils the design of mechanical device commonly known as bioreactor in tissues engineering and biotech field, use for proliferation of stem cells and imparts the proper growing condition for stem cells. This high functional bioreactor provides automation mixing of cell culture and stem cells. This design operates in conjunction with mechanism of reciprocating motion. Compare to commercial bioreactors, this proposed design is more convenient, easy to operate and less maintenance is required as bioreactor culture bag is made of polyethylene which is single use purpose. Development of this bioengineering system will be beneficial for better growth and expansion of stem cell

DHAP plus filgrastim as an effective peripheral stem cell mobilization regimen for autologous stem-cell transplantation in patients with relapsed/refractory lymphoma: A single center experience.

PubMed

Berber, Ilhami; Erkurt, Mehmet Ali; Kuku, Irfan; Kaya, Emin; Bag, Harika Gozukara; Nizam, Ilknur; Koroglu, Mustafa; Ozgul, Mustafa

2016-02-01

This study aimed to evaluate the efficiency of DHAP regimen plus filgrastim for mobilization of stem cells in patients with recurrent and/or refractory lymphoma. Thirty-four patients who took DHAP as salvage therapy prior to autologous stem cell transplantation were included. After chemotherapies, 2 cycles of DHAP plus filgrastim were administered to the patients. Stem cells from 32 patients (94%) were collected on median 11th day (8-12), and the median collected CD34(+) cell dose was 9.7 × 10(6)/kg (range 3.8-41.6). DHAP plus filgrastim was found to be an effective chemotherapy regimen in mobilizing CD34(+) stem cells into the peripheral. Copyright © 2016 Elsevier Ltd. All rights reserved.

Characterizing Human Stem Cell–derived Sensory Neurons at the Single-cell Level Reveals Their Ion Channel Expression and Utility in Pain Research

PubMed Central

Young, Gareth T; Gutteridge, Alex; Fox, Heather DE; Wilbrey, Anna L; Cao, Lishuang; Cho, Lily T; Brown, Adam R; Benn, Caroline L; Kammonen, Laura R; Friedman, Julia H; Bictash, Magda; Whiting, Paul; Bilsland, James G; Stevens, Edward B

2014-01-01

The generation of human sensory neurons by directed differentiation of pluripotent stem cells opens new opportunities for investigating the biology of pain. The inability to generate this cell type has meant that up until now their study has been reliant on the use of rodent models. Here, we use a combination of population and single-cell techniques to perform a detailed molecular, electrophysiological, and pharmacological phenotyping of sensory neurons derived from human embryonic stem cells. We describe the evolution of cell populations over 6 weeks of directed differentiation; a process that results in the generation of a largely homogeneous population of neurons that are both molecularly and functionally comparable to human sensory neurons derived from mature dorsal root ganglia. This work opens the prospect of using pluripotent stem-cell–derived sensory neurons to study human neuronal physiology and as in vitro models for drug discovery in pain and sensory disorders. PMID:24832007

Single Cell-Based Vector Tracing in Patients with ADA-SCID Treated with Stem Cell Gene Therapy.

PubMed

Igarashi, Yuka; Uchiyama, Toru; Minegishi, Tomoko; Takahashi, Sirirat; Watanabe, Nobuyuki; Kawai, Toshinao; Yamada, Masafumi; Ariga, Tadashi; Onodera, Masafumi

2017-09-15

Clinical improvement in stem cell gene therapy (SCGT) for primary immunodeficiencies depends on the engraftment levels of genetically corrected cells, and tracing the transgene in each hematopoietic lineage is therefore extremely important in evaluating the efficacy of SCGT. We established a single cell-based droplet digital PCR (sc-ddPCR) method consisting of the encapsulation of a single cell into each droplet, followed by emulsion PCR with primers and probes specific for the transgene. A fluorescent signal in a droplet indicates the presence of a single cell carrying the target gene in its genome, and this system can clearly determine the ratio of transgene-positive cells in the entire population at the genomic level. Using sc-ddPCR, we analyzed the engraftment of vector-transduced cells in two patients with severe combined immunodeficiency (SCID) who were treated with SCGT. Sufficient engraftment of the transduced cells was limited to the T cell lineage in peripheral blood (PB), and a small percentage of CD34 + cells exhibited vector integration in bone marrow, indicating that the transgene-positive cells in PB might have differentiated from a small population of stem cells or lineage-restricted precursor cells. sc-ddPCR is a simplified and powerful tool for the detailed assessment of transgene-positive cell distribution in patients treated with SCGT.

Functional characterization of human pluripotent stem cell-derived arterial endothelial cells.

PubMed

Zhang, Jue; Chu, Li-Fang; Hou, Zhonggang; Schwartz, Michael P; Hacker, Timothy; Vickerman, Vernella; Swanson, Scott; Leng, Ning; Nguyen, Bao Kim; Elwell, Angela; Bolin, Jennifer; Brown, Matthew E; Stewart, Ron; Burlingham, William J; Murphy, William L; Thomson, James A

2017-07-25

Here, we report the derivation of arterial endothelial cells from human pluripotent stem cells that exhibit arterial-specific functions in vitro and in vivo. We combine single-cell RNA sequencing of embryonic mouse endothelial cells with an EFNB2-tdTomato/EPHB4-EGFP dual reporter human embryonic stem cell line to identify factors that regulate arterial endothelial cell specification. The resulting xeno-free protocol produces cells with gene expression profiles, oxygen consumption rates, nitric oxide production levels, shear stress responses, and TNFα-induced leukocyte adhesion rates characteristic of arterial endothelial cells. Arterial endothelial cells were robustly generated from multiple human embryonic and induced pluripotent stem cell lines and have potential applications for both disease modeling and regenerative medicine.

Stem cell-derived models to improve mechanistic understanding and prediction of human drug-induced liver injury.

PubMed

Goldring, Christopher; Antoine, Daniel J; Bonner, Frank; Crozier, Jonathan; Denning, Chris; Fontana, Robert J; Hanley, Neil A; Hay, David C; Ingelman-Sundberg, Magnus; Juhila, Satu; Kitteringham, Neil; Silva-Lima, Beatriz; Norris, Alan; Pridgeon, Chris; Ross, James A; Young, Rowena Sison; Tagle, Danilo; Tornesi, Belen; van de Water, Bob; Weaver, Richard J; Zhang, Fang; Park, B Kevin

2017-02-01

Current preclinical drug testing does not predict some forms of adverse drug reactions in humans. Efforts at improving predictability of drug-induced tissue injury in humans include using stem cell technology to generate human cells for screening for adverse effects of drugs in humans. The advent of induced pluripotent stem cells means that it may ultimately be possible to develop personalized toxicology to determine interindividual susceptibility to adverse drug reactions. However, the complexity of idiosyncratic drug-induced liver injury means that no current single-cell model, whether of primary liver tissue origin, from liver cell lines, or derived from stem cells, adequately emulates what is believed to occur during human drug-induced liver injury. Nevertheless, a single-cell model of a human hepatocyte which emulates key features of a hepatocyte is likely to be valuable in assessing potential chemical risk; furthermore, understanding how to generate a relevant hepatocyte will also be critical to efforts to build complex multicellular models of the liver. Currently, hepatocyte-like cells differentiated from stem cells still fall short of recapitulating the full mature hepatocellular phenotype. Therefore, we convened a number of experts from the areas of preclinical and clinical hepatotoxicity and safety assessment, from industry, academia, and regulatory bodies, to specifically explore the application of stem cells in hepatotoxicity safety assessment and to make recommendations for the way forward. In this short review, we particularly discuss the importance of benchmarking stem cell-derived hepatocyte-like cells to their terminally differentiated human counterparts using defined phenotyping, to make sure the cells are relevant and comparable between labs, and outline why this process is essential before the cells are introduced into chemical safety assessment. (Hepatology 2017;65:710-721). © 2016 by the American Association for the Study of Liver Diseases.

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

Stem Cell Extracellular Vesicles: Extended Messages of Regeneration

PubMed Central

Riazifar, Milad; Pone, Egest J.; Lötvall, Jan; Zhao, Weian

2017-01-01

Stem cells are critical to maintaining steady-state organ homeostasis and regenerating injured tissues. Recent intriguing reports implicate extracellular vesicles (EVs) as carriers for the distribution of morphogens and growth and differentiation factors from tissue parenchymal cells to stem cells, and conversely, stem cell–derived EVs carrying certain proteins and nucleic acids can support healing of injured tissues. We describe approaches to make use of engineered EVs as technology platforms in therapeutics and diagnostics in the context of stem cells. For some regenerative therapies, natural and engineered EVs from stem cells may be superior to single-molecule drugs, biologics, whole cells, and synthetic liposome or nanoparticle formulations because of the ease of bioengineering with multiple factors while retaining superior biocompatibility and biostability and posing fewer risks for abnormal differentiation or neoplastic transformation. Finally, we provide an overview of current challenges and future directions of EVs as potential therapeutic alternatives to cells for clinical applications. PMID:27814025

Replacement of Lost Lgr5-Positive Stem Cells through Plasticity of Their Enterocyte-Lineage Daughters.

PubMed

Tetteh, Paul W; Basak, Onur; Farin, Henner F; Wiebrands, Kay; Kretzschmar, Kai; Begthel, Harry; van den Born, Maaike; Korving, Jeroen; de Sauvage, Frederic; van Es, Johan H; van Oudenaarden, Alexander; Clevers, Hans

2016-02-04

Intestinal crypts display robust regeneration upon injury. The relatively rare secretory precursors can replace lost stem cells, but it is unknown if the abundant enterocyte progenitors that express the Alkaline phosphate intestinal (Alpi) gene also have this capacity. We created an Alpi-IRES-CreERT2 (Alpi(CreER)) knockin allele for lineage tracing. Marked clones consist entirely of enterocytes and are all lost from villus tips within days. Genetic fate-mapping of Alpi(+) cells before or during targeted ablation of Lgr5-expressing stem cells generated numerous long-lived crypt-villus "ribbons," indicative of dedifferentiation of enterocyte precursors into Lgr5(+) stems. By single-cell analysis of dedifferentiating enterocytes, we observed the generation of Paneth-like cells and proliferative stem cells. We conclude that the highly proliferative, short-lived enterocyte precursors serve as a large reservoir of potential stem cells during crypt regeneration. Copyright © 2016 Elsevier Inc. All rights reserved.

Advancing haematopoietic stem and progenitor cell biology through single-cell profiling.

PubMed

Hamey, Fiona K; Nestorowa, Sonia; Wilson, Nicola K; Göttgens, Berthold

2016-11-01

Haematopoietic stem and progenitor cells (HSPCs) sit at the top of the haematopoietic hierarchy, and their fate choices need to be carefully controlled to ensure balanced production of all mature blood cell types. As cell fate decisions are made at the level of the individual cells, recent technological advances in measuring gene and protein expression in increasingly large numbers of single cells have been rapidly adopted to study both normal and pathological HSPC function. In this review we emphasise the importance of combining the correct computational models with single-cell experimental techniques, and illustrate how such integrated approaches have been used to resolve heterogeneities in populations, reconstruct lineage differentiation, identify regulatory relationships and link molecular profiling to cellular function. © 2016 Federation of European Biochemical Societies.

CARDIAC-LIKE OSCILLATION IN LIVER STEM CELLS INDUCE THEIR ACQUISITION OF CARDIAC PHENOTYPE

EPA Science Inventory

We examined in a cardiac microenvironment the plasticity of a liver stem cell line (WB F344) generated from a cloned, single, non-parenchymal epithelial cell from a normal adult male rat. Our previous studies suggested that WB F344 cells acquire a cardiac phenotype in the absenc...

Production of genome-edited pluripotent stem cells and mice by CRISPR/Cas.

PubMed

Horii, Takuro; Hatada, Izuho

2016-01-01

Clustered regularly at interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) nucleases, so-called CRISPR/Cas, was recently developed as an epoch-making genome engineering technology. This system only requires Cas9 nuclease and single-guide RNA complementary to a target locus. CRISPR/Cas enables the generation of knockout cells and animals in a single step. This system can also be used to generate multiple mutations and knockin in a single step, which is not possible using other methods. In this review, we provide an overview of genome editing by CRISPR/Cas in pluripotent stem cells and mice.

Metabolism and the Control of Cell Fate Decisions and Stem Cell Renewal

PubMed Central

Ito, Kyoko; Ito, Keisuke

2016-01-01

Although the stem cells of various tissues remain in the quiescent state to maintain their undifferentiated state, they also undergo cell divisions as required, and if necessary, even a single stem cell is able to provide for lifelong tissue homeostasis. Stem cell populations are precisely controlled by the balance between their symmetric and asymmetric divisions, with their division patterns determined by whether the daughter cells involved retain their self-renewal capacities. Recent studies have reported that metabolic pathways and the distribution of mitochondria are regulators of the division balance of stem cells and that metabolic defects can shift division balance toward symmetric commitment, which leads to stem cell exhaustion. It has also been observed that in asymmetric division, old mitochondria, which are central metabolic organelles, are segregated to the daughter cell fated to cell differentiation, whereas in symmetric division, young and old mitochondria are equally distributed between both daughter cells. Thus, metabolism and mitochondrial biology play important roles in stem cell fate decisions. As these decisions directly affect tissue homeostasis, understanding their regulatory mechanisms in the context of cellular metabolism is critical. PMID:27482603

Metabolism and the Control of Cell Fate Decisions and Stem Cell Renewal.

PubMed

Ito, Kyoko; Ito, Keisuke

2016-10-06

Although the stem cells of various tissues remain in the quiescent state to maintain their undifferentiated state, they also undergo cell divisions as required, and if necessary, even a single stem cell is able to provide for lifelong tissue homeostasis. Stem cell populations are precisely controlled by the balance between their symmetric and asymmetric divisions, with their division patterns determined by whether the daughter cells involved retain their self-renewal capacities. Recent studies have reported that metabolic pathways and the distribution of mitochondria are regulators of the division balance of stem cells and that metabolic defects can shift division balance toward symmetric commitment, which leads to stem cell exhaustion. It has also been observed that in asymmetric division, old mitochondria, which are central metabolic organelles, are segregated to the daughter cell fated to cell differentiation, whereas in symmetric division, young and old mitochondria are equally distributed between both daughter cells. Thus, metabolism and mitochondrial biology play important roles in stem cell fate decisions. As these decisions directly affect tissue homeostasis, understanding their regulatory mechanisms in the context of cellular metabolism is critical.

[Isolation and identification of dog periodontal ligament stem cells].

PubMed

Chang, Xiu-Mei; Liu, Hong-Wei; Jin, Yan; Liu, Yuan; He, Hui-Xia

2009-02-01

To isolate, culture and identify a dog periodontal ligament stem cells (PDLSC) line in vitro. The adult dog periodontal ligament cells were isolated by limited dilution of culture cell for single cell clone. Cells originated from one of these clones were assessed through colony-forming efficiency and immunocytochemistry assay and alkaline phosphatase stain was used to identify the source of adult dog periodontal stem cells, at the same time, PDLSC were induced with mineralizatin solution and was found to have long protrude like an osteoblast. Differentiation of PDLSC were assessed. Mineralized potential was studied by Von-Kossa staining. The dog PDLSC expressed STRO-1, which was the marker of mesenchymal stem cells. Also Vimentin, osteoblast-like marker alkaline phosphatase and Collagen-I expressed weakly. Cells were clonegenic, highly proliferative cells and capable of differentiating into osteoblasts/cementoblasts. The evidence suggests that the cultured cells were stem cells from adult dog periodontal ligament.

Complete disassociation of adult pancreas into viable single cells through cold trypsin-EDTA digestion*

PubMed Central

Li, Dan; Peng, Shi-yun; Zhang, Zhen-wu; Feng, Rui-cheng; Li, Lu; Liang, Jie; Tai, Sheng; Teng, Chun-bo

2013-01-01

The in vitro isolation and analysis of pancreatic stem/progenitor cells are necessary for understanding their properties and function; however, the preparation of high-quality single-cell suspensions from adult pancreas is prerequisite. In this study, we applied a cold trypsin-ethylenediaminetetraacetic acid (EDTA) digestion method to disassociate adult mouse pancreata into single cells. The yield of single cells and the viability of the harvested cells were much higher than those obtained via the two commonly used warm digestion methods. Flow cytometric analysis showed that the ratio of ductal or BCRP1-positive cells in cell suspensions prepared through cold digestion was consistent with that found in vivo. Cell culture tests showed that pancreatic epithelial cells prepared by cold digestion maintained proliferative capacity comparable to those derived from warm collagenase digestion. These results indicate that cold trypsin-EDTA digestion can effectively disassociate an adult mouse pancreas into viable single cells with minimal cell loss, and can be used for the isolation and analysis of pancreatic stem/progenitor cells. PMID:23825145

Soft fibrin gels promote selection and growth of tumorigenic cells

NASA Astrophysics Data System (ADS)

Liu, Jing; Tan, Youhua; Zhang, Huafeng; Zhang, Yi; Xu, Pingwei; Chen, Junwei; Poh, Yeh-Chuin; Tang, Ke; Wang, Ning; Huang, Bo

2012-08-01

The identification of stem-cell-like cancer cells through conventional methods that depend on stem cell markers is often unreliable. We developed a mechanical method for selecting tumorigenic cells by culturing single cancer cells in fibrin matrices of ~100 Pa in stiffness. When cultured within these gels, primary human cancer cells or single cancer cells from mouse or human cancer cell lines grew within a few days into individual round colonies that resembled embryonic stem cell colonies. Subcutaneous or intravenous injection of 10 or 100 fibrin-cultured cells in syngeneic or severe combined immunodeficiency mice led to the formation of solid tumours at the site of injection or at the distant lung organ much more efficiently than control cancer cells selected using conventional surface marker methods or cultured on conventional rigid dishes or on soft gels. Remarkably, as few as ten such cells were able to survive and form tumours in the lungs of wild-type non-syngeneic mice.

Advances in single-cell RNA sequencing and its applications in cancer research.

PubMed

Zhu, Sibo; Qing, Tao; Zheng, Yuanting; Jin, Li; Shi, Leming

2017-08-08

Unlike population-level approaches, single-cell RNA sequencing enables transcriptomic analysis of an individual cell. Through the combination of high-throughput sequencing and bioinformatic tools, single-cell RNA-seq can detect more than 10,000 transcripts in one cell to distinguish cell subsets and dynamic cellular changes. After several years' development, single-cell RNA-seq can now achieve massively parallel, full-length mRNA sequencing as well as in situ sequencing and even has potential for multi-omic detection. One appealing area of single-cell RNA-seq is cancer research, and it is regarded as a promising way to enhance prognosis and provide more precise target therapy by identifying druggable subclones. Indeed, progresses have been made regarding solid tumor analysis to reveal intratumoral heterogeneity, correlations between signaling pathways, stemness, drug resistance, and tumor architecture shaping the microenvironment. Furthermore, through investigation into circulating tumor cells, many genes have been shown to promote a propensity toward stemness and the epithelial-mesenchymal transition, to enhance anchoring and adhesion, and to be involved in mechanisms of anoikis resistance and drug resistance. This review focuses on advances and progresses of single-cell RNA-seq with regard to the following aspects: 1. Methodologies of single-cell RNA-seq 2. Single-cell isolation techniques 3. Single-cell RNA-seq in solid tumor research 4. Single-cell RNA-seq in circulating tumor cell research 5.

Advances in single-cell RNA sequencing and its applications in cancer research

PubMed Central

Zhu, Sibo; Qing, Tao; Zheng, Yuanting; Jin, Li; Shi, Leming

2017-01-01

Unlike population-level approaches, single-cell RNA sequencing enables transcriptomic analysis of an individual cell. Through the combination of high-throughput sequencing and bioinformatic tools, single-cell RNA-seq can detect more than 10,000 transcripts in one cell to distinguish cell subsets and dynamic cellular changes. After several years’ development, single-cell RNA-seq can now achieve massively parallel, full-length mRNA sequencing as well as in situ sequencing and even has potential for multi-omic detection. One appealing area of single-cell RNA-seq is cancer research, and it is regarded as a promising way to enhance prognosis and provide more precise target therapy by identifying druggable subclones. Indeed, progresses have been made regarding solid tumor analysis to reveal intratumoral heterogeneity, correlations between signaling pathways, stemness, drug resistance, and tumor architecture shaping the microenvironment. Furthermore, through investigation into circulating tumor cells, many genes have been shown to promote a propensity toward stemness and the epithelial-mesenchymal transition, to enhance anchoring and adhesion, and to be involved in mechanisms of anoikis resistance and drug resistance. This review focuses on advances and progresses of single-cell RNA-seq with regard to the following aspects: 1. Methodologies of single-cell RNA-seq 2. Single-cell isolation techniques 3. Single-cell RNA-seq in solid tumor research 4. Single-cell RNA-seq in circulating tumor cell research 5. Perspectives PMID:28881849

Generation of functional organs from stem cells.

PubMed

Liu, Yunying; Yang, Ru; He, Zuping; Gao, Wei-Qiang

2013-01-01

We are now well entering the exciting era of stem cells. Potential stem cell therapy holds great promise for the treatment of many diseases such as stroke, traumatic brain injury, Alzheimer's disease, Parkinson's disease, amyotrophic lateral-sclerosis, myocardial infarction, muscular dystrophy, diabetes, and etc. It is generally believed that transplantation of specific stem cells into the injured tissue to replace the lost cells is an effective way to repair the tissue. In fact, organ transplantation has been successfully practiced in clinics for liver or kidney failure. However, the severe shortage of donor organs has been a major obstacle for the expansion of organ transplantation programs. Toward that direction, generation of transplantable organs using stem cells is a desirable approach for organ replacement and would be of great interest for both basic and clinical scientists. Here we review recent progress in the field of organ generation using various methods including single adult tissue stem cells, a blastocyst complementation system, tissue decellularization/recellularization and a combination of stem cells and tissue engineering.

Optimizing cryopreservation of human spermatogonial stem cells: comparing the effectiveness of testicular tissue and single cell suspension cryopreservation

PubMed Central

Yango, Pamela; Altman, Eran; Smith, James F.; Klatsky, Peter C.; Tran, Nam D.

2015-01-01

Objective To determine whether optimal human spermatogonial stem cell (SSC) cryopreservation is best achieved with testicular tissue or single cell suspension cryopreservation. This study compares the effectiveness between these two approaches by using testicular SSEA-4+ cells, a known population containing SSCs. Design In vitro human testicular tissues. Setting Academic research unit. Patients Adult testicular tissues (n = 4) collected from subjects with normal spermatogenesis and normal fetal testicular tissues (n = 3). Intervention(s) Testicular tissue vs. single cell suspension cryopreservation. Main Outcome Measures Cell viability, total cell recovery per milligram of tissue, as well as, viable and SSEA-4+ cell recovery. Results Single cell suspension cryopreservation yielded higher recovery of SSEA-4+ cells enriched in adult SSCs whereas fetal SSEA-4+ cell recovery was similar between testicular tissue and single cell suspension cryopreservation. Conclusions Adult and fetal human SSEA-4+ populations exhibited differential sensitivity to cryopreservation based on whether they were cryopreserved in situ as testicular tissues or as single cells. Thus, optimal preservation of human SSCs depends on the patient age, type of samples cryopreserved, and end points of therapeutic applications. PMID:25241367

Photothermal optical coherence tomography for depth-resolved imaging of mesenchymal stem cells via single wall carbon nanotubes

NASA Astrophysics Data System (ADS)

Subhash, Hrebesh M.; Connolly, Emma; Murphy, Mary; Barron, Valerie; Leahy, Martin

2014-03-01

The progress in stem cell research over the past decade holds promise and potential to address many unmet clinical therapeutic needs. Tracking stem cell with modern imaging modalities are critically needed for optimizing stem cell therapy, which offers insight into various underlying biological processes such as cell migration, engraftment, homing, differentiation, and functions etc. In this study we report the feasibility of photothermal optical coherence tomography (PT-OCT) to image human mesenchymal stem cells (hMSCs) labeled with single-walled carbon nanotubes (SWNTs) for in vitro cell tracking in three dimensional scaffolds. PT-OCT is a functional extension of conventional OCT with extended capability of localized detection of absorbing targets from scattering background to provide depth-resolved molecular contrast imaging. A 91 kHz line rate, spectral domain PT-OCT system at 1310nm was developed to detect the photothermal signal generated by 800nm excitation laser. In general, MSCs do not have obvious optical absorption properties and cannot be directly visualized using PT-OCT imaging. However, the optical absorption properties of hMSCs can me modified by labeling with SWNTs. Using this approach, MSC were labeled with SWNT and the cell distribution imaged in a 3D polymer scaffold using PT-OCT.

Single Targeted Exon Mutation Creates a True Congenic Mouse for Competitive Hematopoietic Stem Cell Transplantation: The C57BL/6-CD45.1(STEM) Mouse.

PubMed

Mercier, Francois E; Sykes, David B; Scadden, David T

2016-06-14

Defining the molecular regulators of hematopoietic stem and progenitor cells (HSPCs) requires in vivo functional analyses. Competitive bone marrow transplants (BMTs) compare control and test HSPCs to demonstrate the functional role of a genetic change or chemical perturbation. Competitive BMT is enabled by antibodies that specifically recognize hematopoietic cells from congenic mouse strains due to variants of the cell surface protein CD45, designated CD45.1 and CD45.2. The current congenic competitor strain, B6.SJL-Ptprc(a) Pepc(b)/BoyJ (CD45.1), has a substantial inherent disadvantage in competition against the C57BL/6 (CD45.2) strain, confounding experimental interpretation. Despite backcrossing, the congenic interval over which the B6.SJL-Ptprc(a) Pepc(b)/BoyJ strain differs is almost 40 Mb encoding ∼300 genes. Here, we demonstrate that a single amino acid change determines the CD45.1 epitope. Further, we report on the single targeted exon mutant (STEM) mouse strain, CD45.1(STEM), which is functionally equivalent to CD45.2 cells in competitive BMT. This strain will permit the precise definition of functional roles for candidate genes using in vivo HSPC assays. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Induced Pluripotent Stem Cells: Generation, Characterization, and Differentiation--Methods and Protocols.

PubMed

Graversen, Veronica Kon; Chavala, Sai H

2016-01-01

Reprogramming fibroblasts into induced pluripotent stem cells (iPSC) remains a promising technique for cell replacement therapy. Diverse populations of somatic cells have been examined for their reprogramming potential. Recently, ocular ciliary body epithelial cells (CECs) have been reprogrammed with high reprogramming efficiency and single transcription factor reprogramming, making them an exciting candidate for cellular reprogramming strategies.

Hematopoietic stem cell origin of connective tissues.

PubMed

Ogawa, Makio; Larue, Amanda C; Watson, Patricia M; Watson, Dennis K

2010-07-01

Connective tissue consists of "connective tissue proper," which is further divided into loose and dense (fibrous) connective tissues and "specialized connective tissues." Specialized connective tissues consist of blood, adipose tissue, cartilage, and bone. In both loose and dense connective tissues, the principal cellular element is fibroblasts. It has been generally believed that all cellular elements of connective tissue, including fibroblasts, adipocytes, chondrocytes, and bone cells, are generated solely by mesenchymal stem cells. Recently, a number of studies, including those from our laboratory based on transplantation of single hematopoietic stem cells, strongly suggested a hematopoietic stem cell origin of these adult mesenchymal tissues. This review summarizes the experimental evidence for this new paradigm and discusses its translational implications. Copyright 2010 ISEH - Society for Hematology and Stem Cells. All rights reserved.

A single-cell and feeder-free culture system for monkey embryonic stem cells.

PubMed

Ono, Takashi; Suzuki, Yutaka; Kato, Yosuke; Fujita, Risako; Araki, Toshihiro; Yamashita, Tomoko; Kato, Hidemasa; Torii, Ryuzo; Sato, Naoya

2014-01-01

Primate pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), hold great potential for research and application in regenerative medicine and drug discovery. To maximize primate PSC potential, a practical system is required for generating desired functional cells and reproducible differentiation techniques. Much progress regarding their culture systems has been reported to date; however, better methods would still be required for their practical use, particularly in industrial and clinical fields. Here we report a new single-cell and feeder-free culture system for primate PSCs, the key feature of which is an originally formulated serum-free medium containing FGF and activin. In this culture system, cynomolgus monkey ESCs can be passaged many times by single-cell dissociation with traditional trypsin treatment and can be propagated with a high proliferation rate as a monolayer without any feeder cells; further, typical PSC properties and genomic stability can be retained. In addition, it has been demonstrated that monkey ESCs maintained in the culture system can be used for various experiments such as in vitro differentiation and gene manipulation. Thus, compared with the conventional culture system, monkey ESCs grown in the aforementioned culture system can serve as a cell source with the following practical advantages: simple, stable, and easy cell maintenance; gene manipulation; cryopreservation; and desired differentiation. We propose that this culture system can serve as a reliable platform to prepare primate PSCs useful for future research and application.

Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells.

PubMed

Klein, Allon M; Mazutis, Linas; Akartuna, Ilke; Tallapragada, Naren; Veres, Adrian; Li, Victor; Peshkin, Leonid; Weitz, David A; Kirschner, Marc W

2015-05-21

It has long been the dream of biologists to map gene expression at the single-cell level. With such data one might track heterogeneous cell sub-populations, and infer regulatory relationships between genes and pathways. Recently, RNA sequencing has achieved single-cell resolution. What is limiting is an effective way to routinely isolate and process large numbers of individual cells for quantitative in-depth sequencing. We have developed a high-throughput droplet-microfluidic approach for barcoding the RNA from thousands of individual cells for subsequent analysis by next-generation sequencing. The method shows a surprisingly low noise profile and is readily adaptable to other sequencing-based assays. We analyzed mouse embryonic stem cells, revealing in detail the population structure and the heterogeneous onset of differentiation after leukemia inhibitory factor (LIF) withdrawal. The reproducibility of these high-throughput single-cell data allowed us to deconstruct cell populations and infer gene expression relationships. VIDEO ABSTRACT. Copyright © 2015 Elsevier Inc. All rights reserved.

Computational Tools for Stem Cell Biology

PubMed Central

Bian, Qin; Cahan, Patrick

2016-01-01

For over half a century, the field of developmental biology has leveraged computation to explore mechanisms of developmental processes. More recently, computational approaches have been critical in the translation of high throughput data into knowledge of both developmental and stem cell biology. In the last several years, a new sub-discipline of computational stem cell biology has emerged that synthesizes the modeling of systems-level aspects of stem cells with high-throughput molecular data. In this review, we provide an overview of this new field and pay particular attention to the impact that single-cell transcriptomics is expected to have on our understanding of development and our ability to engineer cell fate. PMID:27318512

Computational Tools for Stem Cell Biology.

PubMed

Bian, Qin; Cahan, Patrick

2016-12-01

For over half a century, the field of developmental biology has leveraged computation to explore mechanisms of developmental processes. More recently, computational approaches have been critical in the translation of high throughput data into knowledge of both developmental and stem cell biology. In the past several years, a new subdiscipline of computational stem cell biology has emerged that synthesizes the modeling of systems-level aspects of stem cells with high-throughput molecular data. In this review, we provide an overview of this new field and pay particular attention to the impact that single cell transcriptomics is expected to have on our understanding of development and our ability to engineer cell fate. Copyright © 2016 Elsevier Ltd. All rights reserved.

Comparison of the transcriptpmes of long-tern label retaining-cells and C cells microdissected from mammary epithelium: an initial study to character potential stem/progenitor cells

USDA-ARS?s Scientific Manuscript database

Mammary stem cells (MaSC) account for the cell lineage of mammary epithelia and provide for mammary growth, development and tissue homeostasis. The presence of MaSC was clearly demonstrated by the generation of an entire mammary gland from a single cell implanted into epithelium-ablated mammary fat...

Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single-cell resolution using RNA sequencing.

PubMed

Tang, Qin; Iyer, Sowmya; Lobbardi, Riadh; Moore, John C; Chen, Huidong; Lareau, Caleb; Hebert, Christine; Shaw, McKenzie L; Neftel, Cyril; Suva, Mario L; Ceol, Craig J; Bernards, Andre; Aryee, Martin; Pinello, Luca; Drummond, Iain A; Langenau, David M

2017-10-02

Recent advances in single-cell, transcriptomic profiling have provided unprecedented access to investigate cell heterogeneity during tissue and organ development. In this study, we used massively parallel, single-cell RNA sequencing to define cell heterogeneity within the zebrafish kidney marrow, constructing a comprehensive molecular atlas of definitive hematopoiesis and functionally distinct renal cells found in adult zebrafish. Because our method analyzed blood and kidney cells in an unbiased manner, our approach was useful in characterizing immune-cell deficiencies within DNA-protein kinase catalytic subunit ( prkdc ), interleukin-2 receptor γ a ( il2rga ), and double-homozygous-mutant fish, identifying blood cell losses in T, B, and natural killer cells within specific genetic mutants. Our analysis also uncovered novel cell types, including two classes of natural killer immune cells, classically defined and erythroid-primed hematopoietic stem and progenitor cells, mucin-secreting kidney cells, and kidney stem/progenitor cells. In total, our work provides the first, comprehensive, single-cell, transcriptomic analysis of kidney and marrow cells in the adult zebrafish. © 2017 Tang et al.

Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single-cell resolution using RNA sequencing

PubMed Central

Iyer, Sowmya; Lobbardi, Riadh; Chen, Huidong; Hebert, Christine; Shaw, McKenzie L.; Neftel, Cyril; Suva, Mario L.; Bernards, Andre; Aryee, Martin; Drummond, Iain A.

2017-01-01

Recent advances in single-cell, transcriptomic profiling have provided unprecedented access to investigate cell heterogeneity during tissue and organ development. In this study, we used massively parallel, single-cell RNA sequencing to define cell heterogeneity within the zebrafish kidney marrow, constructing a comprehensive molecular atlas of definitive hematopoiesis and functionally distinct renal cells found in adult zebrafish. Because our method analyzed blood and kidney cells in an unbiased manner, our approach was useful in characterizing immune-cell deficiencies within DNA–protein kinase catalytic subunit (prkdc), interleukin-2 receptor γ a (il2rga), and double-homozygous–mutant fish, identifying blood cell losses in T, B, and natural killer cells within specific genetic mutants. Our analysis also uncovered novel cell types, including two classes of natural killer immune cells, classically defined and erythroid-primed hematopoietic stem and progenitor cells, mucin-secreting kidney cells, and kidney stem/progenitor cells. In total, our work provides the first, comprehensive, single-cell, transcriptomic analysis of kidney and marrow cells in the adult zebrafish. PMID:28878000

Dynamics of lineage commitment revealed by single-cell transcriptomics of differentiating embryonic stem cells.

PubMed

Semrau, Stefan; Goldmann, Johanna E; Soumillon, Magali; Mikkelsen, Tarjei S; Jaenisch, Rudolf; van Oudenaarden, Alexander

2017-10-23

Gene expression heterogeneity in the pluripotent state of mouse embryonic stem cells (mESCs) has been increasingly well-characterized. In contrast, exit from pluripotency and lineage commitment have not been studied systematically at the single-cell level. Here we measure the gene expression dynamics of retinoic acid driven mESC differentiation from pluripotency to lineage commitment, using an unbiased single-cell transcriptomics approach. We find that the exit from pluripotency marks the start of a lineage transition as well as a transient phase of increased susceptibility to lineage specifying signals. Our study reveals several transcriptional signatures of this phase, including a sharp increase of gene expression variability and sequential expression of two classes of transcriptional regulators. In summary, we provide a comprehensive analysis of the exit from pluripotency and lineage commitment at the single cell level, a potential stepping stone to improved lineage manipulation through timing of differentiation cues.

Crispr-mediated Gene Targeting of Human Induced Pluripotent Stem Cells.

PubMed

Byrne, Susan M; Church, George M

2015-01-01

CRISPR/Cas9 nuclease systems can create double-stranded DNA breaks at specific sequences to efficiently and precisely disrupt, excise, mutate, insert, or replace genes. However, human embryonic stem or induced pluripotent stem cells (iPSCs) are more difficult to transfect and less resilient to DNA damage than immortalized tumor cell lines. Here, we describe an optimized protocol for genome engineering of human iPSCs using a simple transient transfection of plasmids and/or single-stranded oligonucleotides. With this protocol, we achieve transfection efficiencies greater than 60%, with gene disruption efficiencies from 1-25% and gene insertion/replacement efficiencies from 0.5-10% without any further selection or enrichment steps. We also describe how to design and assess optimal sgRNA target sites and donor targeting vectors; cloning individual iPSC by single cell FACS sorting, and genotyping successfully edited cells.

s-SHIP expression identifies a subset of murine basal prostate cells as neonatal stem cells

PubMed Central

Brocqueville, Guillaume; Chmelar, Renee S.; Bauderlique-Le Roy, Hélène; Deruy, Emeric; Tian, Lu; Vessella, Robert L.; Greenberg, Norman M.; Bourette, Roland P.

2016-01-01

Isolation of prostate stem cells (PSCs) is crucial for understanding their biology during normal development and tumorigenesis. In this aim, we used a transgenic mouse model expressing GFP from the stem cell-specific s-SHIP promoter to mark putative stem cells during postnatal prostate development. Here we show that cells identified by GFP expression are present transiently during early prostate development and localize to the basal cell layer of the epithelium. These prostate GFP+ cells are a subpopulation of the Lin− CD24+ Sca-1+ CD49f+ cells and are capable of self-renewal together with enhanced growth potential in sphere-forming assay in vitro, a phenotype consistent with that of a PSC population. Transplantation assays of prostate GFP+ cells demonstrate reconstitution of prostate ducts containing both basal and luminal cells in renal grafts. Altogether, these results demonstrate that s-SHIP promoter expression is a new marker for neonatal basal prostate cells exhibiting stem cell properties that enables PSCs in situ identification and isolation via a single consistent parameter. Transcriptional profiling of these GFP+ neonatal stem cells showed an increased expression of several components of the Wnt signaling pathway. It also identified stem cell regulators with potential applications for further analyses of normal and cancer stem cells. PMID:27081082

In Vivo Imaging and Monitoring of Transplanted Stem Cells: Clinical Applications

PubMed Central

Rodriguez-Porcel, Martin

2010-01-01

Regenerative medicine using stem cells has appeared as a potential therapeutic alternative for coronary artery disease, and stem cell clinical studies are currently on their way. However, initial results of these studies have provided mixed information, in part because of the inability to correlate organ functional information with the presence/absence of transplanted stem cells. Recent advances in molecular biology and imaging have allowed the successful noninvasive monitoring of transplanted stem cells in the living subject. In this article, different imaging strategies (direct labeling, indirect labeling with reporter genes) to study the viability and biology of stem cells are discussed. In addition, the limitations of each approach and imaging modality (eg, single photon emission computed tomography, positron emission tomography, and MRI) and their requirements for clinical use are addressed. Use of these strategies will be critical as the different regenerative therapies are being tested for clinical use. PMID:20425184

Hydrogel microstructure live-cell array for multiplexed analyses of cancer stem cells, tumor heterogeneity and differential drug response at single-element resolution.

PubMed

Afrimzon, E; Botchkina, G; Zurgil, N; Shafran, Y; Sobolev, M; Moshkov, S; Ravid-Hermesh, O; Ojima, I; Deutsch, M

2016-03-21

Specific phenotypic subpopulations of cancer stem cells (CSCs) are responsible for tumor development, production of heterogeneous differentiated tumor mass, metastasis, and resistance to therapies. The development of therapeutic approaches based on targeting rare CSCs has been limited partially due to the lack of appropriate experimental models and measurement approaches. The current study presents new tools and methodologies based on a hydrogel microstructure array (HMA) for identification and multiplex analyses of CSCs. Low-melt agarose integrated with type I collagen, a major component of the extracellular matrix (ECM), was used to form a solid hydrogel array with natural non-adhesive characteristics and high optical quality. The array contained thousands of individual pyramidal shaped, nanoliter-volume micro-chambers (MCs), allowing concomitant generation and measurement of large populations of free-floating CSC spheroids from single cells, each in an individual micro-chamber (MC). The optical live cell platform, based on an imaging plate patterned with HMA, was validated using CSC-enriched prostate and colon cancer cell lines. The HMA methodology and quantitative image analysis at single-element resolution clearly demonstrates several levels of tumor cell heterogeneity, including morphological and phenotypic variability, differences in proliferation capacity and in drug response. Moreover, the system facilitates real-time examination of single stem cell (SC) fate, as well as drug-induced alteration in expression of stemness markers. The technology may be applicable in personalized cancer treatment, including multiplex ex vivo analysis of heterogeneous patient-derived tumor specimens, precise detection and characterization of potentially dangerous cell phenotypes, and for representative evaluation of drug sensitivity of CSCs and other types of tumor cells.

Autologous Pluripotent Stem Cell-Derived β-Like Cells for Diabetes Cellular Therapy.

PubMed

Millman, Jeffrey R; Pagliuca, Felicia W

2017-05-01

Development of stem cell technologies for cell replacement therapy has progressed rapidly in recent years. Diabetes has long been seen as one of the first applications for stem cell-derived cells because of the loss of only a single cell type-the insulin-producing β-cell. Recent reports have detailed strategies that overcome prior hurdles to generate functional β-like cells from human pluripotent stem cells in vitro, including from human induced pluripotent stem cells (hiPSCs). Even with this accomplishment, addressing immunological barriers to transplantation remains a major challenge for the field. The development of clinically relevant hiPSC derivation methods from patients and demonstration that these cells can be differentiated into β-like cells presents a new opportunity to treat diabetes without immunosuppression or immunoprotective encapsulation or with only targeted protection from autoimmunity. This review focuses on the current status in generating and transplanting autologous β-cells for diabetes cell therapy, highlighting the unique advantages and challenges of this approach. © 2017 by the American Diabetes Association.

PW1 gene/paternally expressed gene 3 (PW1/Peg3) identifies multiple adult stem and progenitor cell populations

PubMed Central

Besson, Vanessa; Smeriglio, Piera; Wegener, Amélie; Relaix, Frédéric; Nait Oumesmar, Brahim; Sassoon, David A.; Marazzi, Giovanna

2011-01-01

A variety of markers are invaluable for identifying and purifying stem/progenitor cells. Here we report the generation of a murine reporter line driven by Pw1 that reveals cycling and quiescent progenitor/stem cells in all adult tissues thus far examined, including the intestine, blood, testis, central nervous system, bone, skeletal muscle, and skin. Neurospheres generated from the adult PW1-reporter mouse show near 100% reporter-gene expression following a single passage. Furthermore, epidermal stem cells can be purified solely on the basis of reporter-gene expression. These cells are clonogenic, repopulate the epidermal stem-cell niches, and give rise to new hair follicles. Finally, we demonstrate that only PW1 reporter-expressing epidermal cells give rise to follicles that are capable of self-renewal following injury. Our data demonstrate that PW1 serves as an invaluable marker for competent self-renewing stem cells in a wide array of adult tissues, and the PW1-reporter mouse serves as a tool for rapid stem cell isolation and characterization. PMID:21709251

Comparison of EpCAMhighCD44+ cancer stem cells with EpCAMhighCD44- tumor cells in colon cancer by single-cell sequencing.

PubMed

Liu, Mingshan; Di, Jiabo; Liu, Yang; Su, Zhe; Jiang, Beihai; Wang, Zaozao; Su, Xiangqian

2018-03-26

Cancer stem cells (CSCs) are considered to be responsible for tumorigenesis and cancer relapse. EpCAM high CD44 + tumor cells are putative colorectal CSCs that express high levels of stem cell genes, while the EpCAM high CD44 - population mostly contains differentiated tumor cells (DTCs). This study aims to determine whether single CSC (EpCAM high CD44 + ) and DTC (EpCAM high CD44 - ) can be distinguished in terms of somatic copy number alterations (SCNAs). We applied fluorescence-activated cell sorting to isolate the CD45 - EpCAM high CD44 + and CD45 - EpCAM high CD44 - populations from two primary colon tumors, on which low-coverage single-cell whole-genome sequencing (WGS) was then performed ∼0.1x depth. We compared the SCNAs of the CSCs and DTCs at single-cell resolution. In total, 47 qualified single cells of the two populations underwent WGS. The single-cell SCNA profiles showed that there were obvious SCNAs in both the CSCs and DTCs of each patient, and each patient had a specific copy number alteration pattern. Hierarchical clustering and correlation analysis both showed that the SCNA profiles of CSCs and DTCs from the same patient had similar SCNA pattern, while there were regional differences in the CSCs and DTCs in certain patient. SCNAs of CSCs in the same patient were highly reproducible. Our data suggest that major SCNAs occurred at an early stage and were inherited steadily. The similarity of ubiquitous SCNAs between the CSCs and DTCs might have arisen from lineage differentiation. CSCs from the same patient had reproducible SCNA profiles, indicating that gain or loss in certain chromosome is required for colon cancer development.

Single-cell transcriptomics uncovers distinct molecular signatures of stem cells in chronic myeloid leukemia.

PubMed

Giustacchini, Alice; Thongjuea, Supat; Barkas, Nikolaos; Woll, Petter S; Povinelli, Benjamin J; Booth, Christopher A G; Sopp, Paul; Norfo, Ruggiero; Rodriguez-Meira, Alba; Ashley, Neil; Jamieson, Lauren; Vyas, Paresh; Anderson, Kristina; Segerstolpe, Åsa; Qian, Hong; Olsson-Strömberg, Ulla; Mustjoki, Satu; Sandberg, Rickard; Jacobsen, Sten Eirik W; Mead, Adam J

2017-06-01

Recent advances in single-cell transcriptomics are ideally placed to unravel intratumoral heterogeneity and selective resistance of cancer stem cell (SC) subpopulations to molecularly targeted cancer therapies. However, current single-cell RNA-sequencing approaches lack the sensitivity required to reliably detect somatic mutations. We developed a method that combines high-sensitivity mutation detection with whole-transcriptome analysis of the same single cell. We applied this technique to analyze more than 2,000 SCs from patients with chronic myeloid leukemia (CML) throughout the disease course, revealing heterogeneity of CML-SCs, including the identification of a subgroup of CML-SCs with a distinct molecular signature that selectively persisted during prolonged therapy. Analysis of nonleukemic SCs from patients with CML also provided new insights into cell-extrinsic disruption of hematopoiesis in CML associated with clinical outcome. Furthermore, we used this single-cell approach to identify a blast-crisis-specific SC population, which was also present in a subclone of CML-SCs during the chronic phase in a patient who subsequently developed blast crisis. This approach, which might be broadly applied to any malignancy, illustrates how single-cell analysis can identify subpopulations of therapy-resistant SCs that are not apparent through cell-population analysis.

Female mice lack adult germ-line stem cells but sustain oogenesis using stable primordial follicles.

PubMed

Lei, Lei; Spradling, Allan C

2013-05-21

Whether or not mammalian females generate new oocytes during adulthood from germ-line stem cells to sustain the ovarian follicle pool has recently generated controversy. We used a sensitive lineage-labeling system to determine whether stem cells are needed in female adult mice to compensate for follicular losses and to directly identify active germ-line stem cells. Primordial follicles generated during fetal life are highly stable, with a half-life during adulthood of 10 mo, and thus are sufficient to sustain adult oogenesis without a source of renewal. Moreover, in normal mice or following germ-cell depletion with Busulfan, only stable, single oocytes are lineage-labeled, rather than cell clusters indicative of new oocyte formation. Even one germ-line stem cell division per 2 wk would have been detected by our method, based on the kinetics of fetal follicle formation. Thus, adult female mice neither require nor contain active germ-line stem cells or produce new oocytes in vivo.

Micro- and nanoengineering for stem cell biology: the promise with a caution.

PubMed

Kshitiz; Kim, Deok-Ho; Beebe, David J; Levchenko, Andre

2011-08-01

Current techniques used in stem cell research only crudely mimic the physiological complexity of the stem cell niches. Recent advances in the field of micro- and nanoengineering have brought an array of in vitro cell culture models that have enabled development of novel, highly precise and standardized tools that capture physiological details in a single platform, with greater control, consistency, and throughput. In this review, we describe the micro- and nanotechnology-driven modern toolkit for stem cell biologists to design novel experiments in more physiological microenvironments with increased precision and standardization, and caution them against potential challenges that the modern technologies might present. Copyright © 2011 Elsevier Ltd. All rights reserved.

Induced Pluripotent Stem Cells: A novel frontier in the study of human primary immunodeficiencies

PubMed Central

Pessach, Itai M.; Ordovas-Montanes, Jose; Zhang, Shen-Ying; Casanova, Jean-Laurent; Giliani, Silvia; Gennery, Andrew R.; Al-Herz, Waleed; Manos, Philip D.; Schlaeger, Thorsten M.; Park, In-Hyun; Rucci, Francesca; Agarwal, Suneet; Mostoslavsky, Gustavo; Daley, George Q.; Notarangelo, Luigi D.

2010-01-01

Background The novel ability to epigenetically reprogram somatic cells into induced pluripotent stem cells through the exogenous expression of transcription promises to revolutionize the study of human diseases. Objective Here we report on the generation of 25 induced pluripotent stem cell lines from 6 patients with various forms of Primary Immunodeficiencies, affecting adaptive and/or innate immunity. Methods Patients’ dermal fibroblasts were reprogrammed by expression of four transcription factors, OCT4, SOX2, KLF4, and c-MYC using a single excisable polycistronic lentiviral vector. Results Induced pluripotent stem cells derived from patients with primary immunodeficiencies show a stemness profile that is comparable to that observed in human embryonic stem cells. Following in vitro differentiation into embryoid bodies, pluripotency of the patient-derived indiced pluripotent stem cells lines was demonstrated by expression of genes characteristic of each of the three embryonic layers. We have confirmed the patient-specific origin of the induced pluripotent stem cell lines, and ascertained maintenance of karyotypic integrity. Conclusion By providing a limitless source of diseased stem cells that can be differentiated into various cell types in vitro, the repository of induced pluripotent stem cell lines from patients with primary immunodeficiencies represents a unique resource to investigate the pathophysiology of hematopoietic and extra-hematopoietic manifestations of these diseases, and may assist in the development of novel therapeutic approaches based on gene correction. PMID:21185069

Automatic Stem Cell Detection in Microscopic Whole Mouse Cryo-imaging

PubMed Central

Wuttisarnwattana, Patiwet; Gargesha, Madhusudhana; Hof, Wouter van’t; Cooke, Kenneth R.

2016-01-01

With its single cell sensitivity over volumes as large as or larger than a mouse, cryo-imaging enables imaging of stem cell biodistribution, homing, engraftment, and molecular mechanisms. We developed and evaluated a highly automated software tool to detect fluorescently labeled stem cells within very large (~200GB) cryo-imaging datasets. Cell detection steps are: preprocess, remove immaterial regions, spatially filter to create features, identify candidate pixels, classify pixels using bagging decision trees, segment cell patches, and perform 3D labeling. There are options for analysis and visualization. To train the classifier, we created synthetic images by placing realistic digital cell models onto cryo-images of control mice devoid of cells. Very good cell detection results were (precision=98.49%, recall=99.97%) for synthetic cryo-images, (precision=97.81%, recall=97.71%) for manually evaluated, actual cryo-images, and <1% false positives in control mice. An α-multiplier applied to features allows one to correct for experimental variations in cell brightness due to labeling. On dim cells (37% of standard brightness), with correction, we improved recall (49.26%→99.36%) without a significant drop in precision (99.99%→99.75%). With tail vein injection, multipotent adult progenitor cells in a graft-versus-host-disease model in the first days post injection were predominantly found in lung, liver, spleen, and bone marrow. Distribution was not simply related to blood flow. The lung contained clusters of cells while other tissues contained single cells. Our methods provided stem cell distribution anywhere in mouse with single cell sensitivity. Methods should provide a rational means of evaluating dosing, delivery methods, cell enhancements, and mechanisms for therapeutic cells. PMID:26552080

Electrical Stimulation Followed by Mesenchymal Stem Cells Improves Anal Sphincter Anatomy and Function in a Rat Model at a Time Remote From Injury.

PubMed

Sun, Li; Yeh, Judy; Xie, Zhuojun; Kuang, Mei; Damaser, Margot S; Zutshi, Massarat

2016-05-01

We have explored cell-based therapy to aid anal sphincter repair, but a conditioning injury is required to direct stem cells to the site of injury because symptoms usually manifest at a time remote from injury. We aimed to investigate the effect of local electrical stimulation followed by mesenchymal stem cell delivery on anal sphincter regeneration at a time remote from injury. With the use of a rat model, electrical stimulation parameters and cell delivery route were selected based on in vivo cytokine expression and luciferase-labeled cell imaging of the anal sphincter complex. Three weeks after a partial anal sphincter excision, rats were randomly allocated to 4 groups based on different local interventions: no treatment, daily electrical stimulation for 3 days, daily stimulation for 3 days followed by stem cell injection on the third day, and daily electrical stimulation followed by stem cell injection on the first and third days. Histology-assessed anatomy and anal manometry evaluated physiology 4 weeks after intervention. The electrical stimulation parameters that significantly upregulated gene expression of homing cytokines also achieved mesenchymal stem cell retention when injected directly in the anal sphincter complex in comparison with intravascular and intraperitoneal injections. Four weeks after intervention, there was significantly more new muscle in the area of injury and significantly improved anal resting pressure in the group that received daily electrical stimulation for 3 days followed by a single injection of 1 million stem cells on the third day at the site of injury. This was a pilot study and therefore was not powered for functional outcome. In this rat injury model with optimized parameters, electrical stimulation with a single local mesenchymal stem cell injection administered 3 weeks after injury significantly improved both new muscle formation in the area of injury and anal sphincter pressures.

Prospect of Stem Cells in Bone Tissue Engineering: A Review

PubMed Central

Yousefi, Azizeh-Mitra; James, Paul F.; Akbarzadeh, Rosa; Subramanian, Aswati; Flavin, Conor; Oudadesse, Hassane

2016-01-01

Mesenchymal stem cells (MSCs) have been the subject of many studies in recent years, ranging from basic science that looks into MSCs properties to studies that aim for developing bioengineered tissues and organs. Adult bone marrow-derived mesenchymal stem cells (BM-MSCs) have been the focus of most studies due to the inherent potential of these cells to differentiate into various cell types. Although, the discovery of induced pluripotent stem cells (iPSCs) represents a paradigm shift in our understanding of cellular differentiation. These cells are another attractive stem cell source because of their ability to be reprogramed, allowing the generation of multiple cell types from a single cell. This paper briefly covers various types of stem cell sources that have been used for tissue engineering applications, with a focus on bone regeneration. Then, an overview of some recent studies making use of MSC-seeded 3D scaffold systems for bone tissue engineering has been presented. The emphasis has been placed on the reported scaffold properties that tend to improve MSCs adhesion, proliferation, and osteogenic differentiation outcomes. PMID:26880976

[Research progress of Lgr5-positive stem cells in the formation of organoid in 3D culture].

PubMed

He, Q Q; Li, A; Wang, M H; Gao, X

2018-06-07

Stem cell is critical to regeneration of tissue or organ of human. How to promote repair or regeneration in the tissues/organ using its pluripotency is always an important issue. Lgr5-possitive cell is one type of the stem cell-like cells capable of pluripotent differentiation in various tissues/organs of both humans and mice. Current study showed that single or small amount Lgr5-possitive stem cells can grow and form a plurality of organs in 3D culture system, and some organs can present similar biological and physiological properties with the progenitor they were derived. These studies provided new insight into future orientation, for example, Lgr5-possitive inner ear cells were confirmed as inner ear pluripotent cells population, the experiences obtained from organoid studies of Lgr5-possitive cells have certainly showed potential in the future study of inner ear stem cells. This review will focus on the recent progress associated with Lgr 5-positive stem cells forming organoids in the 3D culture.

Neural stem cell heterogeneity through time and space in the ventricular-subventricular zone.

PubMed

Rushing, Gabrielle; Ihrie, Rebecca A

2016-08-01

The origin and classification of neural stem cells (NSCs) has been a subject of intense investigation for the past two decades. Efforts to categorize NSCs based on their location, function and expression have established that these cells are a heterogeneous pool in both the embryonic and adult brain. The discovery and additional characterization of adult NSCs has introduced the possibility of using these cells as a source for neuronal and glial replacement following injury or disease. To understand how one could manipulate NSC developmental programs for therapeutic use, additional work is needed to elucidate how NSCs are programmed and how signals during development are interpreted to determine cell fate. This review describes the identification, classification and characterization of NSCs within the large neurogenic niche of the ventricular-subventricular zone (V-SVZ). A literature search was conducted using Pubmed including the keywords "ventricular-subventricular zone," "neural stem cell," "heterogeneity," "identity" and/or "single cell" to find relevant manuscripts to include within the review. A special focus was placed on more recent findings using single-cell level analyses on neural stem cells within their niche(s). This review discusses over 20 research articles detailing findings on V-SVZ NSC heterogeneity, over 25 articles describing fate determinants of NSCs, and focuses on 8 recent publications using distinct single-cell analyses of neural stem cells including flow cytometry and RNA-seq. Additionally, over 60 manuscripts highlighting the markers expressed on cells within the NSC lineage are included in a chart divided by cell type. Investigation of NSC heterogeneity and fate decisions is ongoing. Thus far, much research has been conducted in mice however, findings in human and other mammalian species are also discussed here. Implications of NSC heterogeneity established in the embryo for the properties of NSCs in the adult brain are explored, including how these cells may be redirected after injury or genetic manipulation.

Reconstitution of a Patterned Neural Tube from Single Mouse Embryonic Stem Cells.

PubMed

Ishihara, Keisuke; Ranga, Adrian; Lutolf, Matthias P; Tanaka, Elly M; Meinhardt, Andrea

2017-01-01

The recapitulation of tissue development and patterning in three-dimensional (3D) culture is an important dimension of stem cell research. Here, we describe a 3D culture protocol in which single mouse ES cells embedded in Matrigel under neural induction conditions clonally form a lumen containing, oval-shaped epithelial structure within 3 days. By Day 7 an apicobasally polarized neuroepithelium with uniformly dorsal cell identity forms. Treatment with retinoic acid at Day 2 results in posteriorization and self-organization of dorsal-ventral neural tube patterning. Neural tube organoid growth is also supported by pure laminin gels as well as poly(ethylene glycol) (PEG)-based artificial extracellular matrix hydrogels, which can be fine-tuned for key microenvironment characteristics. The rapid generation of a simple, patterned tissue in well-defined culture conditions makes the neural tube organoid a tractable model for studying neural stem cell self-organization.

Directing stem cell fate on hydrogel substrates by controlling cell geometry, matrix mechanics and adhesion ligand composition.

PubMed

Lee, Junmin; Abdeen, Amr A; Zhang, Douglas; Kilian, Kristopher A

2013-11-01

There is a dynamic relationship between physical and biochemical signals presented in the stem cell microenvironment to guide cell fate determination. Model systems that modulate cell geometry, substrate stiffness or matrix composition have proved useful in exploring how these signals influence stem cell fate. However, the interplay between these physical and biochemical cues during differentiation remains unclear. Here, we demonstrate a microengineering strategy to vary single cell geometry and the composition of adhesion ligands - on substrates that approximate the mechanical properties of soft tissues - to study adipogenesis and neurogenesis in adherent mesenchymal stem cells. Cells cultured in small circular islands show elevated expression of adipogenesis markers while cells that spread in anisotropic geometries tend to express elevated neurogenic markers. Arraying different combinations of matrix protein in a myriad of 2D and pseudo-3D geometries reveals optimal microenvironments for controlling the differentiation of stem cells to these "soft" lineages without the use of media supplements. © 2013 Elsevier Ltd. All rights reserved.

Technology advancement for integrative stem cell analyses.

PubMed

Jeong, Yoon; Choi, Jonghoon; Lee, Kwan Hyi

2014-12-01

Scientists have endeavored to use stem cells for a variety of applications ranging from basic science research to translational medicine. Population-based characterization of such stem cells, while providing an important foundation to further development, often disregard the heterogeneity inherent among individual constituents within a given population. The population-based analysis and characterization of stem cells and the problems associated with such a blanket approach only underscore the need for the development of new analytical technology. In this article, we review current stem cell analytical technologies, along with the advantages and disadvantages of each, followed by applications of these technologies in the field of stem cells. Furthermore, while recent advances in micro/nano technology have led to a growth in the stem cell analytical field, underlying architectural concepts allow only for a vertical analytical approach, in which different desirable parameters are obtained from multiple individual experiments and there are many technical challenges that limit vertically integrated analytical tools. Therefore, we propose--by introducing a concept of vertical and horizontal approach--that there is the need of adequate methods to the integration of information, such that multiple descriptive parameters from a stem cell can be obtained from a single experiment.

Technology Advancement for Integrative Stem Cell Analyses

PubMed Central

Jeong, Yoon

2014-01-01

Scientists have endeavored to use stem cells for a variety of applications ranging from basic science research to translational medicine. Population-based characterization of such stem cells, while providing an important foundation to further development, often disregard the heterogeneity inherent among individual constituents within a given population. The population-based analysis and characterization of stem cells and the problems associated with such a blanket approach only underscore the need for the development of new analytical technology. In this article, we review current stem cell analytical technologies, along with the advantages and disadvantages of each, followed by applications of these technologies in the field of stem cells. Furthermore, while recent advances in micro/nano technology have led to a growth in the stem cell analytical field, underlying architectural concepts allow only for a vertical analytical approach, in which different desirable parameters are obtained from multiple individual experiments and there are many technical challenges that limit vertically integrated analytical tools. Therefore, we propose—by introducing a concept of vertical and horizontal approach—that there is the need of adequate methods to the integration of information, such that multiple descriptive parameters from a stem cell can be obtained from a single experiment. PMID:24874188

Local calcium signalling is mediated by mechanosensitive ion channels in mesenchymal stem cells

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

Chubinskiy-Nadezhdin, Vladislav I., E-mail: vchubinskiy@gmail.com; Vasileva, Valeria Y.; Pugovkina, Natalia A.

Mechanical forces are implicated in key physiological processes in stem cells, including proliferation, differentiation and lineage switching. To date, there is an evident lack of understanding of how external mechanical cues are coupled with calcium signalling in stem cells. Mechanical reactions are of particular interest in adult mesenchymal stem cells because of their promising potential for use in tissue remodelling and clinical therapy. Here, single channel patch-clamp technique was employed to search for cation channels involved in mechanosensitivity in mesenchymal endometrial-derived stem cells (hMESCs). Functional expression of native mechanosensitive stretch-activated channels (SACs) and calcium-sensitive potassium channels of different conductances inmore » hMESCs was shown. Single current analysis of stretch-induced channel activity revealed functional coupling of SACs and BK channels in plasma membrane. The combination of cell-attached and inside-out experiments have indicated that highly localized Ca{sup 2+} entry via SACs triggers BK channel activity. At the same time, SK channels are not coupled with SACs despite of high calcium sensitivity as compared to BK. Our data demonstrate novel mechanism controlling BK channel activity in native cells. We conclude that SACs and BK channels are clusterized in functional mechanosensitive domains in the plasma membrane of hMESCs. Co-clustering of ion channels may significantly contribute to mechano-dependent calcium signalling in stem cells. - Highlights: • Stretch-induced channel activity in human mesenchymal stem cells was analyzed. • Functional expression of SACs and Ca{sup 2+}-sensitive BK and SK channels was shown. • Local Ca{sup 2+} influx via stretch-activated channels triggers BK channel activity. • SK channels are not coupled with SACs despite higher sensitivity to [Ca{sup 2+}]{sub i}. • Functional clustering of SACs and BK channels in stem cell membrane is proposed.« less

Design guidelines for an umbilical cord blood stem cell therapy quality assessment model

NASA Astrophysics Data System (ADS)

Januszewski, Witold S.; Michałek, Krzysztof; Yagensky, Oleksandr; Wardzińska, Marta

The paper enlists the pivotal guidelines for producing an empirical umbilical cord blood stem cell therapy quality assessment model. The methodology adapted was single equation linear model with domain knowledge derived from MEDAFAR classification. The resulting model is ready for therapeutical application.

Characterization of stem/progenitor cell cycle using murine circumvallate papilla taste bud organoid.

PubMed

Aihara, Eitaro; Mahe, Maxime M; Schumacher, Michael A; Matthis, Andrea L; Feng, Rui; Ren, Wenwen; Noah, Taeko K; Matsu-ura, Toru; Moore, Sean R; Hong, Christian I; Zavros, Yana; Herness, Scott; Shroyer, Noah F; Iwatsuki, Ken; Jiang, Peihua; Helmrath, Michael A; Montrose, Marshall H

2015-11-24

Leucine-rich repeat-containing G-protein coupled receptor 5-expressing (Lgr5(+)) cells have been identified as stem/progenitor cells in the circumvallate papillae, and single cultured Lgr5(+) cells give rise to taste cells. Here we use circumvallate papilla tissue to establish a three-dimensional culture system (taste bud organoids) that develops phenotypic characteristics similar to native tissue, including a multilayered epithelium containing stem/progenitor in the outer layers and taste cells in the inner layers. Furthermore, characterization of the cell cycle of the taste bud progenitor niche reveals striking dynamics of taste bud development and regeneration. Using this taste bud organoid culture system and FUCCI2 transgenic mice, we identify the stem/progenitor cells have at least 5 distinct cell cycle populations by tracking within 24-hour synchronized oscillations of proliferation. Additionally, we demonstrate that stem/progenitor cells have motility to form taste bud organoids. Taste bud organoids provides a system for elucidating mechanisms of taste signaling, disease modeling, and taste tissue regeneration.

Characterization of stem/progenitor cell cycle using murine circumvallate papilla taste bud organoid

PubMed Central

Aihara, Eitaro; Mahe, Maxime M.; Schumacher, Michael A.; Matthis, Andrea L.; Feng, Rui; Ren, Wenwen; Noah, Taeko K.; Matsu-ura, Toru; Moore, Sean R.; Hong, Christian I.; Zavros, Yana; Herness, Scott; Shroyer, Noah F.; Iwatsuki, Ken; Jiang, Peihua; Helmrath, Michael A.; Montrose, Marshall H.

2015-01-01

Leucine-rich repeat-containing G-protein coupled receptor 5-expressing (Lgr5+) cells have been identified as stem/progenitor cells in the circumvallate papillae, and single cultured Lgr5+ cells give rise to taste cells. Here we use circumvallate papilla tissue to establish a three-dimensional culture system (taste bud organoids) that develops phenotypic characteristics similar to native tissue, including a multilayered epithelium containing stem/progenitor in the outer layers and taste cells in the inner layers. Furthermore, characterization of the cell cycle of the taste bud progenitor niche reveals striking dynamics of taste bud development and regeneration. Using this taste bud organoid culture system and FUCCI2 transgenic mice, we identify the stem/progenitor cells have at least 5 distinct cell cycle populations by tracking within 24-hour synchronized oscillations of proliferation. Additionally, we demonstrate that stem/progenitor cells have motility to form taste bud organoids. Taste bud organoids provides a system for elucidating mechanisms of taste signaling, disease modeling, and taste tissue regeneration. PMID:26597788

Suspension culture of pluripotent stem cells: effect of shear on stem cell fate.

PubMed

Keller, Kevin C; Rodrigues, Beatriz; zur Nieden, Nicole I

2014-01-01

Despite significant promise, the routine usage of suspension cell culture to manufacture stem cell-derived differentiated cells has progressed slowly. Suspension culture is an innovative way of either expanding or differentiating cells and sometimes both are combined into a single bioprocess. Its advantages over static 2D culturing include a homogeneous and controllable culture environment and producing a large quantity of cells in a fraction of time. This feature makes suspension cell culture ideal for use in stem cell research and eventually ideal in the large-scale production of differentiated cells for regenerative medicine. Because of their tremendous differentiation capacities and unlimited growth properties, pluripotent stem cells (PSCs) in particular are considered potential sources for future cell-replacement therapies. Currently, expansion of PSCs is accomplished in 2D, which only permits a limited amount of cell growth per culture flask before cells need to be passaged. However, before stem cells can be applied clinically, several aspects of their expansion, such as directed growth, but also differentiation, need to be better controlled. This review will summarize recent advantages in suspension culture of PSCs, while at the same time highlighting current challenges.

Strand displacement amplification for ultrasensitive detection of human pluripotent stem cells.

PubMed

Wu, Wei; Mao, Yiping; Zhao, Shiming; Lu, Xuewen; Liang, Xingguo; Zeng, Lingwen

2015-06-30

Human pluripotent stem cells (hPSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), provide a powerful model system for studies of cellular identity and early mammalian development, which hold great promise for regenerative medicine. It is necessary to develop a convenient method to discriminate hPSCs from other cells in clinics and basic research. Herein, a simple and reliable biosensor for stem cell detection was established. In this biosensor system, stage-specific embryonic antigen-3 (SSEA-3) and stage-specific embryonic antigen-4 (SSEA-4) were used to mark human pluripotent stem cells (hPSCs). Antibody specific for SSEA-3 was coated onto magnetic beads for hPSCs enrichment, and antibody specific for SSEA-4 was conjugated with carboxyl-modified tDNA sequence which was used as template for strand displacement amplification (SDA). The amplified single strand DNA (ssDNA) was detected with a lateral flow biosensor (LFB). This biosensor is capable of detecting a minimum of 19 human embryonic stem cells by a strip reader and 100 human embryonic stem cells by the naked eye within 80min. This approach has also shown excellent specificity to distinguish hPSCs from other types of cells, showing that it is promising for specific and handy detection of human pluripotent stem cells. Copyright © 2015 Elsevier B.V. All rights reserved.

Isolation and morphology of Stem Cells from Deciduous Tooth (SHED) and Human Dental Pulp Stem Cells (hDPSC)

NASA Astrophysics Data System (ADS)

Ariffin, Shahrul Hisham Zainal; Manogaran, Thanaletchumi; Abidin, Intan Zarina Zainol; Senafi, Sahidan; Wahab, Rohaya Megat Abdul

2016-11-01

Dental pulp is a tissue obtained from pulp chamber of deciduous and permanent tooth which contain stem cells. Stem cell isolation procedure is performed to obtain cells from tissue using enzymatic digestion. The aim of this study is to isolate and observe the morphology of stem cells during passage 0 and passage 3. Dental pulp from deciduous and permanent tooth was enzymatically digested using collagenase Type I and cells obtained were cultured in DMEM-KO that contains 10% fetal bovine serum, 1% antibiotic-antimycotic solution and 0.001× GlutaMax®. During culture, cell morphology was observed under the microscope on day 3, 16 and 33 and captured using cellB software. Giemsa staining was conducted on cells at passage 3. Cells attached at the bottom of the flask on day 3 and started forming small colonies. Cells became confluent after approximately 4 weeks. Both Stem Cells from Deciduous Tooth (SHED) and Human Dental Pulp Stem Cells (hDPSC) exhibited fibroblast-like morphology during passage 0 and passage 3. Meanwhile, Giemsa staining at passage 3 revealed single intact nucleus surrounded by fibroblastic cytoplasm structure. It can be concluded that SHED and hDPSC showed consistent fibroblast-like morphology throughout culture period.

Direct Reprogramming of Human Amniotic Fluid Stem Cells by OCT4 and Application in Repairing of Cerebral Ischemia Damage

PubMed Central

Qin, Mingde; Chen, Ruihua; Li, Hong; Liang, Hansi; Xue, Qun; Li, Fang; Chen, Ying; Zhang, Xueguang

2016-01-01

Amniotic fluid stem cells (AFSCs) are a type of fetal stem cell whose stemness encompasses both embryonic and adult stem cells, suggesting that they may be easily and efficiently reprogrammed into induced pluripotent stem cells (iPSCs). To further simplify the reprogramming process, the creation of AFSC-derived iPSCs using a single factor is desirable. Here we report the generation of one-factor human AFSC-iPSCs (AiPSCs) from human AFSCs by ectopic expression of the transcription factor OCT4. Just like human embryonic stem cells, AiPSCs exhibited similar epigenetic status, global gene expression profiles, teratoma formation and in vitro & in vivo pluripotency. Our results indicate that the OCT4 is necessary and sufficient to directly reprogram human AFSCs into pluripotent AiPSCs. Moreover, reflecting the similar memory characteristics of AFSCs and neural stem cells, we show that AiPSC membrane-derived vesicles (MVs) repair cerebral ischemia damage. We anticipate that the successful generation of one-factor AiPSCs will facilitate the creation of patient-specific pluripotent stem cells without the need for transgenic expression of oncogenes. Moreover, MVs from tissue-specific AiPSCs have potential in tissue repair, representing a novel application of iPSCs. PMID:27019637

Intrinsic and extrinsic mechanical properties related to the differentiation of mesenchymal stem cells.

PubMed

Lee, Jin-Ho; Park, Hun-Kuk; Kim, Kyung Sook

2016-05-06

Diverse intrinsic and extrinsic mechanical factors have a strong influence on the regulation of stem cell fate. In this work, we examined recent literature on the effects of mechanical environments on stem cells, especially on differentiation of mesenchymal stem cells (MSCs). We provide a brief review of intrinsic mechanical properties of single MSC and examined the correlation between the intrinsic mechanical property of MSC and the differentiation ability. The effects of extrinsic mechanical factors relevant to the differentiation of MSCs were considered separately. The effect of nanostructure and elasticity of the matrix on the differentiation of MSCs were summarized. Finally, we consider how the extrinsic mechanical properties transfer to MSCs and then how the effects on the intrinsic mechanical properties affect stem cell differentiation. Copyright © 2015 Elsevier Inc. All rights reserved.

Optimality in the Development of Intestinal Crypts

PubMed Central

Itzkovitz, Shalev; Blat, Irene C.; Jacks, Tyler; Clevers, Hans; van Oudenaarden, Alexander

2012-01-01

SUMMARY Intestinal crypts in mammals are comprised of long-lived stem cells and shorter-lived progenies. These two populations are maintained in specific proportions during adult life. Here, we investigate the design principles governing the dynamics of these proportions during crypt morphogenesis. Using optimal control theory, we show that a proliferation strategy known as a “bang-bang” control minimizes the time to obtain a mature crypt. This strategy consists of a surge of symmetric stem cell divisions, establishing the entire stem cell pool first, followed by a sharp transition to strictly asymmetric stem cell divisions, producing nonstem cells with a delay. We validate these predictions using lineage tracing and single-molecule fluorescence in situ hybridization of intestinal crypts in infant mice, uncovering small crypts that are entirely composed of Lgr5-labeled stem cells, which become a minority as crypts continue to grow. Our approach can be used to uncover similar design principles in other developmental systems. PMID:22304925

Quantifying intrinsic and extrinsic control of single-cell fates in cancer and stem/progenitor cell pedigrees with competing risks analysis

PubMed Central

Cornwell, J. A.; Hallett, R. M.; der Mauer, S. Auf; Motazedian, A.; Schroeder, T.; Draper, J. S.; Harvey, R. P.; Nordon, R. E.

2016-01-01

The molecular control of cell fate and behaviour is a central theme in biology. Inherent heterogeneity within cell populations requires that control of cell fate is studied at the single-cell level. Time-lapse imaging and single-cell tracking are powerful technologies for acquiring cell lifetime data, allowing quantification of how cell-intrinsic and extrinsic factors control single-cell fates over time. However, cell lifetime data contain complex features. Competing cell fates, censoring, and the possible inter-dependence of competing fates, currently present challenges to modelling cell lifetime data. Thus far such features are largely ignored, resulting in loss of data and introducing a source of bias. Here we show that competing risks and concordance statistics, previously applied to clinical data and the study of genetic influences on life events in twins, respectively, can be used to quantify intrinsic and extrinsic control of single-cell fates. Using these statistics we demonstrate that 1) breast cancer cell fate after chemotherapy is dependent on p53 genotype; 2) granulocyte macrophage progenitors and their differentiated progeny have concordant fates; and 3) cytokines promote self-renewal of cardiac mesenchymal stem cells by symmetric divisions. Therefore, competing risks and concordance statistics provide a robust and unbiased approach for evaluating hypotheses at the single-cell level. PMID:27250534

Self-Renewal of Single Mouse Hematopoietic Stem Cells Is Reduced by JAK2V617F Without Compromising Progenitor Cell Expansion

PubMed Central

Kent, David G.; Li, Juan; Tanna, Hinal; Fink, Juergen; Kirschner, Kristina; Pask, Dean C.; Silber, Yvonne; Hamilton, Tina L.; Sneade, Rachel; Simons, Benjamin D.; Green, Anthony R.

2013-01-01

Recent descriptions of significant heterogeneity in normal stem cells and cancers have altered our understanding of tumorigenesis, emphasizing the need to understand how single stem cells are subverted to cause tumors. Human myeloproliferative neoplasms (MPNs) are thought to reflect transformation of a hematopoietic stem cell (HSC) and the majority harbor an acquired V617F mutation in the JAK2 tyrosine kinase, making them a paradigm for studying the early stages of tumor establishment and progression. The consequences of activating tyrosine kinase mutations for stem and progenitor cell behavior are unclear. In this article, we identify a distinct cellular mechanism operative in stem cells. By using conditional knock-in mice, we show that the HSC defect resulting from expression of heterozygous human JAK2V617F is both quantitative (reduced HSC numbers) and qualitative (lineage biases and reduced self-renewal per HSC). The defect is intrinsic to individual HSCs and their progeny are skewed toward proliferation and differentiation as evidenced by single cell and transplantation assays. Aged JAK2V617F show a more pronounced defect as assessed by transplantation, but mice that transform reacquire competitive self-renewal ability. Quantitative analysis of HSC-derived clones was used to model the fate choices of normal and JAK2-mutant HSCs and indicates that JAK2V617F reduces self-renewal of individual HSCs but leaves progenitor expansion intact. This conclusion is supported by paired daughter cell analyses, which indicate that JAK2-mutant HSCs more often give rise to two differentiated daughter cells. Together these data suggest that acquisition of JAK2V617F alone is insufficient for clonal expansion and disease progression and causes eventual HSC exhaustion. Moreover, our results show that clonal expansion of progenitor cells provides a window in which collaborating mutations can accumulate to drive disease progression. Characterizing the mechanism(s) of JAK2V617F subclinical clonal expansions and the transition to overt MPNs will illuminate the earliest stages of tumor establishment and subclone competition, fundamentally shifting the way we treat and manage cancers. PMID:23750118

Micro-Raman Spectroscopy of Silver Nanoparticle Induced Stress on Optically-Trapped Stem Cells

PubMed Central

Bankapur, Aseefhali; Krishnamurthy, R. Sagar; Zachariah, Elsa; Santhosh, Chidangil; Chougule, Basavaraj; Praveen, Bhavishna; Valiathan, Manna; Mathur, Deepak

2012-01-01

We report here results of a single-cell Raman spectroscopy study of stress effects induced by silver nanoparticles in human mesenchymal stem cells (hMSCs). A high-sensitivity, high-resolution Raman Tweezers set-up has been used to monitor nanoparticle-induced biochemical changes in optically-trapped single cells. Our micro-Raman spectroscopic study reveals that hMSCs treated with silver nanoparticles undergo oxidative stress at doping levels in excess of 2 µg/ml, with results of a statistical analysis of Raman spectra suggesting that the induced stress becomes more dominant at nanoparticle concentration levels above 3 µg/ml. PMID:22514708

A Preliminary Study: Human Fibroid Stro-1+/CD44+ Stem Cells Isolated From Uterine Fibroids Demonstrate Decreased DNA Repair and Genomic Integrity Compared to Adjacent Myometrial Stro-1+/CD44+ Cells.

PubMed

Prusinski Fernung, Lauren E; Al-Hendy, Ayman; Yang, Qiwei

2018-01-01

Although uterine fibroids (UFs) continue to place a major burden on female reproductive health, the mechanisms behind their origin remain undetermined. Normal myometrial stem cells may be transformed into tumor-initiating stem cells, causing UFs, due to unknown causes of somatic mutations in MED12, found in up to 85% of sporadically formed UFs. It is well established in other tumor types that defective DNA repair increases the risk of such tumorigenic somatic mutations, mechanisms not yet studied in UFs. To examine the putative cause(s) of this stem cell transformation, we analyzed DNA repair within stem cells from human UFs compared to those from adjacent myometrium to determine whether DNA repair in fibroid stem cells is compromised. Human fibroid (F) and adjacent myometrial (Myo) stem cells were isolated from fresh tissues, and gene expression relating to DNA repair was analyzed. Fibroid stem cells differentially expressed DNA repair genes related to DNA double- (DSBs) and single-strand breaks. DNA damage was measured using alkaline comet assay. Additionally, DNA DSBs were induced in these stem cells and DNA DSB repair evaluated (1) by determining changes in phosphorylation of DNA DSB-related proteins and (2) by determining differences in γ-H2AX foci formation and relative DNA repair protein RAD50 expression. Overall, F stem cells demonstrated increased DNA damage and altered DNA repair gene expression and signaling, suggesting that human F stem cells demonstrate impaired DNA repair. Compromised F stem cell DNA repair may contribute to further mutagenesis and, consequently, further growth and propagation of UF tumors.

Chromosomal aberrations and deoxyribonucleic acid single-strand breaks in adipose-derived stem cells during long-term expansion in vitro.

PubMed

Froelich, Katrin; Mickler, Johannes; Steusloff, Gudrun; Technau, Antje; Ramos Tirado, Mario; Scherzed, Agmal; Hackenberg, Stephan; Radeloff, Andreas; Hagen, Rudolf; Kleinsasser, Norbert

2013-07-01

Adipose-derived stem cells (ASCs) are a promising mesenchymal cell source for tissue engineering approaches. To obtain an adequate cell amount, in vitro expansion of the cells may be required in some cases. To monitor potential contraindications for therapeutic applications in humans, DNA strand breaks and chromosomal aberrations in ASCs during in vitro expansion were examined. After isolation of ASC from human lipoaspirates of seven patients, in vitro expansion over 10 passages was performed. Cells from passages 1, 2, 3, 5 and 10 were used for the alkaline single-cell microgel electrophoresis (comet) assay to detect DNA single-strand breaks and alkali labile as well as incomplete excision repair sites. Chromosomal changes were examined by means of the chromosomal aberration test. During in vitro expansion, ASC showed no DNA single-strand breaks in the comet assay. With the chromosomal aberration test, however, a significant increase in chromosomal aberrations were detected. The study showed that although no DNA fragmentation could be determined, the safety of ASC cannot be ensured with respect to chromosome stability during in vitro expansion. Thus, reliable analyses for detecting ASC populations, which accumulate chromosomal aberrations or even undergo malignant transformation during extensive in vitro expansion, must be implemented as part of the safety evaluation of these cells for stem cell-based therapy. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

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

PubMed Central

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

2009-01-01

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

Long-Term Culture of Genome-Stable Bipotent Stem Cells from Adult Human Liver

PubMed Central

Huch, Meritxell; Gehart, Helmuth; van Boxtel, Ruben; Hamer, Karien; Blokzijl, Francis; Verstegen, Monique M.A.; Ellis, Ewa; van Wenum, Martien; Fuchs, Sabine A.; de Ligt, Joep; van de Wetering, Marc; Sasaki, Nobuo; Boers, Susanne J.; Kemperman, Hans; de Jonge, Jeroen; Ijzermans, Jan N.M.; Nieuwenhuis, Edward E.S.; Hoekstra, Ruurdtje; Strom, Stephen; Vries, Robert R.G.; van der Laan, Luc J.W.; Cuppen, Edwin; Clevers, Hans

2015-01-01

Summary Despite the enormous replication potential of the human liver, there are currently no culture systems available that sustain hepatocyte replication and/or function in vitro. We have shown previously that single mouse Lgr5+ liver stem cells can be expanded as epithelial organoids in vitro and can be differentiated into functional hepatocytes in vitro and in vivo. We now describe conditions allowing long-term expansion of adult bile duct-derived bipotent progenitor cells from human liver. The expanded cells are highly stable at the chromosome and structural level, while single base changes occur at very low rates. The cells can readily be converted into functional hepatocytes in vitro and upon transplantation in vivo. Organoids from α1-antitrypsin deficiency and Alagille syndrome patients mirror the in vivo pathology. Clonal long-term expansion of primary adult liver stem cells opens up experimental avenues for disease modeling, toxicology studies, regenerative medicine, and gene therapy. PMID:25533785

Clonal analysis of synovial fluid stem cells to characterize and identify stable mesenchymal stromal cell/mesenchymal progenitor cell phenotypes in a porcine model: a cell source with enhanced commitment to the chondrogenic lineage.

PubMed

Ando, Wataru; Kutcher, Josh J; Krawetz, Roman; Sen, Arindom; Nakamura, Norimasa; Frank, Cyril B; Hart, David A

2014-06-01

Previous studies have demonstrated that porcine synovial membrane stem cells can adhere to a cartilage defect in vivo through the use of a tissue-engineered construct approach. To optimize this model, we wanted to compare effectiveness of tissue sources to determine whether porcine synovial fluid, synovial membrane, bone marrow and skin sources replicate our understanding of synovial fluid mesenchymal stromal cells or mesenchymal progenitor cells from humans both at the population level and the single-cell level. Synovial fluid clones were subsequently isolated and characterized to identify cells with a highly characterized optimal phenotype. The chondrogenic, osteogenic and adipogenic potentials were assessed in vitro for skin, bone marrow, adipose, synovial fluid and synovial membrane-derived stem cells. Synovial fluid cells then underwent limiting dilution analysis to isolate single clonal populations. These clonal populations were assessed for proliferative and differentiation potential by use of standardized protocols. Porcine-derived cells demonstrated the same relationship between cell sources as that demonstrated previously for humans, suggesting that the pig may be an ideal preclinical animal model. Synovial fluid cells demonstrated the highest chondrogenic potential that was further characterized, demonstrating the existence of a unique clonal phenotype with enhanced chondrogenic potential. Porcine stem cells demonstrate characteristics similar to those in human-derived mesenchymal stromal cells from the same sources. Synovial fluid-derived stem cells contain an inherent phenotype that may be optimal for cartilage repair. This must be more fully investigated for future use in the in vivo tissue-engineered construct approach in this physiologically relevant preclinical porcine model. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Mammary stem cells: angels or demons in mammary gland?

PubMed

Chen, Xueman; Liu, Qiang; Song, Erwei

2017-01-01

A highly dynamic development process exits within the epithelia of mammary gland, featuring morphogenetic variation during puberty, pregnancy, lactation, and regression. The identification of mammary stem cells (MaSCs) via lineage-tracing studies has substantiated a hierarchical organization of the mammary epithelia. A single MaSC is capable of reconstituting the entirely functional mammary gland upon orthotopic transplantation. Although different mammary cell subpopulations can be candidate cells-of-origin for distinct breast tumor subtypes, it still lacks experimental proofs whether MaSCs, the most primitive cells, are the 'seeds' of malignant transformation during most, if not all, tumorigenesis in the breast. Here, we review current knowledge of mammary epithelial hierarchy, highlighting the roles of mammary stem/progenitor cells and breast cancer stem cells (BCSCs) along with their key molecular regulators in organ development and cancer evolution. Clarifying these issues will pave the way for developing novel interventions toward stem/progenitor cells in either prevention or treatment of breast cancer (BrCa).

Mammary stem cells: angels or demons in mammary gland?

PubMed Central

Chen, Xueman; Liu, Qiang; Song, Erwei

2017-01-01

A highly dynamic development process exits within the epithelia of mammary gland, featuring morphogenetic variation during puberty, pregnancy, lactation, and regression. The identification of mammary stem cells (MaSCs) via lineage-tracing studies has substantiated a hierarchical organization of the mammary epithelia. A single MaSC is capable of reconstituting the entirely functional mammary gland upon orthotopic transplantation. Although different mammary cell subpopulations can be candidate cells-of-origin for distinct breast tumor subtypes, it still lacks experimental proofs whether MaSCs, the most primitive cells, are the ‘seeds’ of malignant transformation during most, if not all, tumorigenesis in the breast. Here, we review current knowledge of mammary epithelial hierarchy, highlighting the roles of mammary stem/progenitor cells and breast cancer stem cells (BCSCs) along with their key molecular regulators in organ development and cancer evolution. Clarifying these issues will pave the way for developing novel interventions toward stem/progenitor cells in either prevention or treatment of breast cancer (BrCa). PMID:29263909

Tracking the engraftment and regenerative capabilities of transplanted lung stem cells using fluorescent nanodiamonds

NASA Astrophysics Data System (ADS)

Wu, Tsai-Jung; Tzeng, Yan-Kai; Chang, Wei-Wei; Cheng, Chi-An; Kuo, Yung; Chien, Chin-Hsiang; Chang, Huan-Cheng; Yu, John

2013-09-01

Lung stem/progenitor cells are potentially useful for regenerative therapy, for example in repairing damaged or lost lung tissue in patients. Several optical imaging methods and probes have been used to track how stem cells incorporate and regenerate themselves in vivo over time. However, these approaches are limited by photobleaching, toxicity and interference from background tissue autofluorescence. Here we show that fluorescent nanodiamonds, in combination with fluorescence-activated cell sorting, fluorescence lifetime imaging microscopy and immunostaining, can identify transplanted CD45-CD54+CD157+ lung stem/progenitor cells in vivo, and track their engraftment and regenerative capabilities with single-cell resolution. Fluorescent nanodiamond labelling did not eliminate the cells' properties of self-renewal and differentiation into type I and type II pneumocytes. Time-gated fluorescence imaging of tissue sections of naphthalene-injured mice indicates that the fluorescent nanodiamond-labelled lung stem/progenitor cells preferentially reside at terminal bronchioles of the lungs for 7 days after intravenous transplantation.

A special issue on reviews in biomedical applications of nanomaterials, tissue engineering, stem cells, bioimaging, and toxicity.

PubMed

Nalwa, Hari Singh

2014-10-01

This second special issue of the Journal of Biomedical Nanotechnology in a series contains another 30 state-of-the-art reviews focused on the biomedical applications of nanomaterials, biosensors, bone tissue engineering, MRI and bioimaging, single-cell detection, stem cells, endothelial progenitor cells, toxicity and biosafety of nanodrugs, nanoparticle-based new therapeutic approaches for cancer, hepatic and cardiovascular disease.

In vitro generation of three-dimensional substrate-adherent embryonic stem cell-derived neural aggregates for application in animal models of neurological disorders.

PubMed

Hargus, Gunnar; Cui, Yi-Fang; Dihné, Marcel; Bernreuther, Christian; Schachner, Melitta

2012-05-01

In vitro-differentiated embryonic stem (ES) cells comprise a useful source for cell replacement therapy, but the efficiency and safety of a translational approach are highly dependent on optimized protocols for directed differentiation of ES cells into the desired cell types in vitro. Furthermore, the transplantation of three-dimensional ES cell-derived structures instead of a single-cell suspension may improve graft survival and function by providing a beneficial microenvironment for implanted cells. To this end, we have developed a new method to efficiently differentiate mouse ES cells into neural aggregates that consist predominantly (>90%) of postmitotic neurons, neural progenitor cells, and radial glia-like cells. When transplanted into the excitotoxically lesioned striatum of adult mice, these substrate-adherent embryonic stem cell-derived neural aggregates (SENAs) showed significant advantages over transplanted single-cell suspensions of ES cell-derived neural cells, including improved survival of GABAergic neurons, increased cell migration, and significantly decreased risk of teratoma formation. Furthermore, SENAs mediated functional improvement after transplantation into animal models of Parkinson's disease and spinal cord injury. This unit describes in detail how SENAs are efficiently derived from mouse ES cells in vitro and how SENAs are isolated for transplantation. Furthermore, methods are presented for successful implantation of SENAs into animal models of Huntington's disease, Parkinson's disease, and spinal cord injury to study the effects of stem cell-derived neural aggregates in a disease context in vivo.

The role of CD133 in normal human prostate stem cells and malignant cancer-initiating cells.

PubMed

Vander Griend, Donald J; Karthaus, Wouter L; Dalrymple, Susan; Meeker, Alan; DeMarzo, Angelo M; Isaacs, John T

2008-12-01

Resolving the specific cell of origin for prostate cancer is critical to define rational targets for therapeutic intervention and requires the isolation and characterization of both normal human prostate stem cells and prostate cancer-initiating cells (CIC). Single epithelial cells from fresh normal human prostate tissue and prostate epithelial cell (PrEC) cultures derived from them were evaluated for the presence of subpopulations expressing stem cell markers and exhibiting stem-like growth characteristics. When epithelial cell suspensions containing cells expressing the stem cell marker CD133+ are inoculated in vivo, regeneration of stratified human prostate glands requires inductive prostate stromal cells. PrEC cultures contain a small subpopulation of CD133+ cells, and fluorescence-activated cell sorting-purified CD133+ PrECs self-renew and regenerate cell populations expressing markers of transit-amplifying cells (DeltaNp63), intermediate cells (prostate stem cell antigen), and neuroendocrine cells (CD56). Using a series of CD133 monoclonal antibodies, attachment and growth of CD133+ PrECs requires surface expression of full-length glycosylated CD133 protein. Within a series of androgen receptor-positive (AR+) human prostate cancer cell lines, CD133+ cells are present at a low frequency, self-renew, express AR, generate phenotypically heterogeneous progeny negative for CD133, and possess an unlimited proliferative capacity, consistent with CD133+ cells being CICs. Unlike normal adult prostate stem cells, prostate CICs are AR+ and do not require functional CD133. This suggests that (a) AR-expressing prostate CICs are derived from a malignantly transformed intermediate cell that acquires "stem-like activity" and not from a malignantly transformed normal stem cell and (b) AR signaling pathways are a therapeutic target for prostate CICs.

Mapping human pluripotent stem cell differentiation pathways using high throughput single-cell RNA-sequencing.

PubMed

Han, Xiaoping; Chen, Haide; Huang, Daosheng; Chen, Huidong; Fei, Lijiang; Cheng, Chen; Huang, He; Yuan, Guo-Cheng; Guo, Guoji

2018-04-05

Human pluripotent stem cells (hPSCs) provide powerful models for studying cellular differentiations and unlimited sources of cells for regenerative medicine. However, a comprehensive single-cell level differentiation roadmap for hPSCs has not been achieved. We use high throughput single-cell RNA-sequencing (scRNA-seq), based on optimized microfluidic circuits, to profile early differentiation lineages in the human embryoid body system. We present a cellular-state landscape for hPSC early differentiation that covers multiple cellular lineages, including neural, muscle, endothelial, stromal, liver, and epithelial cells. Through pseudotime analysis, we construct the developmental trajectories of these progenitor cells and reveal the gene expression dynamics in the process of cell differentiation. We further reprogram primed H9 cells into naïve-like H9 cells to study the cellular-state transition process. We find that genes related to hemogenic endothelium development are enriched in naïve-like H9. Functionally, naïve-like H9 show higher potency for differentiation into hematopoietic lineages than primed cells. Our single-cell analysis reveals the cellular-state landscape of hPSC early differentiation, offering new insights that can be harnessed for optimization of differentiation protocols.

Mesenchymal Stem Cell Therapy for the Treatment of Vocal Fold Scarring: A Systematic Review of Preclinical Studies

PubMed Central

Wingstrand, Vibe Lindeblad; Jensen, David H.; Bork, Kristian; Sebbesen, Lars; Balle, Jesper; Fischer-Nielsen, Anne; von Buchwald, Christian

2016-01-01

Objectives Therapy with mesenchymal stem cells exhibits potential for the development of novel interventions for many diseases and injuries. The use of mesenchymal stem cells in regenerative therapy for vocal fold scarring exhibited promising results to reduce stiffness and enhance the biomechanical properties of injured vocal folds. This study evaluated the biomechanical effects of mesenchymal stem cell therapy for the treatment of vocal fold scarring. Data Sources PubMed, Embase, the Cochrane Library and Google Scholar were searched. Methods Controlled studies that assessed the biomechanical effects of mesenchymal stem cell therapy for the treatment of vocal fold scarring were included. Primary outcomes were viscoelastic properties and mucosal wave amplitude. Results Seven preclinical animal studies (n = 152 single vocal folds) were eligible for inclusion. Evaluation of viscoelastic parameters revealed a decreased dynamic viscosity (η’) and elastic modulus (G’), i.e., decreased resistance and stiffness, in scarred vocal folds treated with mesenchymal stem cells compared to non-treated scarred vocal folds. Mucosal wave amplitude was increased in scarred vocal folds treated with mesenchymal stem cells vs. non-treated scarred vocal folds. Conclusion The results from these studies suggest an increased regenerative effect of therapy with mesenchymal stem cells for scarred vocal folds and are encouraging for further clinical studies. PMID:27631373

Mesenchymal Stem Cell Therapy for the Treatment of Vocal Fold Scarring: A Systematic Review of Preclinical Studies.

PubMed

Wingstrand, Vibe Lindeblad; Grønhøj Larsen, Christian; Jensen, David H; Bork, Kristian; Sebbesen, Lars; Balle, Jesper; Fischer-Nielsen, Anne; von Buchwald, Christian

2016-01-01

Therapy with mesenchymal stem cells exhibits potential for the development of novel interventions for many diseases and injuries. The use of mesenchymal stem cells in regenerative therapy for vocal fold scarring exhibited promising results to reduce stiffness and enhance the biomechanical properties of injured vocal folds. This study evaluated the biomechanical effects of mesenchymal stem cell therapy for the treatment of vocal fold scarring. PubMed, Embase, the Cochrane Library and Google Scholar were searched. Controlled studies that assessed the biomechanical effects of mesenchymal stem cell therapy for the treatment of vocal fold scarring were included. Primary outcomes were viscoelastic properties and mucosal wave amplitude. Seven preclinical animal studies (n = 152 single vocal folds) were eligible for inclusion. Evaluation of viscoelastic parameters revealed a decreased dynamic viscosity (η') and elastic modulus (G'), i.e., decreased resistance and stiffness, in scarred vocal folds treated with mesenchymal stem cells compared to non-treated scarred vocal folds. Mucosal wave amplitude was increased in scarred vocal folds treated with mesenchymal stem cells vs. non-treated scarred vocal folds. The results from these studies suggest an increased regenerative effect of therapy with mesenchymal stem cells for scarred vocal folds and are encouraging for further clinical studies.

High-Throughput Microfluidic Labyrinth for the Label-free Isolation of Circulating Tumor Cells.

PubMed

Lin, Eric; Rivera-Báez, Lianette; Fouladdel, Shamileh; Yoon, Hyeun Joong; Guthrie, Stephanie; Wieger, Jacob; Deol, Yadwinder; Keller, Evan; Sahai, Vaibhav; Simeone, Diane M; Burness, Monika L; Azizi, Ebrahim; Wicha, Max S; Nagrath, Sunitha

2017-09-27

We present "Labyrinth," a label-free microfluidic device to isolate circulating tumor cells (CTCs) using the combination of long loops and sharp corners to focus both CTCs and white blood cells (WBCs) at a high throughput of 2.5 mL/min. The high yield (>90%) and purity (600 WBCs/mL) of Labyrinth enabled us to profile gene expression in CTCs. As proof of principle, we used previously established cancer stem cell gene signatures to profile single cells isolated from the blood of breast cancer patients. We observed heterogeneous subpopulations of CTCs expressing genes for stem cells, epithelial cells, mesenchymal cells, and cells transitioning between epithelial and mesenchymal. Labyrinth offers a cell-surface marker-independent single-cell isolation platform to study heterogeneous CTC subpopulations. Copyright © 2017 Elsevier Inc. All rights reserved.

Reconstructing blood stem cell regulatory network models from single-cell molecular profiles

PubMed Central

Hamey, Fiona K.; Nestorowa, Sonia; Kinston, Sarah J.; Kent, David G.; Wilson, Nicola K.

2017-01-01

Adult blood contains a mixture of mature cell types, each with specialized functions. Single hematopoietic stem cells (HSCs) have been functionally shown to generate all mature cell types for the lifetime of the organism. Differentiation of HSCs toward alternative lineages must be balanced at the population level by the fate decisions made by individual cells. Transcription factors play a key role in regulating these decisions and operate within organized regulatory programs that can be modeled as transcriptional regulatory networks. As dysregulation of single HSC fate decisions is linked to fatal malignancies such as leukemia, it is important to understand how these decisions are controlled on a cell-by-cell basis. Here we developed and applied a network inference method, exploiting the ability to infer dynamic information from single-cell snapshot expression data based on expression profiles of 48 genes in 2,167 blood stem and progenitor cells. This approach allowed us to infer transcriptional regulatory network models that recapitulated differentiation of HSCs into progenitor cell types, focusing on trajectories toward megakaryocyte–erythrocyte progenitors and lymphoid-primed multipotent progenitors. By comparing these two models, we identified and subsequently experimentally validated a difference in the regulation of nuclear factor, erythroid 2 (Nfe2) and core-binding factor, runt domain, alpha subunit 2, translocated to, 3 homolog (Cbfa2t3h) by the transcription factor Gata2. Our approach confirms known aspects of hematopoiesis, provides hypotheses about regulation of HSC differentiation, and is widely applicable to other hierarchical biological systems to uncover regulatory relationships. PMID:28584094

CCR5 Disruption in Induced Pluripotent Stem Cells Using CRISPR/Cas9 Provides Selective Resistance of Immune Cells to CCR5-tropic HIV-1 Virus.

PubMed

Kang, HyunJun; Minder, Petra; Park, Mi Ae; Mesquitta, Walatta-Tseyon; Torbett, Bruce E; Slukvin, Igor I

2015-12-15

The chemokine (C-C motif) receptor 5 (CCR5) serves as an HIV-1 co-receptor and is essential for cell infection with CCR5-tropic viruses. Loss of functional receptor protects against HIV infection. Here, we report the successful targeting of CCR5 in GFP-marked human induced pluripotent stem cells (iPSCs) using CRISPR/Cas9 with single and dual guide RNAs (gRNAs). Following CRISPER/Cas9-mediated gene editing using a single gRNA, 12.5% of cell colonies demonstrated CCR5 editing, of which 22.2% showed biallelic editing as determined by a Surveyor nuclease assay and direct sequencing. The use of dual gRNAs significantly increased the efficacy of CCR5 editing to 27% with a biallelic gene alteration frequency of 41%. To ensure the homogeneity of gene editing within cells, we used single cell sorting to establish clonal iPSC lines. Single cell-derived iPSC lines with homozygous CCR5 mutations displayed the typical characteristics of pluripotent stem cells and differentiated efficiently into hematopoietic cells, including macrophages. Although macrophages from both wild-type and CCR5-edited iPSCs supported CXCR4-tropic virus replication, macrophages from CCR5-edited iPSCs were uniquely resistant to CCR5-tropic virus challenge. This study demonstrates the feasibility of applying iPSC technology for the study of the role of CCR5 in HIV infection in vitro, and generation of HIV-resistant cells for potential therapeutic applications.

Molecular Programs Underlying Asymmetric Stem Cell Division and Their Disruption in Malignancy.

PubMed

Mukherjee, Subhas; Brat, Daniel J

2017-01-01

Asymmetric division of stem cells is a highly conserved and tightly regulated process by which a single stem cell produces two unequal daughter cells. One retains its stem cell identity while the other becomes specialized through a differentiation program and loses stem cell properties. Coordinating these events requires control over numerous intra- and extracellular biological processes and signaling networks. In the initial stages, critical events include the compartmentalization of fate determining proteins within the mother cell and their subsequent passage to the appropriate daughter cell in order to direct their destiny. Disturbance of these events results in an altered dynamic of self-renewing and differentiation within the cell population, which is highly relevant to the growth and progression of cancer. Other critical events include proper asymmetric spindle assembly, extrinsic regulation through micro-environmental cues, and non-canonical signaling networks that impact cell division and fate determination. In this review, we discuss mechanisms that maintain the delicate balance of asymmetric cell division in normal tissues and describe the current understanding how some of these mechanisms are deregulated in cancer.

Mammary stem cells have myoepithelial cell properties

PubMed Central

Prater, Michael D.; Petit, Valérie; Russell, I. Alasdair; Giraddi, Rajshekhar; Shehata, Mona; Menon, Suraj; Schulte, Reiner; Kalajzic, Ivo; Rath, Nicola; Olson, Michael F.; Metzger, Daniel; Faraldo, Marisa M.; Deugnier, Marie-Ange; Glukhova, Marina A.; Stingl, John

2014-01-01

Contractile myoepithelial cells dominate the basal layer of the mammary epithelium and are considered to be differentiated cells. However, we observe that up to 54% of single basal cells can form colonies when seeded into adherent culture in the presence of agents that disrupt acin-myosin interactions, and on average, 65% of the single-cell-derived basal colonies can repopulate a mammary gland when transplanted in vivo. This indicates that a high proportion of basal myoepithelial cells can give rise to a mammary repopulating unit (MRU). We demonstrate that myoepithelial cells, flow-sorted using 2 independent myoepithelial-specific reporter strategies, have MRU capacity. Using an inducible lineage tracing approach we follow the progeny of α-smooth muscle actin-expressing myoepithelial cells and show that they function as long-lived lineage-restricted stem cells in the virgin state and during pregnancy. PMID:25173976

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

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

Lineage-specific enhancers activate self-renewal genes in macrophages and embryonic stem cells

PubMed Central

Soucie, Erinn L.; Weng, Ziming; Geirsdóttir, Laufey; Molawi, Kaaweh; Maurizio, Julien; Fenouil, Romain; Mossadegh-Keller, Noushine; Gimenez, Gregory; VanHille, Laurent; Beniazza, Meryam; Favret, Jeremy; Berruyer, Carole; Perrin, Pierre; Hacohen, Nir; Andrau, J.-C.; Ferrier, Pierre; Dubreuil, Patrice; Sidow, Arend; Sieweke, Michael H.

2016-01-01

Differentiated macrophages can self-renew in tissues and expand long-term in culture, but the gene regulatory mechanisms that accomplish self-renewal in the differentiated state have remained unknown. Here we show that in mice, the transcription factors MafB and c-Maf repress a macrophage-specific enhancer repertoire associated with a gene network controlling self-renewal. Single cell analysis revealed that, in vivo, proliferating resident macrophages can access this network by transient down-regulation of Maf transcription factors. The network also controls embryonic stem cell self-renewal but is associated with distinct embryonic stem cell-specific enhancers. This indicates that distinct lineage-specific enhancer platforms regulate a shared network of genes that control self-renewal potential in both stem and mature cells. PMID:26797145

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.

Symmetric vs. Asymmetric Stem Cell Divisions: An Adaptation against Cancer?

PubMed Central

Shahriyari, Leili; Komarova, Natalia L.

2013-01-01

Traditionally, it has been held that a central characteristic of stem cells is their ability to divide asymmetrically. Recent advances in inducible genetic labeling provided ample evidence that symmetric stem cell divisions play an important role in adult mammalian homeostasis. It is well understood that the two types of cell divisions differ in terms of the stem cells' flexibility to expand when needed. On the contrary, the implications of symmetric and asymmetric divisions for mutation accumulation are still poorly understood. In this paper we study a stochastic model of a renewing tissue, and address the optimization problem of tissue architecture in the context of mutant production. Specifically, we study the process of tumor suppressor gene inactivation which usually takes place as a consequence of two “hits”, and which is one of the most common patterns in carcinogenesis. We compare and contrast symmetric and asymmetric (and mixed) stem cell divisions, and focus on the rate at which double-hit mutants are generated. It turns out that symmetrically-dividing cells generate such mutants at a rate which is significantly lower than that of asymmetrically-dividing cells. This result holds whether single-hit (intermediate) mutants are disadvantageous, neutral, or advantageous. It is also independent on whether the carcinogenic double-hit mutants are produced only among the stem cells or also among more specialized cells. We argue that symmetric stem cell divisions in mammals could be an adaptation which helps delay the onset of cancers. We further investigate the question of the optimal fraction of stem cells in the tissue, and quantify the contribution of non-stem cells in mutant production. Our work provides a hypothesis to explain the observation that in mammalian cells, symmetric patterns of stem cell division seem to be very common. PMID:24204602

Evolution of Energy Metabolism, Stem Cells and Cancer Stem Cells: How the Warburg and Barker Hypotheses Might Be Linked

PubMed Central

Trosko, James E.; Kang, Kyung-Sun

2012-01-01

The evolutionary transition from single cells to the metazoan forced the appearance of adult stem cells and a hypoxic niche, when oxygenation of the environment forced the appearance of oxidative phosphorylation from that of glycolysis. The prevailing paradigm in the cancer field is that cancers start from the “immortalization” or “re-programming” of a normal, differentiated cell with many mitochondria, that metabolize via oxidative phosphorylation. This paradigm has been challenged with one that assumes that the target cell for carcinogenesis is the normal, immortal adult stem cell, with few mitochondria. This adult organ-specific stem cell is blocked from “mortalizing” or from “programming” to be terminally differentiated. Two hypotheses have been offered to explain cancers, namely, the “stem cell theory” and the “de-differentiation” or “re-programming” theory. This Commentary postulates that the paleochemistry of the oceans, which, initially, provided conditions for life’ s energy to arise via glycolysis, changed to oxidative phosphorylation for life’ s processes. In doing so, stem cells evolved, within hypoxic niches, to protect the species germinal and somatic genomes. This Commentary provides support for the “stem cell theory”, in that cancer cells, which, unlike differentiated cells, have few mitochondria and metabolize via glycolysis. The major argument against the “de-differentiation theory” is that, if re-programming of a differentiated cell to an “induced pluri-potent stem cell” happened in an adult, teratomas, rather than carcinomas, should be the result. PMID:24298354

Genome stability of programmed stem cell products.

PubMed

Martin, Ulrich

2017-10-01

Inherited and acquired genomic abnormalities are known to cause genetic diseases and contribute to cancer formation. Recent studies demonstrated a substantial mutational load in mouse and human embryonic and induced pluripotent stem cells (ESCs and iPSCs). Single nucleotide variants, copy number variations, and larger chromosomal abnormalities may influence the differentiation capacity of pluripotent stem cells and the functionality of their derivatives in disease modeling and drug screening, and are considered a serious risk for cellular therapies based on ESC or iPSC derivatives. This review discusses the types and origins of different genetic abnormalities in pluripotent stem cells, methods for their detection, and the mechanisms of development and enrichment during reprogramming and culture expansion. Copyright © 2017 Elsevier B.V. All rights reserved.

Bioengineering of injectable encapsulated aggregates of pluripotent stem cells for therapy of myocardial infarction

NASA Astrophysics Data System (ADS)

Zhao, Shuting; Xu, Zhaobin; Wang, Hai; Reese, Benjamin E.; Gushchina, Liubov V.; Jiang, Meng; Agarwal, Pranay; Xu, Jiangsheng; Zhang, Mingjun; Shen, Rulong; Liu, Zhenguo; Weisleder, Noah; He, Xiaoming

2016-10-01

It is difficult to achieve minimally invasive injectable cell delivery while maintaining high cell retention and animal survival for in vivo stem cell therapy of myocardial infarction. Here we show that pluripotent stem cell aggregates pre-differentiated into the early cardiac lineage and encapsulated in a biocompatible and biodegradable micromatrix, are suitable for injectable delivery. This method significantly improves the survival of the injected cells by more than six-fold compared with the conventional practice of injecting single cells, and effectively prevents teratoma formation. Moreover, this method significantly enhances cardiac function and survival of animals after myocardial infarction, as a result of a localized immunosuppression effect of the micromatrix and the in situ cardiac regeneration by the injected cells.

FACS-based Isolation of Neural and Glioma Stem Cell Populations from Fresh Human Tissues Utilizing EGF Ligand

PubMed Central

Tome-Garcia, Jessica; Doetsch, Fiona; Tsankova, Nadejda M.

2018-01-01

Direct isolation of human neural and glioma stem cells from fresh tissues permits their biological study without prior culture and may capture novel aspects of their molecular phenotype in their native state. Recently, we demonstrated the ability to prospectively isolate stem cell populations from fresh human germinal matrix and glioblastoma samples, exploiting the ability of cells to bind the Epidermal Growth Factor (EGF) ligand in fluorescence-activated cell sorting (FACS). We demonstrated that FACS-isolated EGF-bound neural and glioblastoma populations encompass the sphere-forming colonies in vitro, and are capable of both self-renewal and multilineage differentiation. Here we describe in detail the purification methodology of EGF-bound (i.e., EGFR+) human neural and glioma cells with stem cell properties from fresh postmortem and surgical tissues. The ability to prospectively isolate stem cell populations using native ligand-binding ability opens new doors for understanding both normal and tumor cell biology in uncultured conditions, and is applicable for various downstream molecular sequencing studies at both population and single-cell resolution. PMID:29516026

A CD133-expressing murine liver oval cell population with bilineage potential.

PubMed

Rountree, C Bart; Barsky, Lora; Ge, Shundi; Zhu, Judy; Senadheera, Shantha; Crooks, Gay M

2007-10-01

Although oval cells are postulated to be adult liver stem cells, a well-defined phenotype of a bipotent liver stem cell remains elusive. The heterogeneity of cells within the oval cell fraction has hindered lineage potential studies. Our goal was to identify an enriched population of bipotent oval cells using a combination of flow cytometry and single cell gene expression in conjunction with lineage-specific liver injury models. Expression of cell surface markers on nonparenchymal, nonhematopoietic (CD45-) cells were characterized. Cell populations were isolated by flow cytometry for gene expression studies. 3,5-Diethoxycarbonyl-1,4-dihydrocollidine toxic injury induced cell cycling and expansion specifically in the subpopulation of oval cells in the periportal zone that express CD133. CD133+CD45- cells expressed hepatoblast and stem cell-associated genes, and single cells coexpressed both hepatocyte and cholangiocyte-associated genes, indicating bilineage potential. CD133+CD45- cells proliferated in response to liver injury. Following toxic hepatocyte damage, CD133+CD45- cells demonstrated upregulated expression of the hepatocyte gene Albumin. In contrast, toxic cholangiocyte injury resulted in upregulation of the cholangiocyte gene Ck19. After 21-28 days in culture, CD133+CD45- cells continued to generate cells of both hepatocyte and cholangiocyte lineages. Thus, CD133 expression identifies a population of oval cells in adult murine liver with the gene expression profile and function of primitive, bipotent liver stem cells. In response to lineage-specific injury, these cells demonstrate a lineage-appropriate genetic response. Disclosure of potential conflicts of interest is found at the end of this article.

Mammalian skin cell biology: at the interface between laboratory and clinic.

PubMed

Watt, Fiona M

2014-11-21

Mammalian skin research represents the convergence of three complementary disciplines: cell biology, mouse genetics, and dermatology. The skin provides a paradigm for current research in cell adhesion, inflammation, and tissue stem cells. Here, I discuss recent insights into the cell biology of skin. Single-cell analysis has revealed that human epidermal stem cells are heterogeneous and differentiate in response to multiple extrinsic signals. Live-cell imaging, optogenetics, and cell ablation experiments show skin cells to be remarkably dynamic. High-throughput, genome-wide approaches have yielded unprecedented insights into the circuitry that controls epidermal stem cell fate. Last, integrative biological analysis of human skin disorders has revealed unexpected functions for elements of the skin that were previously considered purely structural. Copyright © 2014, American Association for the Advancement of Science.

Dynamics of embryonic stem cell differentiation inferred from single-cell transcriptomics show a series of transitions through discrete cell states

PubMed Central

Jang, Sumin; Choubey, Sandeep; Furchtgott, Leon; Zou, Ling-Nan; Doyle, Adele; Menon, Vilas; Loew, Ethan B; Krostag, Anne-Rachel; Martinez, Refugio A; Madisen, Linda; Levi, Boaz P; Ramanathan, Sharad

2017-01-01

The complexity of gene regulatory networks that lead multipotent cells to acquire different cell fates makes a quantitative understanding of differentiation challenging. Using a statistical framework to analyze single-cell transcriptomics data, we infer the gene expression dynamics of early mouse embryonic stem (mES) cell differentiation, uncovering discrete transitions across nine cell states. We validate the predicted transitions across discrete states using flow cytometry. Moreover, using live-cell microscopy, we show that individual cells undergo abrupt transitions from a naïve to primed pluripotent state. Using the inferred discrete cell states to build a probabilistic model for the underlying gene regulatory network, we further predict and experimentally verify that these states have unique response to perturbations, thus defining them functionally. Our study provides a framework to infer the dynamics of differentiation from single cell transcriptomics data and to build predictive models of the gene regulatory networks that drive the sequence of cell fate decisions during development. DOI: http://dx.doi.org/10.7554/eLife.20487.001 PMID:28296635

Effects of umbilical cord blood stem cells on healing factors for diabetic foot injuries.

PubMed

Çil, N; Oğuz, E O; Mete, E; Çetinkaya, A; Mete, G A

2017-01-01

The use of stem or progenitor cells from bone marrow, or peripheral or umbilical cord blood is becoming more common for treatment of diabetic foot problems. These cells promote neovascularization by angiogenic factors and they promote epithelium formation by stimulating cell replication and migration under certain pathological conditions. We investigated the role of CD34 + stem cells from human umbilical cord blood in wound healing using a rat model. Rats were randomly divided into a control group and two groups with diabetes induced by a single dose of 55 mg/kg intraperitoneal streptozocin. Scarred areas 5 mm in diameter were created on the feet of all rats. The diabetic rats constituted the diabetes control group and a diabetes + stem cell group with local injection into the wound site of 0.5 × 106 CD34 + stem cells from human umbilical cord blood. The newly formed skin in the foot wounds following CD34 + stem cell treatment showed significantly improvement by immunohistochemistry and TUNEL staining, and were closer to the wound healing of the control group than the untreated diabetic animals. The increase in FGF expression that accompanied the local injection of CD34 + stem cells indicates that FGF stimulation helped prevent apoptosis. Our findings suggest a promising new treatment approach to diabetic wound healing.

Lycopersicon esculentum lectin is a marker of transient amplifying cells in in vitro cultures of isolated limbal stem cells.

PubMed

Vergallo, C; Fonseca, T; Pizzi, G; Dini, L

2010-08-01

The maintenance of a healthy corneal epithelium under both normal and wound healing conditions is achieved by a population of stem cells (SCs) located in the basal epithelium at the corneoscleral limbus. In the light of the development of strategies for reconstruction of the ocular surface in patients with limbal stem cell deficiency, a major challenge in corneal SCs biology remains the ability to identify stem cells in situ and in vitro. To date, not so much markers exist for the identification of different phenotypes. CESCs (corneal epithelial stem cells) isolated from limbal biopsies were maintained in primary culture for 14 days and stained with Hoechst and a panel of FITC-conjugated lectins. All lectins, with the exception of Lycopersicon esculentum, labelled CESCs irrespective of the degree of differentiation. Lycopersicon esculentum, that binds N-acetylglucosamine oligomers, labelled intensely only the surface of TACs (single corneal epithelial stem cells better than colonial cells). These results suggest that Lycopersicon esculentum lectin is a useful and easy-to-use marker for the in vitro identification of TACs (transient amplifying cells) in cultures of isolated CESCs. Copyright 2010. Published by Elsevier Ltd.

Hematopoietic stem cells derived from human umbilical cord ameliorate cisplatin-induced acute renal failure in rats

PubMed Central

Shalaby, Rokaya H; Rashed, Laila A; Ismaail, Alaa E; Madkour, Naglaa K; Elwakeel, Sherien H

2014-01-01

Injury to a target organ can be sensed by bone marrow stem cells that migrate to the site of damage, undergo differentiation, and promote structural and functional repair. This remarkable stem cell capacity prompted an investigation of the potential of mesenchymal and hematopoietic stem cells to cure acute renal failure. On the basis of the recent demonstration that hematopoietic stem cells (HSCs) can differentiate into renal cells, the current study tested the hypothesis that HSCs can contribute to the regeneration of renal tubular epithelial cells after renal injury. HSCs from human umbilical cord blood which isolated and purified by magnetic activated cell sorting were transplanted intraperitoneal into acute renal failure (ARF) rats which was established by a single dose of cisplatin 5 mg/kg for five days. The Study was carried on 48 male white albino rats, of average weight 120-150 gm. The animals were divided into 4 groups, Group one Served as control and received normal saline throughout the experiments. Group two (model control) received a single dose of cisplatin. Group three and four male-albino rats with induced ARF received interapritoneally (HSCs) at two week and four week respectively. Injection of a single dose of cisplatin resulted in a significant increase in serum creatinine and urea levels, histo-pathological examination of kidney tissue from cisplatin showed severe nephrotoxicity in which 50-75% of glomeruli and renal tubules exhibited massive degenerative change. Four weeks after HSC transplantation, Serum creatinine and urea nitrogen decreased 3.5 times and 2.1 times as well as HGF, IGF-1, VEGF and P53 using quantitative real-time PCR increased 4.3 times, 3.2, 2.4 and 4.2 times compared to ARF groups, respectively. The proliferation of cell nuclear antigen (PCNA)-positive cells (500.083±35.167) was higher than that in the cisplatin groups (58.612±15.743). In addition, the transplanted umbilical cord hematopoietic stem cells UC-HSCs could reside in local injury sites, leading to the relief of hyperemia and inflammation, but no obvious transdifferentiation into renal-like cells. The results lay the foundation for further study on the potential application of UC-HSCs in human disease and Because of their availability; HSC may be useful for cell replacement therapy of acute renal failure. PMID:25232508

Automatic cell cloning assay for determining the clonogenic capacity of cancer and cancer stem-like cells.

PubMed

Fedr, Radek; Pernicová, Zuzana; Slabáková, Eva; Straková, Nicol; Bouchal, Jan; Grepl, Michal; Kozubík, Alois; Souček, Karel

2013-05-01

The clonogenic assay is a well-established in vitro method for testing the survival and proliferative capability of cells. It can be used to determine the cytotoxic effects of various treatments including chemotherapeutics and ionizing radiation. However, this approach can also characterize cells with different phenotypes and biological properties, such as stem cells or cancer stem cells. In this study, we implemented a faster and more precise method for assessing the cloning efficiency of cancer stem-like cells that were characterized and separated using a high-speed cell sorter. Cell plating onto a microplate using an automatic cell deposition unit was performed in a single-cell or dilution rank mode by the fluorescence-activated cell sorting method. We tested the new automatic cell-cloning assay (ACCA) on selected cancer cell lines and compared it with the manual approach. The obtained results were also compared with the results of the limiting dilution assay for different cell lines. We applied the ACCA to analyze the cloning capacity of different subpopulations of prostate and colon cancer cells based on the expression of the characteristic markers of stem (CD44 and CD133) and cancer stem cells (TROP-2, CD49f, and CD44). Our results revealed that the novel ACCA is a straightforward approach for determining the clonogenic capacity of cancer stem-like cells identified in both cell lines and patient samples. Copyright © 2013 International Society for Advancement of Cytometry.

Generation of inner ear organoids containing functional hair cells from human pluripotent stem cells.

PubMed

Koehler, Karl R; Nie, Jing; Longworth-Mills, Emma; Liu, Xiao-Ping; Lee, Jiyoon; Holt, Jeffrey R; Hashino, Eri

2017-06-01

The derivation of human inner ear tissue from pluripotent stem cells would enable in vitro screening of drug candidates for the treatment of hearing and balance dysfunction and may provide a source of cells for cell-based therapies of the inner ear. Here we report a method for differentiating human pluripotent stem cells to inner ear organoids that harbor functional hair cells. Using a three-dimensional culture system, we modulate TGF, BMP, FGF, and WNT signaling to generate multiple otic-vesicle-like structures from a single stem-cell aggregate. Over 2 months, the vesicles develop into inner ear organoids with sensory epithelia that are innervated by sensory neurons. Additionally, using CRISPR-Cas9, we generate an ATOH1-2A-eGFP cell line to detect hair cell induction and demonstrate that derived hair cells exhibit electrophysiological properties similar to those of native sensory hair cells. Our culture system should facilitate the study of human inner ear development and research on therapies for diseases of the inner ear.

Electron microscopy of whole cells in liquid with nanometer resolution

PubMed Central

de Jonge, N.; Peckys, D. B.; Kremers, G. J.; Piston, D. W.

2009-01-01

Single gold-tagged epidermal growth factor (EGF) molecules bound to cellular EGF receptors of fixed fibroblast cells were imaged in liquid with a scanning transmission electron microscope (STEM). The cells were placed in buffer solution in a microfluidic device with electron transparent windows inside the vacuum of the electron microscope. A spatial resolution of 4 nm and a pixel dwell time of 20 μs were obtained. The liquid layer was sufficiently thick to contain the cells with a thickness of 7 ± 1 μm. The experimental findings are consistent with a theoretical calculation. Liquid STEM is a unique approach for imaging single molecules in whole cells with significantly improved resolution and imaging speed over existing methods. PMID:19164524

Hydrogel microfluidics for the patterning of pluripotent stem cells

NASA Astrophysics Data System (ADS)

Cosson, S.; Lutolf, M. P.

2014-03-01

Biomolecular signaling is of utmost importance in governing many biological processes such as the patterning of the developing embryo where biomolecules regulate key cell-fate decisions. In vivo, these factors are presented in a spatiotemporally tightly controlled fashion. Although state-of-the-art microfluidic technologies allow precise biomolecule delivery in time and space, long-term (stem) cell culture at the micro-scale is often far from ideal due to medium evaporation, limited space for cell growth or shear stress. To overcome these challenges, we here introduce a concept based on hydrogel microfluidics for decoupling conventional, macro-scale cell culture from precise biomolecule delivery through a gel layer. We demonstrate the spatiotemporally controlled neuronal commitment of mouse embryonic stem cells via delivery of retinoic acid gradients. This technique should be useful for testing the effect of dose and timing of biomolecules, singly or in combination, on stem cell fate.

Stem cell based anti-HIV Gene therapy

PubMed Central

Kitchen, Scott G.; Shimizu, Saki; An, Dong Sung

2011-01-01

Human stem cell-based therapeutic intervention strategies for treating HIV infection have recently undergone a renaissance as a major focus of investigation. Unlike most conventional antiviral therapies, genetically engineered hematopoietic stem cells possess the capacity for prolonged self-renewal that would continuously produce protected immune cells to fight against HIV. A successful strategy therefore has the potential to stably control and ultimately eradicate HIV from patients by a single or minimal treatment. Recent progress in the development of new technologies and clinical trials sets the stage for the current generation of gene therapy approaches to combat HIV infection. In this review, we will discuss two major approaches that are currently underway in the development of stem cell-based gene therapy to target HIV: One that focuses on the protection of cells from productive infection with HIV, and the other that focuses on targeting immune cells to directly combat HIV infection. PMID:21247612

Molecular imaging in stem cell-based therapies of cardiac diseases.

PubMed

Li, Xiang; Hacker, Marcus

2017-10-01

In the past 15years, despite that regenerative medicine has shown great potential for cardiovascular diseases, the outcome and safety of stem cell transplantation has shown controversial results in the published literature. Medical imaging might be useful for monitoring and quantifying transplanted cells within the heart and to serially characterize the effects of stem cell therapy of the myocardium. From the multiple available noninvasive imaging techniques, magnetic resonance imaging and nuclear imaging by positron (PET) or single photon emission computer tomography (SPECT) are the most used clinical approaches to follow the fate of transplanted stem cells in vivo. In this article, we provide a review on the role of different noninvasive imaging modalities and discuss their advantages and disadvantages. We focus on the different in-vivo labeling and reporter gene imaging strategies for stem cell tracking as well as the concept and reliability to use imaging parameters as noninvasive surrogate endpoints for the evaluation of the post-therapeutic outcome. Copyright © 2017 Elsevier B.V. All rights reserved.

Fluorescence lifetime imaging of induced pluripotent stem cells

NASA Astrophysics Data System (ADS)

Uchugonova, Aisada; Batista, Ana; König, Karsten

2014-02-01

The multiphoton FLIM tomograph MPTflex with its flexible scan head, articulated arm, and the tunable femtosecond laser source was employed to study cell monolayers and 3D cell clusters. FLIM was performed with 250 ps temporal resolution and submicron special resolution using time-correlated single photon counting. The autofluorescence based on NAD(P)H and flavins/flavoproteins has been measured in mouse embryonic fibroblasts, induced pluripotent stem cells (iPS cells) originated from mouse embryonic fibroblasts and non-proliferative mouse embryonic fibroblasts.

Validation of high-throughput single cell analysis methodology.

PubMed

Devonshire, Alison S; Baradez, Marc-Olivier; Morley, Gary; Marshall, Damian; Foy, Carole A

2014-05-01

High-throughput quantitative polymerase chain reaction (qPCR) approaches enable profiling of multiple genes in single cells, bringing new insights to complex biological processes and offering opportunities for single cell-based monitoring of cancer cells and stem cell-based therapies. However, workflows with well-defined sources of variation are required for clinical diagnostics and testing of tissue-engineered products. In a study of neural stem cell lines, we investigated the performance of lysis, reverse transcription (RT), preamplification (PA), and nanofluidic qPCR steps at the single cell level in terms of efficiency, precision, and limit of detection. We compared protocols using a separate lysis buffer with cell capture directly in RT-PA reagent. The two methods were found to have similar lysis efficiencies, whereas the direct RT-PA approach showed improved precision. Digital PCR was used to relate preamplified template copy numbers to Cq values and reveal where low-quality signals may affect the analysis. We investigated the impact of calibration and data normalization strategies as a means of minimizing the impact of inter-experimental variation on gene expression values and found that both approaches can improve data comparability. This study provides validation and guidance for the application of high-throughput qPCR workflows for gene expression profiling of single cells. Copyright © 2014 Elsevier Inc. All rights reserved.

Live-Cell Imaging of the Adult Drosophila Ovary Using Confocal Microscopy.

PubMed

Shalaby, Nevine A; Buszczak, Michael

2017-01-01

The Drosophila ovary represents a key in vivo model used to study germline stem cell (GSC) maintenance and stem cell daughter differentiation because these cells and their somatic cell neighbors can be identified at single-cell resolution within their native environment. Here we describe a fluorescent-based technique for the acquisition of 4D datasets of the Drosophila ovariole for periods that can exceed 12 consecutive hours. Live-cell imaging facilitates the investigation of molecular and cellular dynamics that were not previously possible using still images.

Hydrogel Encapsulation Facilitates Rapid-Cooling Cryopreservation of Stem Cell-Laden Core-Shell Microcapsules as Cell-Biomaterial Constructs.

PubMed

Zhao, Gang; Liu, Xiaoli; Zhu, Kaixuan; He, Xiaoming

2017-12-01

Core-shell structured stem cell microencapsulation in hydrogel has wide applications in tissue engineering, regenerative medicine, and cell-based therapies because it offers an ideal immunoisolative microenvironment for cell delivery and 3D culture. Long-term storage of such microcapsules as cell-biomaterial constructs by cryopreservation is an enabling technology for their wide distribution and ready availability for clinical transplantation. However, most of the existing studies focus on cryopreservation of single cells or cells in microcapsules without a core-shell structure (i.e., hydrogel beads). The goal of this study is to achieve cryopreservation of stem cells encapsulated in core-shell microcapsules as cell-biomaterial constructs or biocomposites. To this end, a capillary microfluidics-based core-shell alginate hydrogel encapsulation technology is developed to produce porcine adipose-derived stem cell-laden microcapsules for vitreous cryopreservation with very low concentration (2 mol L -1 ) of cell membrane penetrating cryoprotective agents (CPAs) by suppressing ice formation. This may provide a low-CPA and cost-effective approach for vitreous cryopreservation of "ready-to-use" stem cell-biomaterial constructs, facilitating their off-the-shelf availability and widespread applications. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

cgCorrect: a method to correct for confounding cell-cell variation due to cell growth in single-cell transcriptomics

NASA Astrophysics Data System (ADS)

Blasi, Thomas; Buettner, Florian; Strasser, Michael K.; Marr, Carsten; Theis, Fabian J.

2017-06-01

Accessing gene expression at a single-cell level has unraveled often large heterogeneity among seemingly homogeneous cells, which remains obscured when using traditional population-based approaches. The computational analysis of single-cell transcriptomics data, however, still imposes unresolved challenges with respect to normalization, visualization and modeling the data. One such issue is differences in cell size, which introduce additional variability into the data and for which appropriate normalization techniques are needed. Otherwise, these differences in cell size may obscure genuine heterogeneities among cell populations and lead to overdispersed steady-state distributions of mRNA transcript numbers. We present cgCorrect, a statistical framework to correct for differences in cell size that are due to cell growth in single-cell transcriptomics data. We derive the probability for the cell-growth-corrected mRNA transcript number given the measured, cell size-dependent mRNA transcript number, based on the assumption that the average number of transcripts in a cell increases proportionally to the cell’s volume during the cell cycle. cgCorrect can be used for both data normalization and to analyze the steady-state distributions used to infer the gene expression mechanism. We demonstrate its applicability on both simulated data and single-cell quantitative real-time polymerase chain reaction (PCR) data from mouse blood stem and progenitor cells (and to quantitative single-cell RNA-sequencing data obtained from mouse embryonic stem cells). We show that correcting for differences in cell size affects the interpretation of the data obtained by typically performed computational analysis.

77 FR 21788 - Center for Scientific Review Notice of Closed Meetings

Federal Register 2010, 2011, 2012, 2013, 2014

2012-04-11

... Development, Stem Cell and Myocardial Regeneration. Date: May 10, 2012. Time: 2 p.m. to 3:30 p.m. Agenda: To... for Scientific Review Special Emphasis Panel; RFA Panel: Single Cell Analysis Reviews. Date: May 8... for Scientific Review Special Emphasis Panel; RFA Panel: Single Cell Analysis Reviews. Date: May 9...

Coregistration of Magnetic Resonance and Single Photon Emission Computed Tomography Images for Noninvasive Localization of Stem Cells Grafted in the Infarcted Rat Myocardium

PubMed Central

Shen, Dinggang; Liu, Dengfeng; Cao, Zixiong; Acton, Paul D.; Zhou, Rong

2008-01-01

This paper demonstrates the application of mutual information based coregistration of radionuclide and magnetic resonance imaging (MRI) in an effort to use multimodality imaging for noninvasive localization of stem cells grafted in the infarcted myocardium in rats. Radionuclide imaging such as single photon emission computed tomography (SPECT) or positron emission tomography (PET) inherently has high sensitivity and is suitable for tracking of labeled stem cells, while high-resolution MRI is able to provide detailed anatomical and functional information of myocardium. Thus, coregistration of PET or SPECT images with MRI will map the location and distribution of stem cells on detailed myocardium structures. To validate this coregistration method, SPECT data were simulated by using a Monte Carlo-based projector that modeled the pinhole-imaging physics assuming nonzero diameter and photon penetration at the edge. Translational and rotational errors of the coregistration were examined with respect to various SPECT activities, and they are on average about 0.50 mm and 0.82°, respectively. Only the rotational error is dependent on activity of SPECT data. Stem cells were labeled with 111 Indium oxyquinoline and grafted in the ischemic myocardium of a rat model. Dual-tracer small-animal SPECT images were acquired, which allowed simultaneous detection of 111In-labeled stem cells and of [99mTc]sestamibi to assess myocardial perfusion deficit. The same animals were subjected to cardiac MRI. A mutual-information-based coregistration method was then applied to the SPECT and MRIs. By coregistration, the 111 In signal from labeled cells was mapped into the akinetic region identified on cine MRIs; the regional perfusion deficit on the SPECT images also coincided with the akinetic region on the MR image. PMID:17053860

Lineage-specific enhancers activate self-renewal genes in macrophages and embryonic stem cells.

PubMed

Soucie, Erinn L; Weng, Ziming; Geirsdóttir, Laufey; Molawi, Kaaweh; Maurizio, Julien; Fenouil, Romain; Mossadegh-Keller, Noushine; Gimenez, Gregory; VanHille, Laurent; Beniazza, Meryam; Favret, Jeremy; Berruyer, Carole; Perrin, Pierre; Hacohen, Nir; Andrau, J-C; Ferrier, Pierre; Dubreuil, Patrice; Sidow, Arend; Sieweke, Michael H

2016-02-12

Differentiated macrophages can self-renew in tissues and expand long term in culture, but the gene regulatory mechanisms that accomplish self-renewal in the differentiated state have remained unknown. Here we show that in mice, the transcription factors MafB and c-Maf repress a macrophage-specific enhancer repertoire associated with a gene network that controls self-renewal. Single-cell analysis revealed that, in vivo, proliferating resident macrophages can access this network by transient down-regulation of Maf transcription factors. The network also controls embryonic stem cell self-renewal but is associated with distinct embryonic stem cell-specific enhancers. This indicates that distinct lineage-specific enhancer platforms regulate a shared network of genes that control self-renewal potential in both stem and mature cells. Copyright © 2016, American Association for the Advancement of Science.

Reactive astrocytes as neural stem or progenitor cells: In vivo lineage, In vitro potential, and Genome‐wide expression analysis

PubMed Central

Sirko, Swetlana; Beckers, Johannes; Irmler, Martin

2015-01-01

Here, we review the stem cell hallmarks of endogenous neural stem cells (NSCs) during development and in some niches of the adult mammalian brain to then compare these with reactive astrocytes acquiring stem cell hallmarks after traumatic and ischemic brain injury. Notably, even endogenous NSCs including the earliest NSCs, the neuroepithelial cells, generate in most cases only a single type of progeny and self‐renew only for a rather short time in vivo. In vitro, however, especially cells cultured under neurosphere conditions reveal a larger potential and long‐term self‐renewal under the influence of growth factors. This is rather well comparable to reactive astrocytes in the traumatic or ischemic brain some of which acquire neurosphere‐forming capacity including multipotency and long‐term self‐renewal in vitro, while they remain within their astrocyte lineage in vivo. Both reactive astrocytes and endogenous NSCs exhibit stem cell hallmarks largely in vitro, but their lineage differs in vivo. Both populations generate largely a single cell type in vivo, but endogenous NSCs generate neurons and reactive astrocytes remain in the astrocyte lineage. However, at some early postnatal stages or in some brain regions reactive astrocytes can be released from this fate restriction, demonstrating that they can also enact neurogenesis. Thus, reactive astrocytes and NSCs share many characteristic hallmarks, but also exhibit key differences. This conclusion is further substantiated by genome‐wide expression analysis comparing NSCs at different stages with astrocytes from the intact and injured brain parenchyma. GLIA 2015;63:1452–1468 PMID:25965557

A microfluidic chaotic mixer platform for cancer stem cell immunocapture and release

NASA Astrophysics Data System (ADS)

Shaner, Sebastian Wesley

Isolation of exceedingly rare and ambiguous cells, like cancer stem cells (CSCs), from a pool of other abundant cells is a daunting task primarily due to the inadequately defined properties of such cells. With phenotypes of different CSCs fairly well-defined, immunocapturing of CSCs is a desirable cell-specific capture technique. A microfluidic device is a proven candidate that offers the platform for user-constrained microenvironments that can be optimized for small-scale volumetric flow experimentation. In this study, we show how a well-known passive micromixer design (staggered herringbone mixer - SHM) can be optimized to induce maximum chaotic mixing within antibody-laced microchannels and, ultimately, promote CSC capture. The device's (Cancer Stem Cell Capture Chip - CSC3 (TM)) principle design configuration is called: Single-Walled Staggered Herringbone (SWaSH). The CSC3 (TM) was constructed of a polydimethylsiloxane (PDMS) foundation and thinly coated with an alginate hydrogel derivatized with streptavidin. The results of our work showed that the non-stickiness of alginate and antigen-specific antibodies allowed for superb target-specific cell isolation and negligible non-specific cell binding. Future engineering design directions include developing new configurations (e.g. Staggered High-Low Herringbone (SHiLoH) and offset SHiLoH) to optimize microvortex generation within the microchannels. This study's qualitative and quantitative results can help stimulate progress into refinements in device design and prospective advancements in cancer stem cell isolation and more comprehensive single-cell and cluster analysis.

Prospective isolation of multipotent pancreatic progenitors using flow-cytometric cell sorting.

PubMed

Suzuki, Atsushi; Nakauchi, Hiromitsu; Taniguchi, Hideki

2004-08-01

During pancreatic development, neogenesis, and regeneration, stem cells might act as a central player to generate endocrine, acinar, and duct cells. Although these cells are well known as pancreatic stem cells (PSCs), indisputable proof of their existence has not been reported. Identification of phenotypic markers for PSCs leads to their prospective isolation and precise characterization to clear whether stem cells exist in the pancreas. By combining flow cytometry and clonal analysis, we show here that a possible pancreatic stem or progenitor cell candidate that resides in the developing and adult mouse pancreas expresses the receptor for the hepatocyte growth factor (HGF) c-Met, but does not express hematopoietic and vascular endothelial antigens such as CD45, TER119, c-Kit, and Flk-1. These cells formed clonal colonies in vitro and differentiated into multiple pancreatic lineage cells from single cells. Some of them could largely expand with self-renewing cell divisions in culture, and, following cell transplantation, they differentiated into pancreatic endocrine and acinar cells in vivo. Furthermore, they produced cells expressing multiple markers of nonpancreatic organs including liver, stomach, and intestine in vitro. Our data strongly suggest that c-Met/HGF signaling plays an important role in stem/progenitor cell function in both developing and adult pancreas. By using this antigen, PSCs could be isolated prospectively, enabling a detailed investigation of stem cell markers and application toward regenerative therapies for diabetes.

Stem cell transplantation (cord blood transplants).

PubMed

Chao, Nelson J; Emerson, Stephen G; Weinberg, Kenneth I

2004-01-01

Allogeneic stem cell transplantation is an accepted treatment modality for selected malignant and non-malignant diseases. However, the ability to identify suitably matched related or unrelated donors can be difficult in some patients. Alternative sources of stem cells such as cord blood provide a readily available graft for such patients. Data accumulated over the past several years have demonstrated that the use of cord blood is an accepted source of stem cells for pediatric patients. Since the cell numbers of hematopoietic progenitors in cord blood is limited and the collection can occur only in a single occasion, its use in adult patients can be more problematic. Here, new developments in the use of cord blood for adults and studies aimed at expansion of cord blood cells and immune reconstitution are described. In Section I, Dr. Nelson Chao describes the early data in cord blood transplantation in adult patients. The patient outcomes are reviewed and analyzed for various factors such as cell dose, HLA typing, and patient selection that could have contributed to the final outcome of these adult patients. Myeloablative as well as nonmyeloablative approaches are presented. Discussion of the various benefits and risks are presented. More recent data from multiple single institutions as well as larger registry data comparisons are also provided. Analyses of these studies suggest methods to improve on the outcome. These newer data should lead to a logical progression in the use of cord blood cells in adult patients. In Section II, Dr. Stephen Emerson describes the historical efforts associated with expansion of hematopoietic stem cells, specifically with cord blood cells. These efforts to expand cord blood cells continue with novel methods. Moreover, a better understanding of stem cell biology and signaling is critical if we are to be able to effectively expand these cells for clinical use. An alternative, more direct, approach to expanding stem cells could be achieved by specific genetic pathways known or believed to support primitive HSC proliferation such as Notch-1 receptor activation, Wnt/LEF-1 pathway induction, telomerase or the Homeobox (Hox) gene products. The clinical experience with the use of expanded cord blood cells is also discussed. In Section III, Dr. Kenneth Weinberg describes immune reconstitution or lack thereof following cord blood transplantation. One of the hallmarks of successful hematopoietic stem cell transplantation is the ability to fully reconstitute the immune system of the recipient. Thus, the relationship between stem cell source and the development of T lymphocyte functions required for protection of the recipient from infection will be described, and cord blood recipients will be compared with those receiving other sources of stem cells. T cell development is described in detail, tracking from prethymic to postthymic lymphocytes with specific attention to umbilical cord blood as the source of stem cells. Moreover, a discussion of the placenta as a special microenvironment for umbilical cord blood is presented. Strategies to overcome the immunological defects are presented to improve the outcome of these recipients.

Cancer stem cell-like cells from a single cell of oral squamous carcinoma cell lines

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

Felthaus, O.; Department of Oral and Maxillofacial Surgery, University of Regensburg; Ettl, T.

2011-04-01

Research highlights: {yields} Four oral squamous cancer cell lines (OSCCL) were analyzed for cancer stem cells (CSCs). {yields} Single cell derived colonies of OSCCL express CSC-marker CD133 differentially. {yields} Monoclonal cell lines showed reduced sensitivity for Paclitaxel. {yields} In situ CD133{sup +} cells are slow cycling (Ki67-) indicating a reduced drug sensitivity. {yields} CD133{sup +} and CSC-like cells can be obtained from single colony forming cells of OSCCL. -- Abstract: Resistance of oral squamous cell carcinomas (OSCC) to conventional chemotherapy or radiation therapy might be due to cancer stem cells (CSCs). The development of novel anticancer drugs requires a simplemore » method for the enrichment of CSCs. CSCs can be enriched from OSCC cell lines, for example, after cultivation in serum-free cell culture medium (SFM). In our study, we analyzed four OSCC cell lines for the presence of CSCs. CSC-like cells could not be enriched with SFM. However, cell lines obtained from holoclone colonies showed CSC-like properties such as a reduced rate of cell proliferation and a reduced sensitivity to Paclitaxel in comparison to cells from the parental lineage. Moreover, these cell lines differentially expressed the CSC-marker CD133, which is also upregulated in OSCC tissues. Interestingly, CD133{sup +} cells in OSCC tissues expressed little to no Ki67, the cell proliferation marker that also indicates reduced drug sensitivity. Our study shows a method for the isolation of CSC-like cell lines from OSCC cell lines. These CSC-like cell lines could be new targets for the development of anticancer drugs under in vitro conditions.« less

Single-cell lineage tracking analysis reveals that an established cell line comprises putative cancer stem cells and their heterogeneous progeny

PubMed Central

Sato, Sachiko; Rancourt, Ann; Sato, Yukiko; Satoh, Masahiko S.

2016-01-01

Mammalian cell culture has been used in many biological studies on the assumption that a cell line comprises putatively homogeneous clonal cells, thereby sharing similar phenotypic features. This fundamental assumption has not yet been fully tested; therefore, we developed a method for the chronological analysis of individual HeLa cells. The analysis was performed by live cell imaging, tracking of every single cell recorded on imaging videos, and determining the fates of individual cells. We found that cell fate varied significantly, indicating that, in contrast to the assumption, the HeLa cell line is composed of highly heterogeneous cells. Furthermore, our results reveal that only a limited number of cells are immortal and renew themselves, giving rise to the remaining cells. These cells have reduced reproductive ability, creating a functionally heterogeneous cell population. Hence, the HeLa cell line is maintained by the limited number of immortal cells, which could be putative cancer stem cells. PMID:27003384

Human skeletal muscle-derived stem cells retain stem cell properties after expansion in myosphere culture

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

Wei, Yan; Department of Otolaryngology, Head and Neck Surgery, The Third Affiliated Hospital of Sun Yat-sen University, Guang Zhou; Li, Yuan

2011-04-15

Human skeletal muscle contains an accessible adult stem-cell compartment in which differentiated myofibers are maintained and replaced by a self-renewing stem cell pool. Previously, studies using mouse models have established a critical role for resident stem cells in skeletal muscle, but little is known about this paradigm in human muscle. Here, we report the reproducible isolation of a population of cells from human skeletal muscle that is able to proliferate for extended periods of time as floating clusters of rounded cells, termed 'myospheres' or myosphere-derived progenitor cells (MDPCs). The phenotypic characteristics and functional properties of these cells were determined usingmore » reverse transcription-polymerase chain reaction (RT-PCR), flow cytometry and immunocytochemistry. Our results showed that these cells are clonogenic, express skeletal progenitor cell markers Pax7, ALDH1, Myod, and Desmin and the stem cell markers Nanog, Sox2, and Oct3/4 significantly elevated over controls. They could be maintained proliferatively active in vitro for more than 20 weeks and passaged at least 18 times, despite an average donor-age of 63 years. Individual clones (4.2%) derived from single cells were successfully expanded showing clonogenic potential and sustained proliferation of a subpopulation in the myospheres. Myosphere-derived cells were capable of spontaneous differentiation into myotubes in differentiation media and into other mesodermal cell lineages in induction media. We demonstrate here that direct culture and expansion of stem cells from human skeletal muscle is straightforward and reproducible with the appropriate technique. These cells may provide a viable resource of adult stem cells for future therapies of disease affecting skeletal muscle or mesenchymal lineage derived cell types.« less

Tumoral stem cell reprogramming as a driver of cancer: Theory, biological models, implications in cancer therapy.

PubMed

Vicente-Dueñas, Carolina; Hauer, Julia; Ruiz-Roca, Lucía; Ingenhag, Deborah; Rodríguez-Meira, Alba; Auer, Franziska; Borkhardt, Arndt; Sánchez-García, Isidro

2015-06-01

Cancer is a clonal malignant disease originated in a single cell and characterized by the accumulation of partially differentiated cells that are phenotypically reminiscent of normal stages of differentiation. According to current models, therapeutic strategies that block oncogene activity are likely to selectively target tumor cells. However, recent evidences have revealed that cancer stem cells could arise through a tumor stem cell reprogramming mechanism, suggesting that genetic lesions that initiate the cancer process might be dispensable for tumor progression and maintenance. This review addresses the impact of these results toward a better understanding of cancer development and proposes new approaches to treat cancer in the future. Copyright © 2014 Elsevier Ltd. All rights reserved.

TOPICAL REVIEW: Stem cell technology using bioceramics: hard tissue regeneration towards clinical application

NASA Astrophysics Data System (ADS)

Ohnishi, Hiroe; Oda, Yasuaki; Ohgushi, Hajime

2010-02-01

Mesenchymal stem cells (MSCs) are adult stem cells which show differentiation capabilities toward various cell lineages. We have already used MSCs for treatments of osteoarthritis, bone necrosis and bone tumor. For this purpose, culture expanded MSCs were combined with various ceramics and then implanted. Because of rejection response to allogeneic MSC implantation, we have utilized patients' own MSCs for the treatment. Bone marrow is a good cell source of MSCs, although the MSCs also exist in adipose tissue. When comparing osteogenic differentiation of these MSCs, bone marrow MSCs show more extensive bone forming capability than adipose MSCs. Thus, the bone marrow MSCs are useful for bone tissue regeneration. However, the MSCs show limited proliferation and differentiation capabilities that hindered clinical applications in some cases. Recent advances reveal that transduction of plural transcription factors into human adult cells results in generation of new type of stem cells called induced pluripotent stem cells (iPS cells). A drawback of the iPS cells for clinical applications is tumor formation after their in vivo implantation; therefore it is difficult to use iPS cells for the treatment. To circumvent the problem, we transduced a single factor of either SOX2 or NANOG into the MSCs and found high proliferation as well as osteogenic differentiation capabilities of the MSCs. The stem cells could be combined with bioceramics for clinical applications. Here, we summarize our recent technologies using adult stem cells in viewpoints of bone tissue regeneration.

Dedifferentiation into blastomere-like cancer stem cells via formation of polyploid giant cancer cells

PubMed Central

Niu, N; Mercado-Uribe, I; Liu, J

2017-01-01

Our recent perplexing findings that polyploid giant cancer cells (PGCCs) acquired embryonic-like stemness and were capable of tumor initiation raised two important unanswered questions: how do PGCCs acquire such stemness, and to which stage of normal development do PGCCs correspond. Intriguingly, formation of giant cells due to failed mitosis/cytokinesis is common in the blastomere stage of the preimplantation embryo. However, the relationship between PGCCs and giant blastomeres has never been studied. Here, we tracked the fate of single PGCCs following paclitaxel-induced mitotic failure. Morphologically, early spheroids derived from PGCCs were indistinguishable from human embryos at the blastomere, polyploid blastomere, compaction, morula and blastocyst-like stages by light, scanning electron or three-dimensional confocal scanning microscopy. Formation of PGCCs was associated with activation of senescence, while budding of daughter cells was associated with senescence escape. PGCCs showed time- and space-dependent activation of expression of the embryonic stem cell markers OCT4, NANOG, SOX2 and SSEA1 and lacked expression of Xist. PGCCs acquired mesenchymal phenotype and were capable of differentiation into all three germ layers in vitro. The embryonic-like stemness of PGCCs was associated with nuclear accumulation of YAP, a key mediator of the Hippo pathway. Spheroids derived from single PGCCs grew into a wide spectrum of human neoplasms, including germ cell tumors, high-grade and low-grade carcinomas and benign tissues. Daughter cells derived from PGCCs showed attenuated capacity for invasion and increased resistance to paclitaxel. We also observed formation of PGCCs and dedifferentiation in ovarian cancer specimens from patients treated with chemotherapy. Taken together, our findings demonstrate that PGCCs represent somatic equivalents of blastomeres, the most primitive cancer stem cells reported to date. Thus, our studies reveal an evolutionarily conserved archaic embryonic program in somatic cells that can be de-repressed for oncogenesis. Our work offers a new paradigm for cancer origin and disease relapse. PMID:28436947

Multi-level characterization of balanced inhibitory-excitatory cortical neuron network derived from human pluripotent stem cells.

PubMed

Nadadhur, Aishwarya G; Emperador Melero, Javier; Meijer, Marieke; Schut, Desiree; Jacobs, Gerbren; Li, Ka Wan; Hjorth, J J Johannes; Meredith, Rhiannon M; Toonen, Ruud F; Van Kesteren, Ronald E; Smit, August B; Verhage, Matthijs; Heine, Vivi M

2017-01-01

Generation of neuronal cultures from induced pluripotent stem cells (hiPSCs) serve the studies of human brain disorders. However we lack neuronal networks with balanced excitatory-inhibitory activities, which are suitable for single cell analysis. We generated low-density networks of hPSC-derived GABAergic and glutamatergic cortical neurons. We used two different co-culture models with astrocytes. We show that these cultures have balanced excitatory-inhibitory synaptic identities using confocal microscopy, electrophysiological recordings, calcium imaging and mRNA analysis. These simple and robust protocols offer the opportunity for single-cell to multi-level analysis of patient hiPSC-derived cortical excitatory-inhibitory networks; thereby creating advanced tools to study disease mechanisms underlying neurodevelopmental disorders.

Application of single-cell technology in cancer research.

PubMed

Liang, Shao-Bo; Fu, Li-Wu

2017-07-01

In this review, we have outlined the application of single-cell technology in cancer research. Single-cell technology has made encouraging progress in recent years and now provides the means to detect rare cancer cells such as circulating tumor cells and cancer stem cells. We reveal how this technology has advanced the analysis of intratumor heterogeneity and tumor epigenetics, and guided individualized treatment strategies. The future prospects now are to bring single-cell technology into the clinical arena. We believe that the clinical application of single-cell technology will be beneficial in cancer diagnostics and treatment, and ultimately improve survival in cancer patients. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

The unregulated commercialization of stem cell treatments: a global perspective.

PubMed

Sipp, Douglas

2011-12-01

Research into the biological properties and clinical potential of stem cells has spurred strong public investment, industry development, media coverage, and patient interest in recent years. To date, however, few clinical applications of demonstrated safety and efficacy have been developed with the exception of uses of hematopoietic stem cells in the treatment of diseases of the blood and immune systems. This lack of an evidence basis notwithstanding, hundreds of companies and private clinics around the world now sell putative stem cell treatments for an enormously broad range of medical and quality-of-life conditions. This represents a major challenge for legitimate scientists working in the field, for authorities seeking to protect their constituencies, and for patients and consumers targeted by such companies' marketing strategies. In this review, I provide an overview of the global industry in pseudomedical stem cell treatments, with an investigation of claims in a single disease area (amyotrophic lateral sclerosis), and make recommendations for the introduction and enforcement of appropriate regulatory responses to this problem.

Genetically Encoded Photoactuators and Photosensors for Characterization and Manipulation of Pluripotent Stem Cells

PubMed Central

Pomeroy, Jordan E.; Nguyen, Hung X.; Hoffman, Brenton D.; Bursac, Nenad

2017-01-01

Our knowledge of pluripotent stem cell biology has advanced considerably in the past four decades, but it has yet to deliver on the great promise of regenerative medicine. The slow progress can be mainly attributed to our incomplete understanding of the complex biologic processes regulating the dynamic developmental pathways from pluripotency to fully-differentiated states of functional somatic cells. Much of the difficulty arises from our lack of specific tools to query, or manipulate, the molecular scale circuitry on both single-cell and organismal levels. Fortunately, the last two decades of progress in the field of optogenetics have produced a variety of genetically encoded, light-mediated tools that enable visualization and control of the spatiotemporal regulation of cellular function. The merging of optogenetics and pluripotent stem cell biology could thus be an important step toward realization of the clinical potential of pluripotent stem cells. In this review, we have surveyed available genetically encoded photoactuators and photosensors, a rapidly expanding toolbox, with particular attention to those with utility for studying pluripotent stem cells. PMID:28912894

Visualizing Intrapopulation Hematopoietic Cell Heterogeneity with Self-Organizing Maps of SIMS Data.

PubMed

Mirshafiee, Vahid; Harley, Brendan A C; Kraft, Mary L

2018-05-07

Characterization of the heterogeneity within stem cell populations, which affects their differentiation potential, is necessary for the design of artificial cultures for stem cell expansion. In this study, we assessed whether self-organizing maps (SOMs) of single-cell time-of-flight secondary ion mass spectrometry (TOF-SIMS) data provide insight into the spectral, and thus the related functional heterogeneity between and within three hematopoietic cell populations. SOMs were created of TOF-SIMS data from individual hematopoietic stem and progenitor cells (HSPCs), lineage-committed common lymphoid progenitors (CLPs), and fully differentiated B cells that had been isolated from murine bone marrow via conventional flow cytometry. The positions of these cells on the SOMs and the spectral variation between adjacent map units, shown on the corresponding unified distance matrix (U-matrix), indicated the CLPs exhibited the highest intrapopulation spectral variation, regardless of the age of the donor mice. SOMs of HSPCs, CLPs, and B cells isolated from young and old mice using the same surface antigen profiles revealed the HSPCs exhibited the most age-related spectral variation, whereas B cells exhibited the least. These results demonstrate that SOMs of single-cell spectra enable characterizing the heterogeneity between and within cell populations that lie along distinct differentiation pathways.

Cell-Type-Specific Predictive Network Yields Novel Insights into Mouse Embryonic Stem Cell Self-Renewal and Cell Fate

PubMed Central

Dowell, Karen G.; Simons, Allen K.; Wang, Zack Z.; Yun, Kyuson; Hibbs, Matthew A.

2013-01-01

Self-renewal, the ability of a stem cell to divide repeatedly while maintaining an undifferentiated state, is a defining characteristic of all stem cells. Here, we clarify the molecular foundations of mouse embryonic stem cell (mESC) self-renewal by applying a proven Bayesian network machine learning approach to integrate high-throughput data for protein function discovery. By focusing on a single stem-cell system, at a specific developmental stage, within the context of well-defined biological processes known to be active in that cell type, we produce a consensus predictive network that reflects biological reality more closely than those made by prior efforts using more generalized, context-independent methods. In addition, we show how machine learning efforts may be misled if the tissue specific role of mammalian proteins is not defined in the training set and circumscribed in the evidential data. For this study, we assembled an extensive compendium of mESC data: ∼2.2 million data points, collected from 60 different studies, under 992 conditions. We then integrated these data into a consensus mESC functional relationship network focused on biological processes associated with embryonic stem cell self-renewal and cell fate determination. Computational evaluations, literature validation, and analyses of predicted functional linkages show that our results are highly accurate and biologically relevant. Our mESC network predicts many novel players involved in self-renewal and serves as the foundation for future pluripotent stem cell studies. This network can be used by stem cell researchers (at http://StemSight.org) to explore hypotheses about gene function in the context of self-renewal and to prioritize genes of interest for experimental validation. PMID:23468881

[18F]FDG labeling of neural stem cells for in vivo cell tracking with positron emission tomography: inhibition of tracer release by phloretin.

PubMed

Stojanov, Katica; de Vries, Erik F J; Hoekstra, Dick; van Waarde, Aren; Dierckx, Rudi A J O; Zuhorn, Inge S

2012-02-01

The introduction of neural stem cells into the brain has promising therapeutic potential for the treatment of neurodegenerative diseases. To monitor the cellular replacement therapy, that is, to determine stem cell migration, survival, and differentiation, in vivo tracking methods are needed. Ideally, these tracking methods are noninvasive. Noninvasive tracking methods that have been successfully used for the visualization of blood-derived progenitor cells include magnetic resonance imaging and radionuclide imaging using single-photon emission computed tomography (SPECT) and positron emission tomography (PET). The SPECT tracer In-111-oxine is suitable for stem cell labeling, but for studies in small animals, the higher sensitivity and facile quantification that can be obtained with PET are preferred. Here the potential of 2'-[18F]fluoro-2'-deoxy-D-glucose ([18F]-FDG), a PET tracer, for tracking of neural stem cell (NSCs) trafficking toward an inflammation site was investigated. [18F]-FDG turns out to be a poor radiopharmaceutical to label NSCs owing to the low labeling efficiency and substantial release of radioactivity from these cells. Efflux of [18F]-FDG from NSCs can be effectively reduced by phloretin in vitro, but inhibition of tracer release is insufficient in vivo for accurate monitoring of stem cell trafficking.

Rapid DNA replication origin licensing protects stem cell pluripotency

PubMed Central

Matson, Jacob Peter; Dumitru, Raluca; Coryell, Philip; Baxley, Ryan M; Chen, Weili; Twaroski, Kirk; Webber, Beau R; Tolar, Jakub; Bielinsky, Anja-Katrin; Purvis, Jeremy E

2017-01-01

Complete and robust human genome duplication requires loading minichromosome maintenance (MCM) helicase complexes at many DNA replication origins, an essential process termed origin licensing. Licensing is restricted to G1 phase of the cell cycle, but G1 length varies widely among cell types. Using quantitative single-cell analyses, we found that pluripotent stem cells with naturally short G1 phases load MCM much faster than their isogenic differentiated counterparts with long G1 phases. During the earliest stages of differentiation toward all lineages, MCM loading slows concurrently with G1 lengthening, revealing developmental control of MCM loading. In contrast, ectopic Cyclin E overproduction uncouples short G1 from fast MCM loading. Rapid licensing in stem cells is caused by accumulation of the MCM loading protein, Cdt1. Prematurely slowing MCM loading in pluripotent cells not only lengthens G1 but also accelerates differentiation. Thus, rapid origin licensing is an intrinsic characteristic of stem cells that contributes to pluripotency maintenance. PMID:29148972

Distinguishing autocrine and paracrine signals in hematopoietic stem cell culture using a biofunctional microcavity platform

NASA Astrophysics Data System (ADS)

Müller, Eike; Wang, Weijia; Qiao, Wenlian; Bornhäuser, Martin; Zandstra, Peter W.; Werner, Carsten; Pompe, Tilo

2016-08-01

Homeostasis of hematopoietic stem cells (HSC) in the mammalian bone marrow stem cell niche is regulated by signals of the local microenvironment. Besides juxtacrine, endocrine and metabolic cues, paracrine and autocrine signals are involved in controlling quiescence, proliferation and differentiation of HSC with strong implications on expansion and differentiation ex vivo as well as in vivo transplantation. Towards this aim, a cell culture analysis on a polymer microcavity carrier platform was combined with a partial least square analysis of a mechanistic model of cell proliferation. We could demonstrate the discrimination of specific autocrine and paracrine signals from soluble factors as stimulating and inhibitory effectors in hematopoietic stem and progenitor cell culture. From that we hypothesize autocrine signals to be predominantly involved in maintaining the quiescent state of HSC in single-cell niches and advocate our analysis platform as an unprecedented option for untangling convoluted signaling mechanisms in complex cell systems being it of juxtacrine, paracrine or autocrine origin.

Ancestral trees for modeling stem cell lineages genetically rather than functionally: understanding mutation accumulation and distinguishing the restrictive cancer stem cell propagation theory and the unrestricted cell propagation theory of human tumorigenesis.

PubMed

Shibata, Darryl K; Kern, Scott E

2008-01-01

Cancer stem cells either could be rare or common in tumors, constituting the major distinction between the two fundamentally opposed theoretical models of tumor progression: A newer and restrictive stem cell propagation model, in which the stem cells are a small and special minority of the tumor cells, and a standard older model, an unrestricted cell proliferation theory, in which many or most tumor cells are capable of indefinite generations of cell division. Stem cells of tumors are difficult to quantitate using functional assays, and the validity of the most common assays is seriously questioned. Nonetheless, stem cells are an essential component of any tumorigenesis model. Alternative approaches to studying tumor stem cells should be explored. Cell populations can be conceived of as having a genealogy, a relationship of cells to their ancestral lineage, from the zygote to the adult cells or neoplasms. Models using ancestral trees thus offer an anatomic and genetic means to "observe" stem cells independent of artificial conditions. Ancestral trees broaden our attention backward along a lineage, to the zygote stage, and thereby add insight into how the mutations of tumors accumulate. It is possible that a large fraction of mutations in a tumor originate from normal, endogenous, replication errors (nearly all being passenger mutations) occurring prior to the emergence of the first transformed cell. Trees can be constructed from experimental measurements - molecular clocks - of real human tissues and tumors. Detailed analysis of single-cell methylation patterns, heritable yet slightly plastic, now can provide this information in the necessary depth. Trees based on observations of molecular clocks may help us to distinguish between competing theories regarding the proliferative properties among cells of actual human tumors, to observe subtle and difficult phenomena such as the extinction of stem lineages, and to address the origins and rates of mutations in various normal, hormone-stimulated, aging, or neoplastic tissues. The simple concept that cancers arise from the transformation of a normal stem cell, the stem cell origination theory, is sometimes superficially and confusingly referred to as "the stem cell theory". This concept is compatible with but not a requisite assumption for both of the major competing theories of tumor progression, and plays essentially no role in clarifying the nature of tumor progression.

Multiple mutant clones in blood rarely coexist

NASA Astrophysics Data System (ADS)

Dingli, David; Pacheco, Jorge M.; Traulsen, Arne

2008-02-01

Leukemias arise due to mutations in the genome of hematopoietic (blood) cells. Hematopoiesis has a multicompartment architecture, with cells exhibiting different rates of replication and differentiation. At the root of this process, one finds a small number of stem cells, and hence the description of the mutation-selection dynamics of blood cells calls for a stochastic approach. We use stochastic dynamics to investigate to which extent acquired hematopoietic disorders are associated with mutations of single or multiple genes within developing blood cells. Our analysis considers the appearance of mutations both in the stem cell compartment as well as in more committed compartments. We conclude that in the absence of genomic instability, acquired hematopoietic disorders due to mutations in multiple genes are most likely very rare events, as multiple mutations typically require much longer development times compared to those associated with a single mutation.

Pancreatic exocrine adult cells and placental stem cells co-culture. Working together is always the best way to go.

PubMed

Suşman, S; Rus-Ciucă, D; Soriţău, Olga; Tomuleasa, C; Buigă, R; Mihu, D; Pop, V I; Mihu, Carmen Mihaela

2011-01-01

The progress made in the last few years have managed to come up withy the possibility of using different stem cell types in an endeavor to correct the alterations that appear in different degenerative diseases. The pancreas, an organ with extremely low regenerative capacity, both for the endocrine and for the exocrine component, is an organ perfect for cell therapy in the hope of restoring its function and cure diabetes mellitus or chronic pancreatitis. One main issue in the stem cell transplantation problem is represented by the influence of the cellular niche, formed by completely differentiated cells, on the phenotype and function of the transplanted cells. In this study, we challenge current knowledge in the field by evaluating the influence of exocrine pancreatic cells on placental stem-like cells using the co-culture technique. In our experiments, we used two different protocols in which adult pancreatic cells were cultured together with mesenchymal stem cells isolated from human placenta. In the case of the first protocol, we seeded pancreatic cells on a pre-adhered single-cell layer of mesenchymal stem cells and in the second one, the seeding of two cell populations in suspension was done at the same time, after passage. During the experiment, we evaluated the alteration of the morphology of the placental cells using and inverted phase microscope and reverse transcriptase-PCR. Based on morphology, in both cases the interaction between epithelial pancreatic cells and placental ones have determined a change in phenotype from mesenchymal to epithelial-like. Taking into consideration the gene expression, placental stem cells have maintained pluripotency gene expression throughout the study. They also expressed pancreatic amylase. These experiments bring out the plasticity of placental stem cells, the cell microenvironment with a decisive part in phenotype and the level of gene expression. The results obtained in vitro can bring a new picture on the effects of the pancreatic stem cell niche.

High incidence of non-random template strand segregation and asymmetric fate determination in dividing stem cells and their progeny.

PubMed

Conboy, Michael J; Karasov, Ariela O; Rando, Thomas A

2007-05-01

Decades ago, the "immortal strand hypothesis" was proposed as a means by which stem cells might limit acquiring mutations that could give rise to cancer, while continuing to proliferate for the life of an organism. Originally based on observations in embryonic cells, and later studied in terms of stem cell self-renewal, this hypothesis has remained largely unaccepted because of few additional reports, the rarity of the cells displaying template strand segregation, and alternative interpretations of experiments involving single labels or different types of labels to follow template strands. Using sequential pulses of halogenated thymidine analogs (bromodeoxyuridine [BrdU], chlorodeoxyuridine [CldU], and iododeoxyuridine [IdU]), and analyzing stem cell progeny during induced regeneration in vivo, we observed extraordinarily high frequencies of segregation of older and younger template strands during a period of proliferative expansion of muscle stem cells. Furthermore, template strand co-segregation was strongly associated with asymmetric cell divisions yielding daughters with divergent fates. Daughter cells inheriting the older templates retained the more immature phenotype, whereas daughters inheriting the newer templates acquired a more differentiated phenotype. These data provide compelling evidence of template strand co-segregation based on template age and associated with cell fate determination, suggest that template strand age is monitored during stem cell lineage progression, and raise important caveats for the interpretation of label-retaining cells.

Telomere shortening in hematopoietic stem cell transplantation: a potential mechanism for late graft failure?

PubMed

Awaya, Norihiro; Baerlocher, Gabriela M; Manley, Thomas J; Sanders, Jean E; Mielcarek, Marco; Torok-Storb, Beverly; Lansdorp, Peter M

2002-01-01

Telomeres serve to maintain the structural integrity of chromosomes, yet each somatic cell division is associated with a decrease in telomere length. The cumulative decrease in telomere length can impose an upper limit for the number of cell divisions that can occur before a cell senesces. When studied in vitro with fibroblasts, this limit is referred to as the Hayflick limit and usually occurs after 40 to 80 cell doublings. In theory, a similar replicative potential in a hematopoietic stem cell could support hematopoiesis in a person for more than 100 years. However, stem cells differentiate, and the telomere length differs among chromosomes within a single cell, among cell types, and among age-matched individuals. This variation in telomere length raises the possibility that long-term hematopoiesis by transplanted stem cells could, depending on the telomere length of the engrafted stem cell and the proliferative demand to which it is subjected, reach a Hayflick limit during the life span of the patient. Although significant shortening of telomeres is reported to occur within the first year posttransplantation, as yet no evidence has indicated that this shortening is associated with marrow function. In this review, we summarize reports on telomere shortening in stem cell transplantation recipients and report 2 cases in which graft failure is associated with significant telomere shortening.

Klotho, stem cells, and aging.

PubMed

Bian, Ao; Neyra, Javier A; Zhan, Ming; Hu, Ming Chang

2015-01-01

Aging is an inevitable and progressive biological process involving dysfunction and eventually destruction of every tissue and organ. This process is driven by a tightly regulated and complex interplay between genetic and acquired factors. Klotho is an antiaging gene encoding a single-pass transmembrane protein, klotho, which serves as an aging suppressor through a wide variety of mechanisms, such as antioxidation, antisenescence, antiautophagy, and modulation of many signaling pathways, including insulin-like growth factor and Wnt. Klotho deficiency activates Wnt expression and activity contributing to senescence and depletion of stem cells, which consequently triggers tissue atrophy and fibrosis. In contrast, the klotho protein was shown to suppress Wnt-signaling transduction, and inhibit cell senescence and preserve stem cells. A better understanding of the potential effects of klotho on stem cells could offer novel insights into the cellular and molecular mechanisms of klotho deficiency-related aging and disease. The klotho protein may be a promising therapeutic agent for aging and aging-related disorders.

Exploiting endogenous fibrocartilage stem cells to regenerate cartilage and repair joint injury

PubMed Central

Embree, Mildred C.; Chen, Mo; Pylawka, Serhiy; Kong, Danielle; Iwaoka, George M.; Kalajzic, Ivo; Yao, Hai; Shi, Chancheng; Sun, Dongming; Sheu, Tzong-Jen; Koslovsky, David A.; Koch, Alia; Mao, Jeremy J.

2016-01-01

Tissue regeneration using stem cell-based transplantation faces many hurdles. Alternatively, therapeutically exploiting endogenous stem cells to regenerate injured or diseased tissue may circumvent these challenges. Here we show resident fibrocartilage stem cells (FCSCs) can be used to regenerate and repair cartilage. We identify FCSCs residing within the superficial zone niche in the temporomandibular joint (TMJ) condyle. A single FCSC spontaneously generates a cartilage anlage, remodels into bone and organizes a haematopoietic microenvironment. Wnt signals deplete the reservoir of FCSCs and cause cartilage degeneration. We also show that intra-articular treatment with the Wnt inhibitor sclerostin sustains the FCSC pool and regenerates cartilage in a TMJ injury model. We demonstrate the promise of exploiting resident FCSCs as a regenerative therapeutic strategy to substitute cell transplantation that could be beneficial for patients suffering from fibrocartilage injury and disease. These data prompt the examination of utilizing this strategy for other musculoskeletal tissues. PMID:27721375

Mechanisms of fate decision and lineage commitment during haematopoiesis.

PubMed

Cvejic, Ana

2016-03-01

Blood stem cells need to both perpetuate themselves (self-renew) and differentiate into all mature blood cells to maintain blood formation throughout life. However, it is unclear how the underlying gene regulatory network maintains this population of self-renewing and differentiating stem cells and how it accommodates the transition from a stem cell to a mature blood cell. Our current knowledge of transcriptomes of various blood cell types has mainly been advanced by population-level analysis. However, a population of seemingly homogenous blood cells may include many distinct cell types with substantially different transcriptomes and abilities to make diverse fate decisions. Therefore, understanding the cell-intrinsic differences between individual cells is necessary for a deeper understanding of the molecular basis of their behaviour. Here we review recent single-cell studies in the haematopoietic system and their contribution to our understanding of the mechanisms governing cell fate choices and lineage commitment.

Single-Cell RNA-Sequencing in Glioma.

PubMed

Johnson, Eli; Dickerson, Katherine L; Connolly, Ian D; Hayden Gephart, Melanie

2018-04-10

In this review, we seek to summarize the literature concerning the use of single-cell RNA-sequencing for CNS gliomas. Single-cell analysis has revealed complex tumor heterogeneity, subpopulations of proliferating stem-like cells and expanded our view of tumor microenvironment influence in the disease process. Although bulk RNA-sequencing has guided our initial understanding of glioma genetics, this method does not accurately define the heterogeneous subpopulations found within these tumors. Single-cell techniques have appealing applications in cancer research, as diverse cell types and the tumor microenvironment have important implications in therapy. High cost and difficult protocols prevent widespread use of single-cell RNA-sequencing; however, continued innovation will improve accessibility and expand our of knowledge gliomas.

High-content image informatics of the structural nuclear protein NuMA parses trajectories for stem/progenitor cell lineages and oncogenic transformation.

PubMed

Vega, Sebastián L; Liu, Er; Arvind, Varun; Bushman, Jared; Sung, Hak-Joon; Becker, Matthew L; Lelièvre, Sophie; Kohn, Joachim; Vidi, Pierre-Alexandre; Moghe, Prabhas V

2017-02-01

Stem and progenitor cells that exhibit significant regenerative potential and critical roles in cancer initiation and progression remain difficult to characterize. Cell fates are determined by reciprocal signaling between the cell microenvironment and the nucleus; hence parameters derived from nuclear remodeling are ideal candidates for stem/progenitor cell characterization. Here we applied high-content, single cell analysis of nuclear shape and organization to examine stem and progenitor cells destined to distinct differentiation endpoints, yet undistinguishable by conventional methods. Nuclear descriptors defined through image informatics classified mesenchymal stem cells poised to either adipogenic or osteogenic differentiation, and oligodendrocyte precursors isolated from different regions of the brain and destined to distinct astrocyte subtypes. Nuclear descriptors also revealed early changes in stem cells after chemical oncogenesis, allowing the identification of a class of cancer-mitigating biomaterials. To capture the metrology of nuclear changes, we developed a simple and quantitative "imaging-derived" parsing index, which reflects the dynamic evolution of the high-dimensional space of nuclear organizational features. A comparative analysis of parsing outcomes via either nuclear shape or textural metrics of the nuclear structural protein NuMA indicates the nuclear shape alone is a weak phenotypic predictor. In contrast, variations in the NuMA organization parsed emergent cell phenotypes and discerned emergent stages of stem cell transformation, supporting a prognosticating role for this protein in the outcomes of nuclear functions. Copyright © 2017 Elsevier Inc. All rights reserved.

Effect of ROCK inhibitor Y-27632 on normal and variant human embryonic stem cells (hESCs) in vitro: its benefits in hESC expansion.

PubMed

Gauthaman, Kalamegam; Fong, Chui-Yee; Bongso, Ariff

2010-03-01

The Rho associated coiled coil protein kinase (ROCK) dependent signaling pathway plays an important role in numerous physiological functions such as cell proliferation, adhesion, migration and inflammation. Human embryonic stem cells (hESCs) undergo differentiation and poor survival after single cell dissociation in culture thus limiting their expansion for cell based therapies. We evaluated the role of the selective ROCK inhibitor Y-27632 on hESC colonies and disassociated single hESCs from two different hESC lines. Karyotypically normal hESCs (HES3) and variant hESCs (BG01V) were treated with Y-27632 at 5, 10 and 20 muM concentrations for 72 h and its effects on hESC self renewal, colony morphology, cell cycle and pluripotency were evaluated. Increased cell proliferation of both HES3 and BG01V were observed for all three concentrations compared to untreated controls following passaging of cell clusters or dissociated single cells and some of these increases were statistically significant. Cell cycle assay demonstrated normal cell cycle progression with no peaks evident of apoptosis. No morphological differentiation was evident following treatment with the highest concentration of Y-27632 (20 muM) and the stemness related genes continued to be highly expressed in both HES3 and BG01V cells compared to untreated controls. The results confirmed that Y-27632 is a useful agent that aids in the expansion of undifferentiated hESC numbers for downstream applications in regenerative medicine.

Programming Morphogenesis through Systems and Synthetic Biology.

PubMed

Velazquez, Jeremy J; Su, Emily; Cahan, Patrick; Ebrahimkhani, Mo R

2018-04-01

Mammalian tissue development is an intricate, spatiotemporal process of self-organization that emerges from gene regulatory networks of differentiating stem cells. A major goal in stem cell biology is to gain a sufficient understanding of gene regulatory networks and cell-cell interactions to enable the reliable and robust engineering of morphogenesis. Here, we review advances in synthetic biology, single cell genomics, and multiscale modeling, which, when synthesized, provide a framework to achieve the ambitious goal of programming morphogenesis in complex tissues and organoids. Copyright © 2017 Elsevier Ltd. All rights reserved.

Limbal Stem Cell Preservation During Proton Beam Irradiation for Diffuse Iris Melanoma.

PubMed

Singh, Arun D; Dupps, William J; Biscotti, Charles V; Suh, John H; Lathrop, Kira L; Nairn, John P; Shih, Helen

2017-01-01

To report the outcome after limbal stem cell preservation during proton beam irradiation for diffuse iris melanoma. This is a single-case report of diffuse iris melanoma that was managed with proton beam radiation (53 Gy), wherein preemptively harvested superior and inferior limbal stem cells before radiation were replaced after irradiation. Regeneration of the palisades of Vogt and the limbal stem cells was documented by an optical coherence tomography-based imaging protocol. At 24 months after radiation therapy, best-corrected visual acuity was 20/25. The cornea was clear without evidence of limbal stem cell dysfunction. Clinical examination (including gonioscopy and ultrasound biomicroscopy [UBM]) was indicative of local control, and systemic surveillance was negative for metastatic disease. At posttransplant (21 months), there were more palisade structures visible in both anterior and posterior regions of the superior and inferior limbus, and the linear presentation of the inferior palisades appears to have regenerated. Diffuse iris melanoma can be managed successfully with proton beam radiation while preserving corneal limbal stem cells by harvesting them before radiation and then replacing them after irradiation. Regeneration of the palisades of Vogt could be documented by an optical coherence tomography-based imaging protocol.

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

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

Chen Shuxun; Wang Xiaolin; Sun Dong

2013-07-15

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

Self-Organizing and Stochastic Behaviors During the Regeneration of Hair Stem Cells

PubMed Central

Plikus, Maksim V.; Baker, Ruth E.; Chen, Chih-Chiang; Fare, Clyde; de la Cruz, Damon; Andl, Thomas; Maini, Philip K.; Millar, Sarah E.; Widelitz, Randall; Chuong, Cheng-Ming

2012-01-01

Stem cells cycle through active and quiescent states. Large populations of stem cells in an organ may cycle randomly or in a coordinated manner. Although stem cell cycling within single hair follicles has been studied, less is known about regenerative behavior in a hair follicle population. By combining predictive mathematical modeling with in vivo studies in mice and rabbits, we show that a follicle progresses through cycling stages by continuous integration of inputs from intrinsic follicular and extrinsic environmental signals based on universal patterning principles. Signaling from the WNT/bone morphogenetic protein activator/inhibitor pair is coopted to mediate interactions among follicles in the population. This regenerative strategy is robust and versatile because relative activator/inhibitor strengths can be modulated easily, adapting the organism to different physiological and evolutionary needs. PMID:21527712

Highly osteogenic PDL stem cell clones specifically express elevated levels of ICAM1, ITGB1 and TERT.

PubMed

Sununliganon, Laddawun; Singhatanadgit, Weerachai

2012-01-01

Cells derived from the periodontal ligament (PDL) have previously been reported to have stem cell-like characteristics (PDL stem cells; PDLSCs) and play an important part in bone engineering, including that of alveolar bone. However, these populations have been heterogeneous, and thus far no specific marker has yet been established from adult human stem cells derived from PDL tissue. We have previously isolated highly purified single cell-derived PDLSC clones and delineated their phenotypic and functional characteristics. In this report, we further obtained three homogeneous and distinct PDLSC clones demonstrating low, moderate and high mineralized matrix forming ability-namely PC12, PC4 and PC3, respectively, and the expression of mesenchymal stem cell pathway-specific genes in these clones was investigated. PCR array revealed that the expression of intercellular adhesion molecule 1 (ICAM1), integrin beta 1 (ITGB1) and telomerase reverse transcriptase (TERT) was associated with highly osteogenic PDLSC clones, as determined by the expression of key osteoblastic markers and their ability to form alizarin red S positive mineralized matrix in vitro. The present results suggest that these three mesenchymal stem cell-associated markers could potentially be used to isolate PDLSCs with high osteogenic capability for engineering new bone.

Stromal cell-derived factor-1 significantly induces proliferation, migration, and collagen type I expression in a human periodontal ligament stem cell subpopulation.

PubMed

Du, Lingqian; Yang, Pishan; Ge, Shaohua

2012-03-01

The pivotal role of chemokine stromal cell-derived factor-1 (SDF-1) in bone marrow mesenchymal stem cells recruitment and tissue regeneration has already been reported. However, its roles in human periodontal ligament stem cells (PDLSCs) remain unknown. PDLSCs are regarded as candidates for periodontal tissue regeneration and are used in stem cell-based periodontal tissue engineering. The expression of chemokine receptors on PDLSCs and the migration of these cells induced by chemokines and their subsequent function in tissue repair may be a crucial procedure for periodontal tissue regeneration. PDL tissues were obtained from clinically healthy premolars extracted for orthodontic reasons and used to isolate single-cell colonies by the limited-dilution method. Immunocytochemical staining was used to detect the expression of the mesenchymal stem cell marker STRO-1. Differentiation potentials were assessed by alizarin-red staining and oil-red O staining. The expression of SDF-1 receptor CXCR4 was evaluated by real-time polymerase chain reaction (PCR) and immunocytochemical staining. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and bromodeoxyuridine incorporation assay were used to determine the viability and proliferation of the PDLSC subpopulation. Expression of collagen type I and alkaline phosphatase was detected by real-time PCR to determine the effect of SDF-1 on cells differentiation. Twenty percent of PDL single-cell colonies expressed STRO-1 positively, and this specific subpopulation was positive for CXCR4 and formed minerals and lipid vacuoles after 4 weeks induction. SDF-1 significantly increased proliferation and stimulated the migration of this PDLSC subpopulation at concentrations between 100 and 400 ng/mL. CXCR4 neutralizing antibody could block cell proliferation and migration, suggesting that SDF-1 exerted its effects on cells through CXCR4. SDF-1 promoted collagen type I level significantly but had little effect on alkaline phosphatase level. SDF-1 may have the potential of promoting periodontal tissue regeneration by the mechanism of guiding PDLSCs to destructive periodontal tissue, promoting their activation and proliferation and influencing the differentiation of these stem cells.

Differentiation and characteristics of the enhanced green fluorescent protein gene transgenic goat neural stem cells cultured in attached and non-attached plates.

PubMed

Zheng, Yue-Mao; Dang, Yong-Hui; Qiu, Shuang; Qi, Ying-Pei; Xu, Yong-Ping; Sai, Wu-Jia-Fu

2011-08-01

The aims of this study were (i) to determine whether NSCs (neural stem cells) could be isolated from the brain of embryonic day 98 fetal goat, (ii) to determine if these stem cells have the capability of multipotent differentiation following transfection with a reporter gene, EGFP (enhanced green fluorescent protein) and (iii) to study the characteristics of the stem cells cultured in attached and non-attached plates. NSCs were isolated from embryonic day 98 fetal goat brain, transfected with EGFP gene using lipofection, and subcultured in attached and non-attached plates respectively. The transgenic stem cells were induced to differentiate into osteogenic and endothelial cells in vitro respectively. Markers associated with undifferentiated NSCs and their differentiated cells were tested by RT-PCR (reverse transcription-PCR). The results demonstrated that stem cells could be isolated from embryonic day 98 fetal goat brain, and EGFP gene could be transfected into the cells. The transgenic NSCs were capable of self-renewal, a defining property of stem cells, and were grown as free-floating neurospheres in non-attached plates. When the neurospheres were transferred and cultured in attached plates, cells migrate from the neurospheres and are grown as spindle cells. The stem cells were grown as quasi-circular cells when the single stem cells were cultured in attached plates. Both the NSCs cultured in non-attached and attached plates could express Hes1 (hairy and enhancer of split 1), Oct4 (octamer-binding protein 4), Nanog, Sox2 [SRY (sex-determining region Y)-box 2] and Nestin, while following differentiation cells expressed markers for osteogenic cells (Osteocalcin+ and Osteonectin+) and endothelium (CD34+ and eNOS+). The results demonstrated that the goat EGFP gene transgenic NSCs have the capability of multipotent differentiation, which means that the transgenic NSCs may be useful in cell transplantation studies in future.

Modeling Inborn Errors of Hepatic Metabolism Using Induced Pluripotent Stem Cells.

PubMed

Pournasr, Behshad; Duncan, Stephen A

2017-11-01

Inborn errors of hepatic metabolism are because of deficiencies commonly within a single enzyme as a consequence of heritable mutations in the genome. Individually such diseases are rare, but collectively they are common. Advances in genome-wide association studies and DNA sequencing have helped researchers identify the underlying genetic basis of such diseases. Unfortunately, cellular and animal models that accurately recapitulate these inborn errors of hepatic metabolism in the laboratory have been lacking. Recently, investigators have exploited molecular techniques to generate induced pluripotent stem cells from patients' somatic cells. Induced pluripotent stem cells can differentiate into a wide variety of cell types, including hepatocytes, thereby offering an innovative approach to unravel the mechanisms underlying inborn errors of hepatic metabolism. Moreover, such cell models could potentially provide a platform for the discovery of therapeutics. In this mini-review, we present a brief overview of the state-of-the-art in using pluripotent stem cells for such studies. © 2017 American Heart Association, Inc.

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

PubMed Central

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

2014-01-01

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

Fusion-independent expression of functional ACh receptors in mouse mesoangioblast stem cells contacting muscle cells

PubMed Central

Grassi, Francesca; Pagani, Francesca; Spinelli, Gabriele; Angelis, Luciana De; Cossu, Giulio; Eusebi, Fabrizio

2004-01-01

Mesoangioblasts are vessel-associated fetal stem cells that can be induced to differentiate into skeletal muscle, both in vitro and in vivo. Whether this is due to fusion or to transdifferentiation into bona fide satellite cells is still an open question, for mesoangioblasts as well as for other types of stem cells. The early steps of satellite cell myogenic differentiation involve MyoD activation, membrane hyperpolarization and the appearance of ACh sensitivity and gap junctional communication. If mesoangioblasts differentiate into satellite cells, these characteristics should be observed in stem cells prior to fusion into multinucleated myotubes. We have investigated the functional properties acquired by mononucleated green fluorescent protein (GFP)-positive mesoangioblasts co-cultured with differentiating C2C12 myogenic cells, using the patch-clamp technique. Mesoangioblasts whose membrane contacted myogenic cells developed a hyperpolarized membrane resting potential and ACh-evoked current responses. Dye and electrical coupling was observed among mesoangioblasts but not between mesoangioblasts and myotubes. Mouse MyoD was detected by RT-PCR both in single, mononucleated mesoangioblasts co-cultured with C2C12 myotubes and in the total mRNA from mouse mesoangioblasts co-cultured with human myotubes, but not in human myotubes or stem cells cultured in isolation. In conclusion, when co-cultured with muscle cells, mesoangioblasts acquire many of the functional characteristics of differentiating satellite cells in the absence of cell fusion, strongly indicating that these stem cells undergo transdifferentiation into satellite cells, when exposed to a myogenic environment. PMID:15319417

An efficient and scalable pipeline for epitope tagging in mammalian stem cells using Cas9 ribonucleoprotein

PubMed Central

Dewari, Pooran Singh; Southgate, Benjamin; Mccarten, Katrina; Monogarov, German; O'Duibhir, Eoghan; Quinn, Niall; Tyrer, Ashley; Leitner, Marie-Christin; Plumb, Colin; Kalantzaki, Maria; Blin, Carla; Finch, Rebecca; Bressan, Raul Bardini; Morrison, Gillian; Jacobi, Ashley M; Behlke, Mark A; von Kriegsheim, Alex; Tomlinson, Simon; Krijgsveld, Jeroen

2018-01-01

CRISPR/Cas9 can be used for precise genetic knock-in of epitope tags into endogenous genes, simplifying experimental analysis of protein function. However, Cas9-assisted epitope tagging in primary mammalian cell cultures is often inefficient and reliant on plasmid-based selection strategies. Here, we demonstrate improved knock-in efficiencies of diverse tags (V5, 3XFLAG, Myc, HA) using co-delivery of Cas9 protein pre-complexed with two-part synthetic modified RNAs (annealed crRNA:tracrRNA) and single-stranded oligodeoxynucleotide (ssODN) repair templates. Knock-in efficiencies of ~5–30%, were achieved without selection in embryonic stem (ES) cells, neural stem (NS) cells, and brain-tumor-derived stem cells. Biallelic-tagged clonal lines were readily derived and used to define Olig2 chromatin-bound interacting partners. Using our novel web-based design tool, we established a 96-well format pipeline that enabled V5-tagging of 60 different transcription factors. This efficient, selection-free and scalable epitope tagging pipeline enables systematic surveys of protein expression levels, subcellular localization, and interactors across diverse mammalian stem cells. PMID:29638216

Pluripotency of adult stem cells derived from human and rat pancreas

NASA Astrophysics Data System (ADS)

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

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

Single-Cell RNA-Sequencing Reveals a Continuous Spectrum of Differentiation in Hematopoietic Cells.

PubMed

Macaulay, Iain C; Svensson, Valentine; Labalette, Charlotte; Ferreira, Lauren; Hamey, Fiona; Voet, Thierry; Teichmann, Sarah A; Cvejic, Ana

2016-02-02

The transcriptional programs that govern hematopoiesis have been investigated primarily by population-level analysis of hematopoietic stem and progenitor cells, which cannot reveal the continuous nature of the differentiation process. Here we applied single-cell RNA-sequencing to a population of hematopoietic cells in zebrafish as they undergo thrombocyte lineage commitment. By reconstructing their developmental chronology computationally, we were able to place each cell along a continuum from stem cell to mature cell, refining the traditional lineage tree. The progression of cells along this continuum is characterized by a highly coordinated transcriptional program, displaying simultaneous suppression of genes involved in cell proliferation and ribosomal biogenesis as the expression of lineage specific genes increases. Within this program, there is substantial heterogeneity in the expression of the key lineage regulators. Overall, the total number of genes expressed, as well as the total mRNA content of the cell, decreases as the cells undergo lineage commitment. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Cell Patterning Chip for Controlling the Stem Cell Microenvironment

PubMed Central

Rosenthal, Adam; Macdonald, Alice; Voldman, Joel

2007-01-01

Cell-cell signaling is an important component of the stem cell microenvironment, affecting both differentiation and self-renewal. However, traditional cell-culture techniques do not provide precise control over cell-cell interactions, while existing cell patterning technologies are limited when used with proliferating or motile cells. To address these limitations, we created the Bio Flip Chip (BFC), a microfabricated polymer chip containing thousands of microwells, each sized to trap down to a single stem cell. We have demonstrated the functionality of the BFC by patterning a 50×50 grid of murine embryonic stem cells (mESCs), with patterning efficiencies > 75%, onto a variety of substrates – a cell-culture dish patterned with gelatin, a 3-D substrate, and even another layer of cells. We also used the BFC to pattern small groups of cells, with and without cell-cell contact, allowing incremental and independent control of contact-mediated signaling. We present quantitative evidence that cell-cell contact plays an important role in depressing mESC colony formation, and show that E-cadherin is involved in this negative regulatory pathway. Thus, by allowing exquisite control of the cellular microenvironment, we provide a technology that enables new applications in tissue engineering and regenerative medicine. PMID:17434582

Induced pluripotent stem cells meet genome editing

PubMed Central

Hockemeyer, Dirk; Jaenisch, Rudolf

2016-01-01

It is extremely rare for a single experiment to be so impactful and timely that it shapes and forecasts the experiments of the next decade. Here, we review how two such experiments --the generation of human induced pluripotent stem cells (iPSCs) and the development of CRISPR/Cas9 technology-- have fundamentally reshaped our approach to biomedical research, stem cell biology and human genetics. We will also highlight the previous knowledge that iPSC and CRISPR/Cas9 technologies were built on as this groundwork demonstrated the need for solutions and the benefits that these technologies provided, and have set the stage for their success. PMID:27152442

Induced Pluripotent Stem Cells Meet Genome Editing.

PubMed

Hockemeyer, Dirk; Jaenisch, Rudolf

2016-05-05

It is extremely rare for a single experiment to be so impactful and timely that it shapes and forecasts the experiments of the next decade. Here, we review how two such experiments-the generation of human induced pluripotent stem cells (iPSCs) and the development of CRISPR/Cas9 technology-have fundamentally reshaped our approach to biomedical research, stem cell biology, and human genetics. We will also highlight the previous knowledge that iPSC and CRISPR/Cas9 technologies were built on as this groundwork demonstrated the need for solutions and the benefits that these technologies provided and set the stage for their success. Copyright © 2016 Elsevier Inc. All rights reserved.

Single-molecule microscopy reveals membrane microdomain organization of cells in a living vertebrate.

PubMed

Schaaf, Marcel J M; Koopmans, Wiepke J A; Meckel, Tobias; van Noort, John; Snaar-Jagalska, B Ewa; Schmidt, Thomas S; Spaink, Herman P

2009-08-19

It has been possible for several years to study the dynamics of fluorescently labeled proteins by single-molecule microscopy, but until now this technology has been applied only to individual cells in culture. In this study, it was extended to stem cells and living vertebrate organisms. As a molecule of interest we used yellow fluorescent protein fused to the human H-Ras membrane anchor, which has been shown to serve as a model for proteins anchored in the plasma membrane. We used a wide-field fluorescence microscopy setup to visualize individual molecules in a zebrafish cell line (ZF4) and in primary embryonic stem cells. A total-internal-reflection microscopy setup was used for imaging in living organisms, in particular in epidermal cells in the skin of 2-day-old zebrafish embryos. Our results demonstrate the occurrence of membrane microdomains in which the diffusion of membrane proteins in a living organism is confined. This membrane organization differed significantly from that observed in cultured cells, illustrating the relevance of performing single-molecule microscopy in living organisms.

The Retinoblastoma pathway regulates stem cell proliferation in freshwater planarians.

PubMed

Zhu, Shu Jun; Pearson, Bret J

2013-01-15

Freshwater planarians are flatworms of the Lophotrochozoan superphylum and are well known for their regenerative abilities, which rely on a large population of pluripotent adult stem cells. However, the mechanisms by which planarians maintain a precise population of adult stem cells while balancing proliferation and cell death, remain to be elucidated. Here we have identified, characterized, and functionally tested the core Retinoblastoma (Rb) pathway components in planarian adult stem cell biology. The Rb pathway is an ancient and conserved mechanism of proliferation control from plants to animals and is composed of three core components: an Rb protein, and a transcription factor heterodimer of E2F and DP proteins. Although the planarian genome contains all components of the Rb pathway, we found that they have undergone gene loss from the ancestral state, similar to other species in their phylum. The single Rb homolog (Smed-Rb) was highly expressed in planarian stem cells and was required for stem cell maintenance, similar to the Rb-homologs p107 and p130 in vertebrates. We show that planarians and their phylum have undergone the most severe reduction in E2F genes observed thus far, and the single remaining E2F was predicted to be a repressive-type E2F (Smed-E2F4-1). Knockdown of either Smed-E2F4-1 or its dimerization partner Dp (Smed-Dp) by RNAi resulted in temporary hyper-proliferation. Finally, we showed that known Rb-interacting genes in other systems, histone deacetylase 1 and cyclinD (Smed-HDAC1; Smed-cycD), were similar to Rb in expression and phenotypes when knocked down by RNAi, suggesting that these established interactions with Rb may also be conserved in planarians. Together, these results showed that planarians use the conserved components of the Rb tumor suppressor pathway to control proliferation and cell survival. Copyright © 2012 Elsevier Inc. All rights reserved.

Isolation, Characterization, Cryopreservation of Human Amniotic Stem Cells and Differentiation to Osteogenic and Adipogenic Cells

PubMed Central

Gholizadeh-Ghaleh Aziz, Shiva; Pashaei-Asl, Fatima; Fardyazar, Zahra; Pashaiasl, Maryam

2016-01-01

Human stem cells and progenitor cells can be used to treat cancer and replace dysfunctional cells within a tissue or organ. The objective of this study was to identify the appropriate cells type in regenerative medicine and targeted therapy. As an alternative to embryonic and bone marrow stem cells, we examined human amniotic fluid stem cells (hAFSCs), one of the potential source of multipotent stem cells isolated from both cell pellet (using single-stage method), and supernatant of human amniotic fluid. Source of isolation and unique property of the cells emphasize that these cells are one of the promising new tools in therapeutic field. Double sources for isolation and availability of the left over samples in diagnostic laboratory at the same time have less legal and ethical concerns compared with embryonic stem cell studies. Cells were isolated, cultured for 18th passage for 6 months and characterized using qPCR and flow cytometry. Cells showed good proliferative ability in culture condition. The cells successfully differentiated into the adipogenic and osteogenic lineages. Based on these findings, amniotic fluid can be considered as an appropriate and convenient source of human amniotic fluid stem cells. These cells provide potential tools for therapeutic applications in the field of regenerative medicine. To get a better understanding of crosstalk between Oct4/NANOG with osteogenesis and adipogenesis, we used network analysis based on Common Targets algorithm and Common Regulators algorithm as well as subnetwork discovery based on gene set enrichment. Network analysis highlighted the possible role of MIR 302A and MIR let-7g. We demonstrated the high expression of MIR 302A and low expression of MIR let7g in hAFSCs by qPCR. PMID:27434028

An RNA-binding protein, Qki5, regulates embryonic neural stem cells through pre-mRNA processing in cell adhesion signaling.

PubMed

Hayakawa-Yano, Yoshika; Suyama, Satoshi; Nogami, Masahiro; Yugami, Masato; Koya, Ikuko; Furukawa, Takako; Zhou, Li; Abe, Manabu; Sakimura, Kenji; Takebayashi, Hirohide; Nakanishi, Atsushi; Okano, Hideyuki; Yano, Masato

2017-09-15

Cell type-specific transcriptomes are enabled by the action of multiple regulators, which are frequently expressed within restricted tissue regions. In the present study, we identify one such regulator, Quaking 5 (Qki5), as an RNA-binding protein (RNABP) that is expressed in early embryonic neural stem cells and subsequently down-regulated during neurogenesis. mRNA sequencing analysis in neural stem cell culture indicates that Qki proteins play supporting roles in the neural stem cell transcriptome and various forms of mRNA processing that may result from regionally restricted expression and subcellular localization. Also, our in utero electroporation gain-of-function study suggests that the nuclear-type Qki isoform Qki5 supports the neural stem cell state. We next performed in vivo transcriptome-wide protein-RNA interaction mapping to search for direct targets of Qki5 and elucidate how Qki5 regulates neural stem cell function. Combined with our transcriptome analysis, this mapping analysis yielded a bona fide map of Qki5-RNA interaction at single-nucleotide resolution, the identification of 892 Qki5 direct target genes, and an accurate Qki5-dependent alternative splicing rule in the developing brain. Last, our target gene list provides the first compelling evidence that Qki5 is associated with specific biological events; namely, cell-cell adhesion. This prediction was confirmed by histological analysis of mice in which Qki proteins were genetically ablated, which revealed disruption of the apical surface of the lateral wall in the developing brain. These data collectively indicate that Qki5 regulates communication between neural stem cells by mediating numerous RNA processing events and suggest new links between splicing regulation and neural stem cell states. © 2017 Hayakawa-Yano et al.; Published by Cold Spring Harbor Laboratory Press.

Droplet Microarray Based on Superhydrophobic-Superhydrophilic Patterns for Single Cell Analysis.

PubMed

Jogia, Gabriella E; Tronser, Tina; Popova, Anna A; Levkin, Pavel A

2016-12-09

Single-cell analysis provides fundamental information on individual cell response to different environmental cues and is a growing interest in cancer and stem cell research. However, current existing methods are still facing challenges in performing such analysis in a high-throughput manner whilst being cost-effective. Here we established the Droplet Microarray (DMA) as a miniaturized screening platform for high-throughput single-cell analysis. Using the method of limited dilution and varying cell density and seeding time, we optimized the distribution of single cells on the DMA. We established culturing conditions for single cells in individual droplets on DMA obtaining the survival of nearly 100% of single cells and doubling time of single cells comparable with that of cells cultured in bulk cell population using conventional methods. Our results demonstrate that the DMA is a suitable platform for single-cell analysis, which carries a number of advantages compared with existing technologies allowing for treatment, staining and spot-to-spot analysis of single cells over time using conventional analysis methods such as microscopy.

Will stem cell therapies be safe and effective for treating spinal cord injuries?

PubMed Central

Thomas, Katharine E.; Moon, Lawrence D. F.

2017-01-01

Introduction A large number of different cells including embryonic and adult stem cells have been transplanted into animal models of spinal cord injury, and in many cases these procedures have resulted in modest sensorimotor benefits. In October 2010 the world’s first clinical trial using human embryonic stem cells began, using stem cells converted into oligodendrocyte precursor cells. Sources of data In this review we examine some of the publically-available pre-clinical evidence that some of these cell types improve outcome in animal models of spinal cord injury. Much evidence is not available for public scrutiny, however, being private commercial property of various stem cell companies. Areas of agreement Transplantation of many different types of stem and progenitor cell enhances spontaneous recovery of function when transplanted acutely after spinal cord injury in animal models. Areas of disagreement The common mechanism(s) whereby the generic procedure of cellular transplantation enhances recovery of function are not well understood, although a range of possibilities are usually cited (including preservation of tissue, remyelination, axon sprouting, glial cell replacement). Only in exceptional cases has it been shown that functional recovery depends causally on the survival and differentiation of the transplanted cells. There is no agreement about the optimal cell type for transplantation: candidate stem cells have not yet been compared with each other or with other cell types (e.g., autologous Schwann cells) in a single study. Areas timely for developing research Transplantation of cells into animals with a long lifespan is important to determine whether or not tumours will eventually form. It will also be important to determine whether long-term survival of cells is required for functional recovery, and if so, how many are optimal. PMID:21586446

A Standard Nomenclature for Referencing and Authentication of Pluripotent Stem Cells.

PubMed

Kurtz, Andreas; Seltmann, Stefanie; Bairoch, Amos; Bittner, Marie-Sophie; Bruce, Kevin; Capes-Davis, Amanda; Clarke, Laura; Crook, Jeremy M; Daheron, Laurence; Dewender, Johannes; Faulconbridge, Adam; Fujibuchi, Wataru; Gutteridge, Alexander; Hei, Derek J; Kim, Yong-Ou; Kim, Jung-Hyun; Kokocinski, Anja Kolb-; Lekschas, Fritz; Lomax, Geoffrey P; Loring, Jeanne F; Ludwig, Tenneille; Mah, Nancy; Matsui, Tohru; Müller, Robert; Parkinson, Helen; Sheldon, Michael; Smith, Kelly; Stachelscheid, Harald; Stacey, Glyn; Streeter, Ian; Veiga, Anna; Xu, Ren-He

2018-01-09

Unambiguous cell line authentication is essential to avoid loss of association between data and cells. The risk for loss of references increases with the rapidity that new human pluripotent stem cell (hPSC) lines are generated, exchanged, and implemented. Ideally, a single name should be used as a generally applied reference for each cell line to access and unify cell-related information across publications, cell banks, cell registries, and databases and to ensure scientific reproducibility. We discuss the needs and requirements for such a unique identifier and implement a standard nomenclature for hPSCs, which can be automatically generated and registered by the human pluripotent stem cell registry (hPSCreg). To avoid ambiguities in PSC-line referencing, we strongly urge publishers to demand registration and use of the standard name when publishing research based on hPSC lines. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Single-cell transcriptional analysis of normal, aberrant, and malignant hematopoiesis in zebrafish.

PubMed

Moore, Finola E; Garcia, Elaine G; Lobbardi, Riadh; Jain, Esha; Tang, Qin; Moore, John C; Cortes, Mauricio; Molodtsov, Aleksey; Kasheta, Melissa; Luo, Christina C; Garcia, Amaris J; Mylvaganam, Ravi; Yoder, Jeffrey A; Blackburn, Jessica S; Sadreyev, Ruslan I; Ceol, Craig J; North, Trista E; Langenau, David M

2016-05-30

Hematopoiesis culminates in the production of functionally heterogeneous blood cell types. In zebrafish, the lack of cell surface antibodies has compelled researchers to use fluorescent transgenic reporter lines to label specific blood cell fractions. However, these approaches are limited by the availability of transgenic lines and fluorescent protein combinations that can be distinguished. Here, we have transcriptionally profiled single hematopoietic cells from zebrafish to define erythroid, myeloid, B, and T cell lineages. We also used our approach to identify hematopoietic stem and progenitor cells and a novel NK-lysin 4(+) cell type, representing a putative cytotoxic T/NK cell. Our platform also quantified hematopoietic defects in rag2(E450fs) mutant fish and showed that these fish have reduced T cells with a subsequent expansion of NK-lysin 4(+) cells and myeloid cells. These data suggest compensatory regulation of the innate immune system in rag2(E450fs) mutant zebrafish. Finally, analysis of Myc-induced T cell acute lymphoblastic leukemia showed that cells are arrested at the CD4(+)/CD8(+) cortical thymocyte stage and that a subset of leukemia cells inappropriately reexpress stem cell genes, including bmi1 and cmyb In total, our experiments provide new tools and biological insights into single-cell heterogeneity found in zebrafish blood and leukemia. © 2016 Moore et al.

Single-cell transcriptional analysis of normal, aberrant, and malignant hematopoiesis in zebrafish

PubMed Central

Garcia, Elaine G.; Lobbardi, Riadh; Jain, Esha; Tang, Qin; Moore, John C.; Cortes, Mauricio; Molodtsov, Aleksey; Kasheta, Melissa; Luo, Christina C.; Garcia, Amaris J.; Mylvaganam, Ravi; Yoder, Jeffrey A.; Blackburn, Jessica S.; Sadreyev, Ruslan I.; Ceol, Craig J.; North, Trista E.

2016-01-01

Hematopoiesis culminates in the production of functionally heterogeneous blood cell types. In zebrafish, the lack of cell surface antibodies has compelled researchers to use fluorescent transgenic reporter lines to label specific blood cell fractions. However, these approaches are limited by the availability of transgenic lines and fluorescent protein combinations that can be distinguished. Here, we have transcriptionally profiled single hematopoietic cells from zebrafish to define erythroid, myeloid, B, and T cell lineages. We also used our approach to identify hematopoietic stem and progenitor cells and a novel NK-lysin 4+ cell type, representing a putative cytotoxic T/NK cell. Our platform also quantified hematopoietic defects in rag2E450fs mutant fish and showed that these fish have reduced T cells with a subsequent expansion of NK-lysin 4+ cells and myeloid cells. These data suggest compensatory regulation of the innate immune system in rag2E450fs mutant zebrafish. Finally, analysis of Myc-induced T cell acute lymphoblastic leukemia showed that cells are arrested at the CD4+/CD8+ cortical thymocyte stage and that a subset of leukemia cells inappropriately reexpress stem cell genes, including bmi1 and cmyb. In total, our experiments provide new tools and biological insights into single-cell heterogeneity found in zebrafish blood and leukemia. PMID:27139488

Assessing similarity to primary tissue and cortical layer identity in induced pluripotent stem cell-derived cortical neurons through single-cell transcriptomics

PubMed Central

Handel, Adam E.; Chintawar, Satyan; Lalic, Tatjana; Whiteley, Emma; Vowles, Jane; Giustacchini, Alice; Argoud, Karene; Sopp, Paul; Nakanishi, Mahito; Bowden, Rory; Cowley, Sally; Newey, Sarah; Akerman, Colin; Ponting, Chris P.; Cader, M. Zameel

2016-01-01

Induced pluripotent stem cell (iPSC)-derived cortical neurons potentially present a powerful new model to understand corticogenesis and neurological disease. Previous work has established that differentiation protocols can produce cortical neurons, but little has been done to characterize these at cellular resolution. In particular, it is unclear to what extent in vitro two-dimensional, relatively disordered culture conditions recapitulate the development of in vivo cortical layer identity. Single-cell multiplex reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) was used to interrogate the expression of genes previously implicated in cortical layer or phenotypic identity in individual cells. Totally, 93.6% of single cells derived from iPSCs expressed genes indicative of neuronal identity. High proportions of single neurons derived from iPSCs expressed glutamatergic receptors and synaptic genes. And, 68.4% of iPSC-derived neurons expressing at least one layer marker could be assigned to a laminar identity using canonical cortical layer marker genes. We compared single-cell RNA-seq of our iPSC-derived neurons to available single-cell RNA-seq data from human fetal and adult brain and found that iPSC-derived cortical neurons closely resembled primary fetal brain cells. Unexpectedly, a subpopulation of iPSC-derived neurons co-expressed canonical fetal deep and upper cortical layer markers. However, this appeared to be concordant with data from primary cells. Our results therefore provide reassurance that iPSC-derived cortical neurons are highly similar to primary cortical neurons at the level of single cells but suggest that current layer markers, although effective, may not be able to disambiguate cortical layer identity in all cells. PMID:26740550

Targeting Notch signalling pathway of cancer stem cells.

PubMed

Venkatesh, Vandana; Nataraj, Raghu; Thangaraj, Gopenath S; Karthikeyan, Murugesan; Gnanasekaran, Ashok; Kaginelli, Shanmukhappa B; Kuppanna, Gobianand; Kallappa, Chandrashekrappa Gowdru; Basalingappa, Kanthesh M

2018-01-01

Cancer stem cells (CSCs) have been defined as cells within tumor that possess the capacity to self-renew and to cause the heterogeneous lineages of cancer cells that comprise the tumor. CSCs have been increasingly identified in blood cancer, prostate, ovarian, lung, melanoma, pancreatic, colon, brain and many more malignancies. CSCs have slow growth rate and are resistant to chemotherapy and radiotherapy that lead to the failure of traditional current therapy. Eradicating the CSCs and recurrence, is promising aspect for the cure of cancer. The CSCs like any other stem cells activate the signal transduction pathways that involve the development and tissue homeostasis, which include Notch signaling pathway. The new treatment targets these pathway that control stem-cell replication, survival and differentiation that are under development. Notch inhibitors either single or in combination with chemotherapy drugs have been developed to treat cancer and its recurrence. This approach of targeting signaling pathway of CSCs represents a promising future direction for the therapeutic strategy to cure cancer.

Distinct mechanisms underlie oral vs aboral regeneration in the cnidarian Hydractinia echinata.

PubMed

Bradshaw, Brian; Thompson, Kerry; Frank, Uri

2015-04-17

Cnidarians possess remarkable powers of regeneration, but the cellular and molecular mechanisms underlying this capability are unclear. Studying the hydrozoan Hydractinia echinata we show that a burst of stem cell proliferation occurs following decapitation, forming a blastema at the oral pole within 24 hr. This process is necessary for head regeneration. Knocking down Piwi1, Vasa, Pl10 or Ncol1 expressed by blastema cells inhibited regeneration but not blastema formation. EdU pulse-chase experiments and in vivo tracking of individual transgenic Piwi1(+) stem cells showed that the cellular source for blastema formation is migration of stem cells from a remote area. Surprisingly, no blastema developed at the aboral pole after stolon removal. Instead, polyps transformed into stolons and then budded polyps. Hence, distinct mechanisms act to regenerate different body parts in Hydractinia. This model, where stem cell behavior can be monitored in vivo at single cell resolution, offers new insights for regenerative biology.

Label-free screening of niche-to-niche variation in satellite stem cells using functionalized pores

NASA Astrophysics Data System (ADS)

Chapman, Matthew R.; Balakrishnan, Karthik; Conboy, Michael J.; Mohanty, Swomitra; Jabart, Eric; Huang, Haiyan; Hack, James; Conboy, Irina M.; Sohn, Lydia L.

2012-02-01

Combinations of surface markers are currently used to identify muscle satellite cells. Using pores functionalized with specific antibodies and measuring the transit time of cells passing through these pores, we discovered remarkable heterogeneity in the expression of these markers in muscle (satellite) stem cells that reside in different single myofibers. Microniche-specific variation in stem cells of the same organ has not been previously described, as bulk analysis does not discriminate between separate myofibers or even separate hind-leg muscle groups. We found a significant population of Sca-1+ satellite cells that form myotubes, thereby demonstrating the myogenic potential of Sca-1+ cells, which are currently excluded in bulk sorting. Finally, using our label-free pore screening technique, we have been able to quantify directly surface expression of Notch1 without activation of the Notch pathway. We show for the first time Notch1-expression heterogeneity in unactivated satellite cells. The discovery of fiber-to-fiber variations prompts new research into the reasons for such diversity in muscle stem cells.

Targeting cancer stem cell plasticity through modulation of epidermal growth factor and insulin-like growth factor receptor signaling in head and neck squamous cell cancer.

PubMed

Leong, Hui Sun; Chong, Fui Teen; Sew, Pui Hoon; Lau, Dawn P; Wong, Bernice H; Teh, Bin-Tean; Tan, Daniel S W; Iyer, N Gopalakrishna

2014-09-01

Emerging data suggest that cancer stem cells (CSCs) exist in equilibrium with differentiated cells and that stochastic transitions between these states can account for tumor heterogeneity and drug resistance. The aim of this study was to establish an in vitro system that recapitulates stem cell plasticity in head and neck squamous cell cancers (HNSCCs) and identify the factors that play a role in the maintenance and repopulation of CSCs. Tumor spheres were established using patient-derived cell lines via anchorage-independent cell culture techniques. These tumor spheres were found to have higher aldehyde dehydrogenase (ALD) cell fractions and increased expression of Kruppel-like factor 4, SRY (sex determining region Y)-box 2, and Nanog and were resistant to γ-radiation, 5-fluorouracil, cisplatin, and etoposide treatment compared with monolayer culture cells. Monolayer cultures were subject to single cell cloning to generate clones with high and low ALD fractions. ALDHigh clones showed higher expression of stem cell and epithelial-mesenchymal transition markers compared with ALDLow clones. ALD fractions, representing stem cell fractions, fluctuated with serial passaging, equilibrating at a level specific to each cell line, and could be augmented by the addition of epidermal growth factor (EGF) and/or insulin. ALDHigh clones showed increased EGF receptor (EGFR) and insulin-like growth factor-1 receptor (IGF-1R) phosphorylation, with increased activation of downstream pathways compared with ALDLow clones. Importantly, blocking these pathways using specific inhibitors against EGFR and IGF-1R reduced stem cell fractions drastically. Taken together, these results show that HNSCC CSCs exhibit plasticity, with the maintenance of the stem cell fraction dependent on the EGFR and IGF-1R pathways and potentially amenable to targeted therapeutics. ©AlphaMed Press.

Symmetry of initial cell divisions among primitive hematopoietic progenitors is independent of ontogenic age and regulatory molecules.

PubMed

Huang, S; Law, P; Francis, K; Palsson, B O; Ho, A D

1999-10-15

We have developed a time-lapse camera system to follow the replication history and the fate of hematopoietic stem cells (HSC) at a single-cell level. Combined with single-cell culture, we correlated the early replication behavior with colony development after 14 days. The membrane dye PKH26 was used to monitor cell division. In addition to multiple, synchronous, and symmetric divisions, single-sorted CD34(+)/CD38(-) cells derived from fetal liver (FLV) also gave rise to a daughter cell that remained quiescent for up to 8 days, whereas the other daughter cell proliferated exponentially. Upon separation and replating as single cells onto medium containing a cytokine cocktail, 60.6% +/- 9.8% of the initially quiescent cells (PKH26 bright) gave rise again to colonies and 15.8% +/- 7.8% to blast colonies that could be replated. We have then determined the effects of various regulatory molecules on symmetry of initial cell divisions. After single-cell sorting, the CD34(+)/CD38(-) cells derived from FLV were exposed to flt3-ligand, thrombopoietin, stem cell factor (SCF), or medium containing a cytokine cocktail (with SCF, interleukin-3, interleukin-6, granulocyte-macrophage colony-stimulating factor, and erythropoietin). Whereas mitotic rate, colony efficiency, and asymmetric divisions could be altered using various regulatory molecules, the asymmetric division index, defined as the number of asymmetric divisions versus the number of dividing cells, was not altered significantly. This observation suggests that, although lineage commitment and cell proliferation can be skewed by extrinsic signaling, symmetry of early divisions is probably under the control of intrinsic factors.

High-content image informatics of the structural nuclear protein NuMA parses trajectories for stem/progenitor cell lineages and oncogenic transformation

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

Vega, Sebastián L.; Liu, Er; Arvind, Varun

Stem and progenitor cells that exhibit significant regenerative potential and critical roles in cancer initiation and progression remain difficult to characterize. Cell fates are determined by reciprocal signaling between the cell microenvironment and the nucleus; hence parameters derived from nuclear remodeling are ideal candidates for stem/progenitor cell characterization. Here we applied high-content, single cell analysis of nuclear shape and organization to examine stem and progenitor cells destined to distinct differentiation endpoints, yet undistinguishable by conventional methods. Nuclear descriptors defined through image informatics classified mesenchymal stem cells poised to either adipogenic or osteogenic differentiation, and oligodendrocyte precursors isolated from different regionsmore » of the brain and destined to distinct astrocyte subtypes. Nuclear descriptors also revealed early changes in stem cells after chemical oncogenesis, allowing the identification of a class of cancer-mitigating biomaterials. To capture the metrology of nuclear changes, we developed a simple and quantitative “imaging-derived” parsing index, which reflects the dynamic evolution of the high-dimensional space of nuclear organizational features. A comparative analysis of parsing outcomes via either nuclear shape or textural metrics of the nuclear structural protein NuMA indicates the nuclear shape alone is a weak phenotypic predictor. In contrast, variations in the NuMA organization parsed emergent cell phenotypes and discerned emergent stages of stem cell transformation, supporting a prognosticating role for this protein in the outcomes of nuclear functions. - Highlights: • High-content analysis of nuclear shape and organization classify stem and progenitor cells poised for distinct lineages. • Early oncogenic changes in mesenchymal stem cells (MSCs) are also detected with nuclear descriptors. • A new class of cancer-mitigating biomaterials was identified based on image informatics. • Textural metrics of the nuclear structural protein NuMA are sufficient to parse emergent cell phenotypes.« less

Scaling and automation of a high-throughput single-cell-derived tumor sphere assay chip.

PubMed

Cheng, Yu-Heng; Chen, Yu-Chih; Brien, Riley; Yoon, Euisik

2016-10-07

Recent research suggests that cancer stem-like cells (CSCs) are the key subpopulation for tumor relapse and metastasis. Due to cancer plasticity in surface antigen and enzymatic activity markers, functional tumorsphere assays are promising alternatives for CSC identification. To reliably quantify rare CSCs (1-5%), thousands of single-cell suspension cultures are required. While microfluidics is a powerful tool in handling single cells, previous works provide limited throughput and lack automatic data analysis capability required for high-throughput studies. In this study, we present the scaling and automation of high-throughput single-cell-derived tumor sphere assay chips, facilitating the tracking of up to ∼10 000 cells on a chip with ∼76.5% capture rate. The presented cell capture scheme guarantees sampling a representative population from the bulk cells. To analyze thousands of single-cells with a variety of fluorescent intensities, a highly adaptable analysis program was developed for cell/sphere counting and size measurement. Using a Pluronic® F108 (poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)) coating on polydimethylsiloxane (PDMS), a suspension culture environment was created to test a controversial hypothesis: whether larger or smaller cells are more stem-like defined by the capability to form single-cell-derived spheres. Different cell lines showed different correlations between sphere formation rate and initial cell size, suggesting heterogeneity in pathway regulation among breast cancer cell lines. More interestingly, by monitoring hundreds of spheres, we identified heterogeneity in sphere growth dynamics, indicating the cellular heterogeneity even within CSCs. These preliminary results highlight the power of unprecedented high-throughput and automation in CSC studies.

Agent-Based Deterministic Modeling of the Bone Marrow Homeostasis.

PubMed

Kurhekar, Manish; Deshpande, Umesh

2016-01-01

Modeling of stem cells not only describes but also predicts how a stem cell's environment can control its fate. The first stem cell populations discovered were hematopoietic stem cells (HSCs). In this paper, we present a deterministic model of bone marrow (that hosts HSCs) that is consistent with several of the qualitative biological observations. This model incorporates stem cell death (apoptosis) after a certain number of cell divisions and also demonstrates that a single HSC can potentially populate the entire bone marrow. It also demonstrates that there is a production of sufficient number of differentiated cells (RBCs, WBCs, etc.). We prove that our model of bone marrow is biologically consistent and it overcomes the biological feasibility limitations of previously reported models. The major contribution of our model is the flexibility it allows in choosing model parameters which permits several different simulations to be carried out in silico without affecting the homeostatic properties of the model. We have also performed agent-based simulation of the model of bone marrow system proposed in this paper. We have also included parameter details and the results obtained from the simulation. The program of the agent-based simulation of the proposed model is made available on a publicly accessible website.

Pluripotent Stem Cell-Based Therapies in Combination with Substrate for the Treatment of Age-Related Macular Degeneration.

PubMed

Pennington, Britney O; Clegg, Dennis O

2016-06-01

Age-related macular degeneration (AMD) is the leading cause of blindness in the western world, which severely decreases the quality of life in the patients and places an economic burden on their families and society. The disease is caused by the dysfunction of a specialized cell layer in the back of the eye called the retinal pigmented epithelium (RPE). Pluripotent stem cells can provide an unlimited source of RPE, and laboratories around the world are investigating their potential as therapies for AMD. To ensure the precise delivery of functional RPE to the diseased site, some groups are developing a therapy composed of mature RPE monolayers on a supportive scaffold for transplantation as an alternative to injecting a single-cell suspension. This review summarizes methods of generating RPE from pluripotent stem cells, compares biodegradable and biostable materials as scaffolds, and describes the specific combination of human embryonic stem cell-derived RPE on Parylene-C membranes, which is scheduled to begin clinical trials in the United Sates in 2016. Stem cell-derived RPE monolayers on scaffolds hold great promise for the treatment of AMD and other retinal diseases.

Identification of Multipotent Stem/Progenitor Cells in Murine Sclera

PubMed Central

Tsai, Chia-Ling; Wu, Pei-Chang; Fini, M. Elizabeth; Shi, Songtao

2011-01-01

Purpose. The sclera forms the fibrous outer coat of the eyeball and acts as a supportive framework. The purpose of this study was to examine whether the sclera contains mesenchymal stem/progenitor cells. Method. Scleral tissue from C57BL6/J mice was separated from the retina and choroid and subsequently enzyme digested to release single cells. Proliferation capacity, self-renewal capacity, and ability for multipotent differentiation were analyzed by BrdU labeling, flow cytometry, reverse transcriptase–polymerase chain reaction, immunocytochemistry, and in vivo transplantation. Results. The scleral stem/progenitor cells (SSPCs) possessed clonogenic and high doubling capacities. These cells were positive for the mesenchymal markers Sca-1, CD90.2, CD44, CD105, and CD73 and negative for the hematopoietic markers CD45, CD11b, Flk1, CD34, and CD117. In addition to expressing stem cell genes ABCG2, Six2, Notch1, and Pax6, SSPCs were able to differentiate to adipogenic, chondrogenic, and neurogenic lineages. Conclusions. This study indicates that the sclera contains multipotent mesenchymal stem cells. Further study of SSPCs may help elucidate the cellular and molecular mechanism of scleral diseases such as scleritis and myopia. PMID:21788434

An "age"-structured model of hematopoietic stem cell organization with application to chronic myeloid leukemia.

PubMed

Roeder, Ingo; Herberg, Maria; Horn, Matthias

2009-04-01

Previously, we have modeled hematopoietic stem cell organization by a stochastic, single cell-based approach. Applications to different experimental systems demonstrated that this model consistently explains a broad variety of in vivo and in vitro data. A major advantage of the agent-based model (ABM) is the representation of heterogeneity within the hematopoietic stem cell population. However, this advantage comes at the price of time-consuming simulations if the systems become large. One example in this respect is the modeling of disease and treatment dynamics in patients with chronic myeloid leukemia (CML), where the realistic number of individual cells to be considered exceeds 10(6). To overcome this deficiency, without losing the representation of the inherent heterogeneity of the stem cell population, we here propose to approximate the ABM by a system of partial differential equations (PDEs). The major benefit of such an approach is its independence from the size of the system. Although this mean field approach includes a number of simplifying assumptions compared to the ABM, it retains the key structure of the model including the "age"-structure of stem cells. We show that the PDE model qualitatively and quantitatively reproduces the results of the agent-based approach.

Dynamic and social behaviors of human pluripotent stem cells.

PubMed

Phadnis, Smruti M; Loewke, Nathan O; Dimov, Ivan K; Pai, Sunil; Amwake, Christine E; Solgaard, Olav; Baer, Thomas M; Chen, Bertha; Reijo Pera, Renee A

2015-09-18

Human pluripotent stem cells (hPSCs) can self-renew or differentiate to diverse cell types, thus providing a platform for basic and clinical applications. However, pluripotent stem cell populations are heterogeneous and functional properties at the single cell level are poorly documented leading to inefficiencies in differentiation and concerns regarding reproducibility and safety. Here, we use non-invasive time-lapse imaging to continuously examine hPSC maintenance and differentiation and to predict cell viability and fate. We document dynamic behaviors and social interactions that prospectively distinguish hPSC survival, self-renewal, and differentiation. Results highlight the molecular role of E-cadherin not only for cell-cell contact but also for clonal propagation of hPSCs. Results indicate that use of continuous time-lapse imaging can distinguish cellular heterogeneity with respect to pluripotency as well as a subset of karyotypic abnormalities whose dynamic properties were monitored.

Dynamic and social behaviors of human pluripotent stem cells

PubMed Central

Phadnis, Smruti M.; Loewke, Nathan O.; Dimov, Ivan K.; Pai, Sunil; Amwake, Christine E.; Solgaard, Olav; Baer, Thomas M.; Chen, Bertha; Pera, Renee A. Reijo

2015-01-01

Human pluripotent stem cells (hPSCs) can self-renew or differentiate to diverse cell types, thus providing a platform for basic and clinical applications. However, pluripotent stem cell populations are heterogeneous and functional properties at the single cell level are poorly documented leading to inefficiencies in differentiation and concerns regarding reproducibility and safety. Here, we use non-invasive time-lapse imaging to continuously examine hPSC maintenance and differentiation and to predict cell viability and fate. We document dynamic behaviors and social interactions that prospectively distinguish hPSC survival, self-renewal, and differentiation. Results highlight the molecular role of E-cadherin not only for cell-cell contact but also for clonal propagation of hPSCs. Results indicate that use of continuous time-lapse imaging can distinguish cellular heterogeneity with respect to pluripotency as well as a subset of karyotypic abnormalities whose dynamic properties were monitored. PMID:26381699

3D Graphene Oxide-encapsulated Gold Nanoparticles to Detect Neural Stem Cell Differentiation

PubMed Central

Kim, Tae-Hyung; Lee, Ki-Bum; Choi, Jeong-Woo

2013-01-01

Monitoring of stem cell differentiation and pluripotency is an important step for the practical use of stem cells in the field of regenerative medicine. Hence, a new non-destructive detection tool capable of in situ monitoring of stem cell differentiation is highly needed. In this study, we report a 3D graphene oxide-encapsulated gold nanoparticle that is very effective for the detection of the differentiation potential of neural stem cells (NSCs) based on surface-enhanced Raman spectroscopy (SERS). A new material, 3D GO-encapsulated gold nanoparticle, is developed to induce the double enhancement effect of graphene oxide and gold nanoparticle on SERS signals which is only effective for undifferentiated NSCs. The Raman peaks achieved from undifferentiated NSCs on the graphene oxide (GO)-encapsulated gold nanoparticles were 3.5 times higher than peaks obtained from normal metal structures and were clearly distinguishable from those of differentiated cells. The number of C=C bonds and the raman instensity at 1656cm−1 was found to show a positive correlation, which matches the differentiation state of the NSCs. Moreover, the substrate composed of 3D GO-encapsulated gold nanoparticles was also effective at distinguishing the differentiation state of single NSC by using electrochemical and electrical techniques. Hence, the proposed technique can be used as a powerful non-destructive in situ monitoring tool for the identification of the differentiation potential of various kinds of stem cells (mesenchymal, hematopoietic, and neural stem cells). PMID:23937915

In Vitro T-Cell Generation From Adult, Embryonic, and Induced Pluripotent Stem Cells: Many Roads to One Destination.

PubMed

Smith, Michelle J; Webber, Beau R; Mohtashami, Mahmood; Stefanski, Heather E; Zúñiga-Pflücker, Juan Carlos; Blazar, Bruce R

2015-11-01

T lymphocytes are critical mediators of the adaptive immune system and have the capacity to serve as therapeutic agents in the areas of transplant and cancer immunotherapy. While T cells can be isolated and expanded from patients, T cells derived in vitro from both hematopoietic stem/progenitor cells (HSPCs) and human pluripotent stem cells (hPSCs) offer great potential advantages in generating a self-renewing source of T cells that can be readily genetically modified. T-cell differentiation in vivo is a complex process requiring tightly regulated signals; providing the correct signals in vitro to induce T-cell lineage commitment followed by their development into mature, functional, single positive T cells, is similarly complex. In this review, we discuss current methods for the in vitro derivation of T cells from murine and human HSPCs and hPSCs that use feeder-cell and feeder-cell-free systems. Furthermore, we explore their potential for adoption for use in T-cell-based therapies. © 2015 AlphaMed Press.

Generation of mature T cells from human hematopoietic stem and progenitor cells in artificial thymic organoids.

PubMed

Seet, Christopher S; He, Chongbin; Bethune, Michael T; Li, Suwen; Chick, Brent; Gschweng, Eric H; Zhu, Yuhua; Kim, Kenneth; Kohn, Donald B; Baltimore, David; Crooks, Gay M; Montel-Hagen, Amélie

2017-05-01

Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3 + TCR-αβ + single-positive CD8 + or CD4 + cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naive phenotypes, a diverse T cell receptor (TCR) repertoire and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen-specific cytotoxicity and near-complete lack of endogenous TCR Vβ expression, consistent with allelic exclusion of Vβ-encoding loci. ATOs provide a robust tool for studying human T cell differentiation and for the future development of stem-cell-based engineered T cell therapies.

Autonomous assembly of epithelial structures by subrenal implantation of dissociated embryonic inner-ear cells.

PubMed

Wang, Li; Zhang, Kaiqing; Zhu, Helen He; Gao, Wei-Qiang

2015-05-27

Microenvironment and cell-cell interactions play an important role during embryogenesis and are required for the stemness and differentiation of stem cells. The inner-ear sensory epithelium, containing hair cells and supporting cells, is derived from the stem cells within the otic vesicle at early embryonic stages. However, whether or not such microenvironment or cell-cell interactions within the embryonic otic tissue have the capacity to regulate the proliferation and differentiation of stem cells and to autonomously reassemble the cells into epithelial structures is unknown. Here, we report that on enzymatic digestion and dissociation to harvest all the single cells from 13.5-day-old rat embryonic (E13.5) inner-ear tissue as well as on implantation of these cells under renal capsules; the dissociated cells are able to reassemble themselves to form epithelial structures as early as 7 days after implantation. By 25 days after implantation, more mature epithelial structures are formed. Immunostaining with cell-type-specific markers reveals that hair cells and supporting cells are not only formed, but are also well aligned with the hair cells located in the apical layer surrounded by the supporting cells. These findings suggest that microenvironment and cell-cell interactions within the embryonic inner-ear tissue have the autonomous signals to induce the formation of sensory epithelial structures. This method may also provide a useful system to study the potential of stem cells to differentiate into hair cells in vivo.

Flow cytometric analysis of normal and neoplastic mast cells: role in diagnosis and follow-up of mast cell disease.

PubMed

Escribano, Luis; Garcia Montero, Andres C; Núñez, Rosa; Orfao, Alberto

2006-08-01

Human mast cells (MCs) are directly derived from human pluripotent CD34+ stem and progenitor hematopoietic cells with stem cell factor being a critical growth factor supporting human MC proliferation, differentiation, and survival. Because of the advantages that flow cytometry offers (it allows rapid, objective, and sensitive multiparameter analysis of high numbers of cells from a sample, with information being provided on the basis of a single cell), it has become the method of choice in the past decade for immunophenotypic identification, enumeration, and characterization of human MCs in bone marrow and other tissue specimens.

Characterization of stem-like cells in a new astroblastoma cell line

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

Coban, Esra Aydemir; Kasikci, Ezgi; Karatas, Omer Faruk

Cell lines established from tumors are the most commonly used models in cancer research, and their use in recent years has enabled a greater understanding of the biology of cancer and the means to develop effective treatment strategies. Astroblastomas are uncommon neuroepithelial tumors of glial origin, predominantly affecting young people, mainly teenagers and children, predominantly females. To date, only a single study has reported that astroblastomas contain a large number of neural stem-like cells, which had only a partial proliferation capacity and differentiation. Our objective was to establish an astroblastoma cell line to investigate the presence of astroblastic cells andmore » cancer stem-like cells. The migratory and invasion abilities of the cells were quantified with invasion and migration assays and compared to a glioblastoma cell line. The presence of stem cells was detected with surface-marker analysis by using flow cytometry, and measuring the differentiation ability with a differentiation assay and the self-renewal capacity with a sphere-forming assay. These characteristics may determine whether this novel cell line is a model for astroblastomas that may have stem-cell characteristics. With this novel cell line, scientists can investigate the molecular pathways underlying astroblastomas and develop new therapeutic strategies for patients with these tumors. - Highlights: • An establishment of a novel astroblastoma cell line was proposed. • The presence of astroblastic cells and cancer stem-like cells was investigated. • The molecular pathways underlying astroblastomas may be investigated. • New therapeutic strategies for patients with astroblastoma may be developed.« less

β-Globin-Expressing Definitive Erythroid Progenitor Cells Generated from Embryonic and Induced Pluripotent Stem Cell-Derived Sacs.

PubMed

Fujita, Atsushi; Uchida, Naoya; Haro-Mora, Juan J; Winkler, Thomas; Tisdale, John

2016-06-01

Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells represent a potential alternative source for red blood cell transfusion. However, when using traditional methods with embryoid bodies, ES cell-derived erythroid cells predominantly express embryonic type ɛ-globin, with lesser fetal type γ-globin and very little adult type β-globin. Furthermore, no β-globin expression is detected in iPS cell-derived erythroid cells. ES cell-derived sacs (ES sacs) have been recently used to generate functional platelets. Due to its unique structure, we hypothesized that ES sacs serve as hemangioblast-like progenitors capable to generate definitive erythroid cells that express β-globin. With our ES sac-derived erythroid differentiation protocol, we obtained ∼120 erythroid cells per single ES cell. Both primitive (ɛ-globin expressing) and definitive (γ- and β-globin expressing) erythroid cells were generated from not only ES cells but also iPS cells. Primitive erythropoiesis is gradually switched to definitive erythropoiesis during prolonged ES sac maturation, concurrent with the emergence of hematopoietic progenitor cells. Primitive and definitive erythroid progenitor cells were selected on the basis of glycophorin A or CD34 expression from cells within the ES sacs before erythroid differentiation. This selection and differentiation strategy represents an important step toward the development of in vitro erythroid cell production systems from pluripotent stem cells. Further optimization to improve expansion should be required for clinical application. Stem Cells 2016;34:1541-1552. © 2016 AlphaMed Press.

Isolation and clonal characterization of hematopoietic and liver stem cells.

PubMed

Nakauchi, Hiromitsu

2004-11-01

Prospective isolation of stem cells is essential to understanding the mechanisms that control their proliferation and differentiation. Using 9 monoclonal antibodies and fluorescence-activated cell sorting (FACS), we have succeeded in prospectively identifying hematopoietic stem cells (HSCs) in adult mouse bone marrow. Mouse HSCs were exclusively enriched in CD34 negative, c-Kit Sca-1 Lineage Marker (CD34 KSL) cells representing 0.004% of bone marrow (BM) mononuclear cells. When single CD34-KSL cells were transplanted individually into a lethally irradiated mouse, 25% of the recipient mice survived and showed long-term reconstitution of the BM, providing evidence for multipotency and a self-renewal capacity of HSCs. Using a similar approach, we also prospectively identified hepatic stem cells with multilineage differentiation potential and self-renewal capability in the c-Met CD49f c-Kit CD45 Ter119 fraction of cells isolated from day 13.5 fetal mouse liver. On cell transplantation, these cells differentiated into hepatocytes and cholangiocytes. As an alternative to the antibody based stem cell isolation, Hoechst33342 staining is useful. To understand the mechanism responsible for SP phenotype, we performed an expression cloning and identified bcrp-1/ABCG2 gene, a member of ATP binding-cassette (ABC) transporter family. Bcrp-1 is almost exclusively expressed in CD34 KSL cells among blood cells; however their expression in other tissue specific stem cells remains to be studied. With the use of FACS and monoclonal antibodies, hematopoietic and liver stem cells were prospectively isolated and characterized. HSCs could also be purified by Hoechst 33342 staining. By expression cloning, we identify bcrp-1/ABCG2 transporter as a molecule responsible for SP phenotype. Elucidation of the physiological role of bcrp-1/ABCG2 in HSCs may provide us with clues to understand the molecular mechanisms of stem cell self-renewal and differentiation.

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.

Diffusion maps for high-dimensional single-cell analysis of differentiation data.

PubMed

Haghverdi, Laleh; Buettner, Florian; Theis, Fabian J

2015-09-15

Single-cell technologies have recently gained popularity in cellular differentiation studies regarding their ability to resolve potential heterogeneities in cell populations. Analyzing such high-dimensional single-cell data has its own statistical and computational challenges. Popular multivariate approaches are based on data normalization, followed by dimension reduction and clustering to identify subgroups. However, in the case of cellular differentiation, we would not expect clear clusters to be present but instead expect the cells to follow continuous branching lineages. Here, we propose the use of diffusion maps to deal with the problem of defining differentiation trajectories. We adapt this method to single-cell data by adequate choice of kernel width and inclusion of uncertainties or missing measurement values, which enables the establishment of a pseudotemporal ordering of single cells in a high-dimensional gene expression space. We expect this output to reflect cell differentiation trajectories, where the data originates from intrinsic diffusion-like dynamics. Starting from a pluripotent stage, cells move smoothly within the transcriptional landscape towards more differentiated states with some stochasticity along their path. We demonstrate the robustness of our method with respect to extrinsic noise (e.g. measurement noise) and sampling density heterogeneities on simulated toy data as well as two single-cell quantitative polymerase chain reaction datasets (i.e. mouse haematopoietic stem cells and mouse embryonic stem cells) and an RNA-Seq data of human pre-implantation embryos. We show that diffusion maps perform considerably better than Principal Component Analysis and are advantageous over other techniques for non-linear dimension reduction such as t-distributed Stochastic Neighbour Embedding for preserving the global structures and pseudotemporal ordering of cells. The Matlab implementation of diffusion maps for single-cell data is available at https://www.helmholtz-muenchen.de/icb/single-cell-diffusion-map. fbuettner.phys@gmail.com, fabian.theis@helmholtz-muenchen.de Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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 mature T cells from human hematopoietic stem/progenitor cells in artificial thymic organoids

PubMed Central

Seet, Christopher S.; He, Chongbin; Bethune, Michael T.; Li, Suwen; Chick, Brent; Gschweng, Eric H.; Zhu, Yuhua; Kim, Kenneth; Kohn, Donald B.; Baltimore, David; Crooks, Gay M.; Montel-Hagen, Amélie

2017-01-01

Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3+TCRab+ single positive (SP) CD8+ or CD4+ cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports highly efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naïve phenotypes, a diverse TCR repertoire, and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen specific cytotoxicity and near complete lack of endogenous TCR Vβ expression, consistent with allelic exclusion of Vβ loci. ATOs provide a robust tool for studying human T cell development and stem cell based approaches to engineered T cell therapies. PMID:28369043

GiniClust: detecting rare cell types from single-cell gene expression data with Gini index.

PubMed

Jiang, Lan; Chen, Huidong; Pinello, Luca; Yuan, Guo-Cheng

2016-07-01

High-throughput single-cell technologies have great potential to discover new cell types; however, it remains challenging to detect rare cell types that are distinct from a large population. We present a novel computational method, called GiniClust, to overcome this challenge. Validation against a benchmark dataset indicates that GiniClust achieves high sensitivity and specificity. Application of GiniClust to public single-cell RNA-seq datasets uncovers previously unrecognized rare cell types, including Zscan4-expressing cells within mouse embryonic stem cells and hemoglobin-expressing cells in the mouse cortex and hippocampus. GiniClust also correctly detects a small number of normal cells that are mixed in a cancer cell population.

Comparative study of allogenic and xenogeneic mesenchymal stem cells on cisplatin-induced acute kidney injury in Sprague-Dawley rats.

PubMed

Ashour, Rehab H; Saad, Mohamed-Ahdy; Sobh, Mohamed-Ahmed; Al-Husseiny, Fatma; Abouelkheir, Mohamed; Awad, Amal; Elghannam, Doaa; Abdel-Ghaffar, Hassan; Sobh, Mohamed

2016-09-01

The paracrine and regenerative activities of mesenchymal stem cells (MSCs) may vary with different stem cell sources. The aim of the present study is to compare the effects of MSCs from different sources on acute kidney injury (AKI) induced by cisplatin and their influence on renal regeneration. A single intraperitoneal injection of cisplatin (5 mg/kg) was used to induce AKI in 120 Sprague-Dawley rats. Rats were treated with either rat bone marrow stem cells (rBMSCs), human adipose tissue-derived stem cells (hADSCs), or human amniotic fluid-derived stem cells (hAFSCs). 5 × 10(6) MSCs of different sources were administered through rat tail vein in a single dose, 24 hours after cisplatin injection. Within each group, rats were sacrificed at the 4th, 7th, 11th, and 30th day after cisplatin injection. Serum creatinine, BUN, and renal tissue oxidative stress parameters were measured. Renal tissue was scored histopathologically for evidence of injury, regeneration, and chronicity. Immunohistochemistry was also done using Ki67 for renal proliferative activity evaluation. MSCs of the three sources were able to ameliorate cisplatin-induced renal function deterioration and tissue damage. The rat BMSCs-treated group had the lowest serum creatinine by day 30 (0.52 ± 0.06) compared to hADSCs and hAFSCs. All MSC-treated groups had nearly equal antioxidant activity as indicated by the decreased renal tissue malondialdehyde (MDA) and increased reduced glutathione (GSH) level and superoxide dismutase (SOD) activity at different time intervals. Additionally, all MSCs improved injury and regenerative scores. Rat BMSCs had the highest count and earliest proliferative activity in the renal cortex by day 7 as identified by Ki67; while, hAFSCs seem to have the greatest improvement in the regenerative and proliferative activities with a higher count of renal cortex Ki67-positive cells at day 11 and with the least necrotic lesions. Rat BMSCs, hADSCs, and hAFSCs, in early single IV dose, had a renoprotective effect against cisplatin-induced AKI, and were able to reduce oxidative stress markers. Rat BMSCs had the earliest proliferative activity by day 7; however, hAFSCs seemed to have the greatest improvement in the regenerative activities. Human ADSCs were the least effective in the terms of proliferative and regenerative activities.

Xeno-free culture of human pluripotent stem cells on oligopeptide-grafted hydrogels with various molecular designs

PubMed Central

Chen, Yen-Ming; Chen, Li-Hua; Li, Meng-Pei; Li, Hsing-Fen; Higuchi, Akon; Kumar, S. Suresh; Ling, Qing-Dong; Alarfaj, Abdullah A.; Munusamy, Murugan A.; Chang, Yung; Benelli, Giovanni; Murugan, Kadarkarai; Umezawa, Akihiro

2017-01-01

Establishing cultures of human embryonic (ES) and induced pluripotent (iPS) stem cells in xeno-free conditions is essential for producing clinical-grade cells. Development of cell culture biomaterials for human ES and iPS cells is critical for this purpose. We designed several structures of oligopeptide-grafted poly (vinyl alcohol-co-itaconic acid) hydrogels with optimal elasticity, and prepared them in formations of single chain, single chain with joint segment, dual chain with joint segment, and branched-type chain. Oligopeptide sequences were selected from integrin- and glycosaminoglycan-binding domains of the extracellular matrix. The hydrogels grafted with vitronectin-derived oligopeptides having a joint segment or a dual chain, which has a storage modulus of 25 kPa, supported the long-term culture of human ES and iPS cells for over 10 passages. The dual chain and/or joint segment with cell adhesion molecules on the hydrogels facilitated the proliferation and pluripotency of human ES and iPS cells. PMID:28332572

Xeno-free culture of human pluripotent stem cells on oligopeptide-grafted hydrogels with various molecular designs.

PubMed

Chen, Yen-Ming; Chen, Li-Hua; Li, Meng-Pei; Li, Hsing-Fen; Higuchi, Akon; Kumar, S Suresh; Ling, Qing-Dong; Alarfaj, Abdullah A; Munusamy, Murugan A; Chang, Yung; Benelli, Giovanni; Murugan, Kadarkarai; Umezawa, Akihiro

2017-03-23

Establishing cultures of human embryonic (ES) and induced pluripotent (iPS) stem cells in xeno-free conditions is essential for producing clinical-grade cells. Development of cell culture biomaterials for human ES and iPS cells is critical for this purpose. We designed several structures of oligopeptide-grafted poly (vinyl alcohol-co-itaconic acid) hydrogels with optimal elasticity, and prepared them in formations of single chain, single chain with joint segment, dual chain with joint segment, and branched-type chain. Oligopeptide sequences were selected from integrin- and glycosaminoglycan-binding domains of the extracellular matrix. The hydrogels grafted with vitronectin-derived oligopeptides having a joint segment or a dual chain, which has a storage modulus of 25 kPa, supported the long-term culture of human ES and iPS cells for over 10 passages. The dual chain and/or joint segment with cell adhesion molecules on the hydrogels facilitated the proliferation and pluripotency of human ES and iPS cells.

Bufalin inhibits the differentiation and proliferation of human osteosarcoma cell line hMG63-derived cancer stem cells.

PubMed

Chang, Yuewen; Zhao, Yongfang; Zhan, Hongsheng; Wei, Xiaoen; Liu, Tianjin; Zheng, Bo

2014-02-01

Cancer stem cells (CSCs) play an important role in drug resistance of tumor and are responsible for high recurrence rates. Agents that can suppress the proliferation and differentiation of CSCs would provide new opportunity to fight against tumor recurrence. In this study, we developed a new strategy to enrich CSCs in human osteosarcoma cell line hMG63. Using these CSCs as model, we tested the effect of bufalin, a traditional Chinese medicine, on the proliferation and differentiation of CSCs. hMG63 cells were cultured in poly-HEMA-treated dish and cancer stem cell-specific medium. In this nonadhesive culture system, hMG63 formed spheres, which were then collected and injected into the immunodeficient mice. Cisplatin was administered every 3 days for five times. The enriched xenograft tumors were cultured in cancer stem cell-specific medium again to form tumor spheres. Expression of cancer stem cell markers of these cells was measured by flow cytometry. These cells were then treated with bufalin, and the proliferation and differentiation ability were indicated by the expression level of molecular markers and the formation of sphere again in vitro. We obtained a low CD133+/CD44 cell population with high-level stem cell marker. When treated with bufalin, the sphere could not get attached to the flask and failed to differentiate, which was indicated by the stable expression of stem cell marker CD133 and OCT-4 in the condition permissive to differentiation. Treatment of bufalin also suppressed the single cells isolated from the sphere to form sphere again in the nonadhesive culture system, and a decreased expression of proliferation marker Ki67 was also detected in these cells. Sphere-formed and chemoresistant colon xenograft tumors in immunodeficient mice could enrich cancer stem cell population. Bufalin could inhibit proliferation and differentiation of CSCs.

Human multipotent adult progenitor cells are nonimmunogenic and exert potent immunomodulatory effects on alloreactive T-cell responses.

PubMed

Jacobs, Sandra A; Pinxteren, Jef; Roobrouck, Valerie D; Luyckx, Ariane; van't Hof, Wouter; Deans, Robert; Verfaillie, Catherine M; Waer, Mark; Billiau, An D; Van Gool, Stefaan W

2013-01-01

Multipotent adult progenitor cells (MAPCs) are bone marrow-derived nonhematopoietic stem cells with a broad differentiation potential and extensive expansion capacity. A comparative study between human mesenchymal stem cells (hMSCs) and human MAPCs (hMAPCs) has shown that hMAPCs have clearly distinct phenotypical and functional characteristics from hMSCs. In particular, hMAPCs express lower levels of MHC class I than hMSCs and cannot only differentiate into typical mesenchymal cell types but can also differentiate in vitro and in vivo into functional endothelial cells. The use of hMSCs as cellular immunomodulatory stem cell products gained much interest since their immunomodulatory capacities in vitro became evident over the last decade. Currently, the clinical grade stem cell product of hMAPCs is already used in clinical trials to prevent graft-versus-host disease (GVHD), as well as for the treatment of acute myocardial infarct, ischemic stroke, and Crohn's disease. Therefore, we studied the immune phenotype, immunogenicity, and immunosuppressive effect of hMAPCs in vitro. We demonstrated that hMAPCs are nonimmunogenic for T-cell proliferation and cytokine production. In addition, hMAPCs exert strong immunosuppressive effects on T-cell alloreactivity and on T-cell proliferation induced by mitogens and recall antigens. This immunomodulatory effect was not MHC restricted, which makes off-the-shelf use promising. The immunosuppressive effect of hMAPCs is partially mediated via soluble factors and dependent on indoleamine 2,3-dioxygenase (IDO) activity. At last, we isolated hMAPCs, the clinical grade stem cell product of hMAPCs, named MultiStem, and hMSCs from one single donor and observed that both the immunogenicity and the immunosuppressive capacities of all three stem cell products are comparable in vitro. In conclusion, hMAPCs have potent immunomodulatory properties in vitro and can serve as a valuable cell source for the clinical use of immunomodulatory cellular stem cell product.

Isolation and culture of rabbit embryonic stem cells.

PubMed

Honda, Arata

2013-01-01

Mammalian stem cells are invaluable research resources for the study of cell and embryonic development as well as practical tools for use in the production of genetically engineered animals and further therapeutics. It is important that we further our knowledge and understanding of a variety of stem cells from several different animal species before trials in humans commence. Here we describe methods for establishing rabbit embryonic stem (rES) cell lines with indefinite proliferation potential. rES cells attain maximum proliferation potential when cultured at a feeder cell density of one-sixth of that of full confluency. Higher and lower densities of feeder cells induced ES cell differentiation or division arrest. Fibroblast growth factor (FGF)2 can maintain the undifferentiated status of rES cells; however leukemia inhibitory factor (LIF) is dispensable. Under optimized conditions, rES cells could be passaged by trypsinization 50 times. This culture system enabled efficient gene transduction and clonal expansion from single cells. rES cells grew as flat monolayer cell colonies, as reported for monkey and human ES cells, and expressed pluripotency markers. Embryoid bodies and teratomas formed readily in vitro and in vivo, respectively. Characterization of ES cells from different species is important for establishing common features of pluripotency. We have demonstrated the similarity of ES cells between rabbit and humans. These cell lines could be applied directly using gene-targeting techniques, or in combination with induced pluripotent stem cells. Thus, rES cells are a suitable model for studying human transplantation therapy and disease treatments.

Recent advances in lineage differentiation from stem cells: hurdles and opportunities?

PubMed Central

Terryn, Joke; Tricot, Tine; Gajjar, Madhavsai; Verfaillie, Catherine

2018-01-01

Pluripotent stem cells have the property of long-term self-renewal and the potential to give rise to descendants of the three germ layers and hence all mature cells in the human body. Therefore, they hold the promise of offering insight not only into human development but also for human disease modeling and regenerative medicine. However, the generation of mature differentiated cells that closely resemble their in vivo counterparts remains challenging. Recent advances in single-cell transcriptomics and computational modeling of gene regulatory networks are revealing a better understanding of lineage commitment and are driving modern genome editing approaches. Additional modification of the chemical microenvironment, as well as the use of bioengineering tools to recreate the cellular, extracellular matrix, and physical characteristics of the niche wherein progenitors and mature cells reside, is now being used to further improve the maturation and functionality of stem cell progeny. PMID:29552337

Self-renewal of a purified Tie2+ hematopoietic stem cell population relies on mitochondrial clearance.

PubMed

Ito, Kyoko; Turcotte, Raphaël; Cui, Jinhua; Zimmerman, Samuel E; Pinho, Sandra; Mizoguchi, Toshihide; Arai, Fumio; Runnels, Judith M; Alt, Clemens; Teruya-Feldstein, Julie; Mar, Jessica C; Singh, Rajat; Suda, Toshio; Lin, Charles P; Frenette, Paul S; Ito, Keisuke

2016-12-02

A single hematopoietic stem cell (HSC) is capable of reconstituting hematopoiesis and maintaining homeostasis by balancing self-renewal and cell differentiation. The mechanisms of HSC division balance, however, are not yet defined. Here we demonstrate, by characterizing at the single-cell level a purified and minimally heterogeneous murine Tie2 + HSC population, that these top hierarchical HSCs preferentially undergo symmetric divisions. The induction of mitophagy, a quality control process in mitochondria, plays an essential role in self-renewing expansion of Tie2 + HSCs. Activation of the PPAR (peroxisome proliferator-activated receptor)-fatty acid oxidation pathway promotes expansion of Tie2 + HSCs through enhanced Parkin recruitment in mitochondria. These metabolic pathways are conserved in human TIE2 + HSCs. Our data thus identify mitophagy as a key mechanism of HSC expansion and suggest potential methods of cell-fate manipulation through metabolic pathways. Copyright © 2016, American Association for the Advancement of Science.

Self-renewal of a purified Tie2+ hematopoietic stem cell population relies on mitochondrial clearance

PubMed Central

Ito, Kyoko; Turcotte, Raphaël; Cui, Jinhua; Zimmerman, Samuel E.; Pinho, Sandra; Mizoguchi, Toshihide; Arai, Fumio; Runnels, Judith M.; Alt, Clemens; Teruya-Feldstein, Julie; Mar, Jessica C.; Singh, Rajat; Suda, Toshio; Lin, Charles P.; Frenette, Paul S.; Ito, Keisuke

2016-01-01

A single hematopoietic stem cell (HSC) is capable of reconstituting hematopoiesis and maintaining homeostasis by balancing self-renewal and cell differentiation. The mechanisms of HSC division balance, however, are not yet defined. Here we demonstrate, by characterizing at the single-cell level a purified and minimally heterogeneous murine Tie2+ HSC population, that these top hierarchical HSCs preferentially undergo symmetric divisions. The induction of mitophagy, a quality control process in mitochondria, plays an essential role in self-renewing expansion of Tie2+ HSCs. Activation of the PPAR (peroxisome proliferator–activated receptor)–fatty acid oxidation pathway promotes expansion of Tie2+ HSCs through enhanced Parkin recruitment in mitochondria. These metabolic pathways are conserved in human TIE2+ HSCs. Our data thus identify mitophagy as a key mechanism of HSC expansion and suggest potential methods of cell-fate manipulation through metabolic pathways. PMID:27738012

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

PubMed

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

2016-10-01

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

Generation and expansion of highly pure motor neuron progenitors from human pluripotent stem cells.

PubMed

Du, Zhong-Wei; Chen, Hong; Liu, Huisheng; Lu, Jianfeng; Qian, Kun; Huang, CindyTzu-Ling; Zhong, Xiaofen; Fan, Frank; Zhang, Su-Chun

2015-03-25

Human pluripotent stem cells (hPSCs) have opened new opportunities for understanding human development, modelling disease processes and developing new therapeutics. However, these applications are hindered by the low efficiency and heterogeneity of cell types, such as motorneurons (MNs), differentiated from hPSCs as well as our inability to maintain the potency of lineage-committed progenitors. Here by using a combination of small molecules that regulate multiple signalling pathways, we develop a method to guide human embryonic stem cells to a near-pure population (>95%) of motor neuron progenitors (MNPs) in 12 days, and an enriched population (>90%) of functionally mature MNs in an additional 16 days. More importantly, the MNPs can be expanded for at least five passages so that a single MNP can be amplified to 1 × 10(4). This method is reproducible in human-induced pluripotent stem cells and is applied to model MN-degenerative diseases and in proof-of-principle drug-screening assays.

In utero hematopoietic stem cell transfer: current status and future strategies.

PubMed

Surbek, D V; Gratwohl, A; Holzgreve, W

1999-07-01

Successful prenatal treatment of severe immunodeficiencies by allogeneic hematopoietic stem cell transplantation in utero has been reported. Though other diseases like hemoglobinopathies or storage diseases are potentially amenable to this novel therapeutic approach, no success has yet been achieved in recipients without severe immunodeficiency. Graft rejection by the developing fetus and/or lack of selective, competitive advantage of donor versus host stem cells preventing stable engraftment seem to be the major obstacles. Several strategies to overcome these hurdles are being explored in preclinical settings, including timing and repeated dosing of stem cell administration to the fetus, ex vivo modification of the transplant, using different fetal compartments as targets for early stem cell transfer, or inducing microchimerism for postnatal transplantation from the same donor. In addition, the exact definition of the basic concept of early fetal immunologic naivete and the understanding of the molecular basics of migration and homing in fetal hematopoiesis system seem mandatory for a successful approach. Gene therapy using ex vivo transduced autologous cord blood cells or direct gene targeting in utero are other potential means to correct hematopoietic and immunologic single gene disorders in utero, though this approach is still away from the stage of clinical trials.

Comparative analysis of single-cell RNA sequencing data from mouse spermatogonial and mesenchymal stem cells to identify differentially expressed genes and transcriptional regulators of germline cells.

PubMed

Sisakhtnezhad, Sajjad; Heshmati, Parvin

2018-07-01

Identifying effective internal factors for regulating germline commitment during development and for maintaining spermatogonial stem cells (SSCs) self-renewal is important to understand the molecular basis of spermatogenesis process, and to develop new protocols for the production of the germline cells from other cell sources. Therefore, this study was designed to investigate single-cell RNA-sequencing data for identification of differentially expressed genes (DEGs) in 12 mouse-derived single SSCs (mSSCs) in compare with 16 mouse-derived single mesenchymal stem cells. We also aimed to find transcriptional regulators of DEGs. Collectively, 1,584 up-regulated DEGs were identified that are associated with 32 biological processes. Moreover, investigation of the expression profiles of genes including in spermatogenesis process revealed that Dazl, Ddx4, Sall4, Fkbp6, Tex15, Tex19.1, Rnf17, Piwil2, Taf7l, Zbtb16, and Cadm1 are presented in the first 30 up-regulated DEGs. We also found 12 basal transcription factors (TFs) and three sequence-specific TFs that control the expression of DEGs. Our findings also indicated that MEIS1, SMC3, TAF1, KAT2A, STAT3, GTF3C2, SIN3A, BDP1, PHC1, and EGR1 are the main central regulators of DEGs in mSSCs. In addition, we collectively detected two significant protein complexes in the protein-protein interactions network for DEGs regulators. Finally, this study introduces the major upstream kinases for the main central regulators of DEGs and the components of core protein complexes. In conclusion, this study provides a molecular blueprint to uncover the molecular mechanisms behind the biology of SSCs and offers a list of candidate factors for cell type conversion approaches and production of germ cells. © 2017 Wiley Periodicals, Inc.

Impact of Feeding Strategies on the Scalable Expansion of Human Pluripotent Stem Cells in Single-Use Stirred Tank Bioreactors.

PubMed

Kropp, Christina; Kempf, Henning; Halloin, Caroline; Robles-Diaz, Diana; Franke, Annika; Scheper, Thomas; Kinast, Katharina; Knorpp, Thomas; Joos, Thomas O; Haverich, Axel; Martin, Ulrich; Zweigerdt, Robert; Olmer, Ruth

2016-10-01

: The routine application of human pluripotent stem cells (hPSCs) and their derivatives in biomedicine and drug discovery will require the constant supply of high-quality cells by defined processes. Culturing hPSCs as cell-only aggregates in (three-dimensional [3D]) suspension has the potential to overcome numerous limitations of conventional surface-adherent (two-dimensional [2D]) cultivation. Utilizing single-use instrumented stirred-tank bioreactors, we showed that perfusion resulted in a more homogeneous culture environment and enabled superior cell densities of 2.85 × 10 6 cells per milliliter and 47% higher cell yields compared with conventional repeated batch cultures. Flow cytometry, quantitative reverse-transcriptase polymerase chain reaction, and global gene expression analysis revealed a high similarity across 3D suspension and 2D precultures, underscoring that matrix-free hPSC culture efficiently supports maintenance of pluripotency. Interestingly, physiological data and gene expression assessment indicated distinct changes of the cells' energy metabolism, suggesting a culture-induced switch from glycolysis to oxidative phosphorylation in the absence of hPSC differentiation. Our data highlight the plasticity of hPSCs' energy metabolism and provide clear physiological and molecular targets for process monitoring and further development. This study paves the way toward more efficient GMP-compliant cell production and underscores the enormous process development potential of hPSCs in suspension culture. Human pluripotent stem cells (hPSCs) are a unique source for the, in principle, unlimited production of functional human cell types in vitro, which are of high value for therapeutic and industrial applications. This study applied single-use, clinically compliant bioreactor technology to develop advanced, matrix-free, and more efficient culture conditions for the mass production of hPSCs in scalable suspension culture. Using extensive analytical tools to compare established conditions with this novel culture strategy, unexpected physiological features of hPSCs were discovered. These data allow a more rational process development, providing significant progress in the field of translational stem cell research and medicine. ©AlphaMed Press.

Clonal analysis of lineage fate in native haematopoiesis.

PubMed

Rodriguez-Fraticelli, Alejo E; Wolock, Samuel L; Weinreb, Caleb S; Panero, Riccardo; Patel, Sachin H; Jankovic, Maja; Sun, Jianlong; Calogero, Raffaele A; Klein, Allon M; Camargo, Fernando D

2018-01-11

Haematopoiesis, the process of mature blood and immune cell production, is functionally organized as a hierarchy, with self-renewing haematopoietic stem cells and multipotent progenitor cells sitting at the very top. Multiple models have been proposed as to what the earliest lineage choices are in these primitive haematopoietic compartments, the cellular intermediates, and the resulting lineage trees that emerge from them. Given that the bulk of studies addressing lineage outcomes have been performed in the context of haematopoietic transplantation, current models of lineage branching are more likely to represent roadmaps of lineage potential than native fate. Here we use transposon tagging to clonally trace the fates of progenitors and stem cells in unperturbed haematopoiesis. Our results describe a distinct clonal roadmap in which the megakaryocyte lineage arises largely independently of other haematopoietic fates. Our data, combined with single-cell RNA sequencing, identify a functional hierarchy of unilineage- and oligolineage-producing clones within the multipotent progenitor population. Finally, our results demonstrate that traditionally defined long-term haematopoietic stem cells are a significant source of megakaryocyte-restricted progenitors, suggesting that the megakaryocyte lineage is the predominant native fate of long-term haematopoietic stem cells. Our study provides evidence for a substantially revised roadmap for unperturbed haematopoiesis, and highlights unique properties of multipotent progenitors and haematopoietic stem cells in situ.

Drop-on-Demand Single Cell Isolation and Total RNA Analysis

PubMed Central

Moon, Sangjun; Kim, Yun-Gon; Dong, Lingsheng; Lombardi, Michael; Haeggstrom, Edward; Jensen, Roderick V.; Hsiao, Li-Li; Demirci, Utkan

2011-01-01

Technologies that rapidly isolate viable single cells from heterogeneous solutions have significantly contributed to the field of medical genomics. Challenges remain both to enable efficient extraction, isolation and patterning of single cells from heterogeneous solutions as well as to keep them alive during the process due to a limited degree of control over single cell manipulation. Here, we present a microdroplet based method to isolate and pattern single cells from heterogeneous cell suspensions (10% target cell mixture), preserve viability of the extracted cells (97.0±0.8%), and obtain genomic information from isolated cells compared to the non-patterned controls. The cell encapsulation process is both experimentally and theoretically analyzed. Using the isolated cells, we identified 11 stem cell markers among 1000 genes and compare to the controls. This automated platform enabling high-throughput cell manipulation for subsequent genomic analysis employs fewer handling steps compared to existing methods. PMID:21412416

IL-6 Inhibition With MEDI5117 Decreases The Fraction of Head and Neck Cancer Stem Cells and Prevents Tumor Recurrence.

PubMed

Finkel, Kelsey A; Warner, Kristy A; Kerk, Samuel; Bradford, Carol R; McLean, Scott A; Prince, Mark E; Zhong, Haihong; Hurt, Elaine M; Hollingsworth, Robert E; Wicha, Max S; Tice, David A; Nör, Jacques E

2016-05-01

Head and neck squamous cell carcinomas (HNSCC) exhibit a small population of uniquely tumorigenic cancer stem cells (CSC) endowed with self-renewal and multipotency. We have recently shown that IL-6 enhances the survival and tumorigenic potential of head and neck cancer stem cells (i.e. ALDH(high)CD44(high) cells). Here, we characterized the effect of therapeutic inhibition of IL-6 with a novel humanized anti-IL-6 antibody (MEDI5117) using three low-passage patient-derived xenograft (PDX) models of HNSCC. We observed that single agent MEDI5117 inhibited the growth of PDX-SCC-M1 tumors (P < .05). This PDX model was generated from a previously untreated HNSCC. In contrast, MEDI5117 was not effective at reducing overall tumor volume for PDX models representing resistant disease (PDX-SCC-M0, PDX-SCC-M11). Low dose MEDI5117 (3 mg/kg) consistently decreased the fraction of cancer stem cells in PDX models of HNSCC when compared to IgG-treated controls, as follows: PDX-SCC-M0 (P < .001), PDX-SCC-M1 (P < .001), PDX-SCC-M11 (P = .04). Interestingly, high dose MEDI5117 (30 mg/kg) decreased the CSC fraction in the PDX-SCC-M11 model (P = .002), but not in PDX-SCC-M0 and PDX-SCC-M1. MEDI5117 mediated a dose-dependent decrease in the number of orospheres generated by ALDH(high)CD44(high) cells cultured in ultra-low attachment plates (P < .05), supporting an inhibitory effect on head and neck cancer stem cells. Notably, single agent MEDI5117 reduced the overall recurrence rate of PDX-SCC-M0, a PDX generated from the local recurrence of human HNSCC. Collectively, these data demonstrate that therapeutic inhibition of IL-6 with low-dose MEDI5117 decreases the fraction of cancer stem cells, and that adjuvant MEDI5117 inhibits recurrence in preclinical models of HNSCC. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Single-cell RNA sequencing highlights transcription activity of autophagy-related genes during hematopoietic stem cell formation in mouse embryos.

PubMed

Hu, Yongfei; Huang, Yan; Yi, Ying; Wang, Hongwei; Liu, Bing; Yu, Jia; Wang, Dong

2017-04-03

Accumulating evidence has demonstrated that macroautophagy/autophagy plays an essential role in self-renewal and differentiation in embryonic hematopoiesis. Here, according to the RNA sequencing data sets of 5 population cells related to hematopoietic stem cell (HSC) formation during mouse embryogenesis (endothelial cells, PTPRC/CD45 - and PTPRC/CD45 + pre-HSCs in the E11 aorta-gonad-mesonephros (AGM) region, mature HSCs in E12 and E14 fetal liver), we explored the dynamic expression of mouse autophagy-related genes in this course at the single-cell level. Our results revealed that the transcription activity of autophagy-related genes had a substantial increase when endothelial cells (ECs) specified into pre-HSCs, and the upregulation of autophagy-essential genes correlated with reduced NOTCH signaling in pre-HSCs, suggesting the autophagy activity may be greatly enhanced during pre-HSC specification from endothelial precursors. In summary, our results presented strong evidence that autophagy plays a critical role in HSC emergence during mouse midgestation.

Single-cell RNA-seq of human induced pluripotent stem cells reveals cellular heterogeneity and cell state transitions between subpopulations.

PubMed

Nguyen, Quan; Lukowski, Samuel; Chiu, Han; Senabouth, Anne; Bruxner, Timothy; Christ, Angelika; Palpant, Nathan; Powell, Joseph

2018-05-11

Heterogeneity of cell states represented in pluripotent cultures have not been described at the transcriptional level. Since gene expression is highly heterogeneous between cells, single-cell RNA sequencing can be used to identify how individual pluripotent cells function. Here, we present results from the analysis of single-cell RNA sequencing data from 18,787 individual WTC CRISPRi human induced pluripotent stem cells. We developed an unsupervised clustering method, and through this identified four subpopulations distinguishable on the basis of their pluripotent state including: a core pluripotent population (48.3%), proliferative (47.8%), early-primed for differentiation (2.8%) and late-primed for differentiation (1.1%). For each subpopulation we were able to identify the genes and pathways that define differences in pluripotent cell states. Our method identified four discrete predictor gene sets comprised of 165 unique genes that denote the specific pluripotency states; and using these sets, we developed a multigenic machine learning prediction method to accurately classify single cells into each of the subpopulations. Compared against a set of established pluripotency markers, our method increases prediction accuracy by 10%, specificity by 20%, and explains a substantially larger proportion of deviance (up to 3-fold) from the prediction model. Finally, we developed an innovative method to predict cells transitioning between subpopulations, and support our conclusions with results from two orthogonal pseudotime trajectory methods. Published by Cold Spring Harbor Laboratory Press.

Stem cells and regenerative medicine: principles, prospects and problems.

PubMed

Gardner, Richard L

2007-01-01

Stem cells have been used routinely for more than three decades to repair tissues and organs damaged by injury or disease, most notably haematopoietic stem cells taken from bone marrow, umbilical cord or, increasingly, from peripheral blood. Other examples, such as grafts of skin to treat severe burns, entail transplantation of stem cells within organized tissue rather than following isolation. The prospect of exploiting stem cells more widely in regenerative medicine was encouraged both by the development of human assisted conception and growing evidence that various adult cells retained greater versatility than had been suspected hitherto. The aim is to employ stem cells as a source of appropriately differentiated cells to replace those lost through physical, chemical or ischaemic injury, or as a result of degenerative disease. This may entail transplantation of just a single type of cell or, more challengingly, require a complex of several different types of cells possessing a defined architecture. Cardiomyocytes, hepatocytes or neuronal cells producing specific transmitters offer promising examples of the former, although how transplanted healthy cells will function in a perturbed tissue environment remains to be established. Recent success in repairing urinary bladder defects with grafts of urothelial and muscle cells seeded on a biodegradable collagen scaffold is an encouraging step towards assembling organs in vitro. Nevertheless, this is still far removed from the level of sophistication required to counter the ever increasing shortfall in supply of kidneys for transplantation. Various problems must be addressed if recent advances in the laboratory are to be translated into clinical practice. In many cases, it has yet to be established that cells derived from adults that retain plasticity are actually stem cells. There is also a pressing need for appropriate assays to ensure that, regardless of source, stem cells maintained in vitro are safe to transplant. Assays currently available for human ES cells are far from ideal. It is, in addition, important to ensure that differentiated cultures are pure and, depending on whether cell renewal is required or to be avoided, retain or lack appropriate stem cells. Neither autografts nor those obtained by so-called 'therapeutic cloning' are options for treating condition with an obvious genetic basis. Moreover, claims that some stem cells are more likely than others to yield successful allografts have yet to be confirmed and explained.

Impact of Feeding Strategies on the Scalable Expansion of Human Pluripotent Stem Cells in Single-Use Stirred Tank Bioreactors

PubMed Central

Kropp, Christina; Kempf, Henning; Halloin, Caroline; Robles-Diaz, Diana; Franke, Annika; Scheper, Thomas; Kinast, Katharina; Knorpp, Thomas; Joos, Thomas O.; Haverich, Axel; Martin, Ulrich; Olmer, Ruth

2016-01-01

The routine application of human pluripotent stem cells (hPSCs) and their derivatives in biomedicine and drug discovery will require the constant supply of high-quality cells by defined processes. Culturing hPSCs as cell-only aggregates in (three-dimensional [3D]) suspension has the potential to overcome numerous limitations of conventional surface-adherent (two-dimensional [2D]) cultivation. Utilizing single-use instrumented stirred-tank bioreactors, we showed that perfusion resulted in a more homogeneous culture environment and enabled superior cell densities of 2.85 × 106 cells per milliliter and 47% higher cell yields compared with conventional repeated batch cultures. Flow cytometry, quantitative reverse-transcriptase polymerase chain reaction, and global gene expression analysis revealed a high similarity across 3D suspension and 2D precultures, underscoring that matrix-free hPSC culture efficiently supports maintenance of pluripotency. Interestingly, physiological data and gene expression assessment indicated distinct changes of the cells’ energy metabolism, suggesting a culture-induced switch from glycolysis to oxidative phosphorylation in the absence of hPSC differentiation. Our data highlight the plasticity of hPSCs’ energy metabolism and provide clear physiological and molecular targets for process monitoring and further development. This study paves the way toward more efficient GMP-compliant cell production and underscores the enormous process development potential of hPSCs in suspension culture. Significance Human pluripotent stem cells (hPSCs) are a unique source for the, in principle, unlimited production of functional human cell types in vitro, which are of high value for therapeutic and industrial applications. This study applied single-use, clinically compliant bioreactor technology to develop advanced, matrix-free, and more efficient culture conditions for the mass production of hPSCs in scalable suspension culture. Using extensive analytical tools to compare established conditions with this novel culture strategy, unexpected physiological features of hPSCs were discovered. These data allow a more rational process development, providing significant progress in the field of translational stem cell research and medicine. PMID:27369897

Comparison of tumor biology of two distinct cell sub-populations in lung cancer stem cells.

PubMed

Wang, Jianyu; Sun, Zhiwei; Liu, Yongli; Kong, Liangsheng; Zhou, Shixia; Tang, Junlin; Xing, Hongmei Rosie

2017-11-14

Characterization of the stem-like properties of cancer stem cells (CSCs) remain indirect and qualitative, especially the ability of CSCs to undergo asymmetric cell division for self renewal and differentiation, a unique property of cells of stem origin. It is partly due to the lack of stable cellular models of CSCs. In this study, we developed a new approach for CSC isolation and purification to derive a CSC-enriched cell line (LLC-SE). By conducting five consecutive rounds of single cell cloning using the LLC-SE cell line, we obtained two distinct sub-population of cells within the Lewis lung cancer CSCs that employed largely symmetric division for self-renewal (LLC-SD) or underwent asymmetric division for differentiation (LLC-ASD). LLC-SD and LLC-ASD cell lines could be stably passaged in culture and be distinguished by cell morphology, stem cell marker, spheroid formation and subcutaneous tumor initiation efficiency, as well as orthotopic lung tumor growth, progression and survival. The ability LLC-ASD cells to undergo asymmetric division was visualized and quantified by the asymmetric segregation of labeled BrdU and NUMB to one of the two daughter cells in anaphase cell division. The more stem-like LLC-SD cells exhibited higher capacity for tumorigenesis and progression and shorter survival. As few as 10 LLC-SD could initiate subcutaneous tumor growth when transplanted to the athymic mice. Collectively, these observations suggest that the SD-type of cells appear to be on the top of the hierarchical order of the CSCs. Furthermore, they have lead to generated cellular models of CSC self-renewal for future mechanistic investigations.

Drug Screening Using a Library of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Reveals Disease Specific Patterns of Cardiotoxicity

PubMed Central

Liang, Ping; Lan, Feng; Lee, Andrew S.; Gong, Tingyu; Sanchez-Freire, Veronica; Wang, Yongming; Diecke, Sebastian; Sallam, Karim; Knowles, Joshua W.; Wang, Paul J.; Nguyen, Patricia K.; Bers, Donald M.; Robbins, Robert C.; Wu, Joseph C.

2013-01-01

Background Cardiotoxicity is a leading cause for drug attrition during pharmaceutical development and has resulted in numerous preventable patient deaths. Incidents of adverse cardiac drug reactions are more common in patients with pre-existing heart disease than the general population. Here we generated a library of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from patients with various hereditary cardiac disorders to model differences in cardiac drug toxicity susceptibility for patients of different genetic backgrounds. Methods and Results Action potential duration (APD) and drug-induced arrhythmia were measured at the single cell level in hiPSC-CMs derived from healthy subjects and patients with hereditary long QT syndrome (LQT), familial hypertrophic cardiomyopathy (HCM), and familial dilated cardiomyopathy (DCM). Disease phenotypes were verified in LQT, HCM, and DCM iPSC-CMs by immunostaining and single cell patch clamp. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and the human ether-a-go-go-related gene (hERG) expressing human embryonic kidney (HEK293) cells were used as controls. Single cell PCR confirmed expression of all cardiac ion channels in patient-specific hiPSC-CMs as well as hESC-CMs, but not in HEK293 cells. Disease-specific hiPSC-CMs demonstrated increased susceptibility to known cardiotoxic drugs as measured by APD and quantification of drug-induced arrhythmias such as early after depolarizations (EADs) and delayed after depolarizations (DADs). Conclusions We have recapitulated drug-induced cardiotoxicity profiles for healthy subjects, LQT, HCM, and DCM patients at the single cell level for the first time. Our data indicate that healthy and diseased individuals exhibit different susceptibilities to cardiotoxic drugs and that use of disease-specific hiPSC-CMs may predict adverse drug responses more accurately than standard hERG test or healthy control hiPSC-CM/hESC-CM screening assays. PMID:23519760

Slow and sustained nitric oxide releasing compounds inhibit multipotent vascular stem cell proliferation and differentiation without causing cell death

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

Curtis, Brandon M.; Leix, Kyle Alexander; Ji, Yajing

Highlights: • Multipotent vascular stem cells (MVSCs) proliferate and differentiate. • Nitric oxide inhibits proliferation of MVSCs. • Nitric oxide inhibits MVSC differentiation to mesenchymal-like stem cells (MSCs). • Smooth muscle cells (SMCs) neither de-differentiate nor proliferate. - Abstract: Atherosclerosis is the leading cause of cerebral and myocardial infarction. It is believed that neointimal growth common in the later stages of atherosclerosis is a result of vascular smooth muscle cell (SMC) de-differentiation in response to endothelial injury. However, the claims of the SMC de-differentiation theory have not been substantiated by monitoring the fate of mature SMCs in response to suchmore » injuries. A recent study suggests that atherosclerosis is a consequence of multipotent vascular stem cell (MVSC) differentiation. Nitric oxide (NO) is a well-known mediator against atherosclerosis, in part because of its inhibitory effect on SMC proliferation. Using three different NO-donors, we have investigated the effects of NO on MVSC proliferation. Results indicate that NO inhibits MVSC proliferation in a concentration dependent manner. A slow and sustained delivery of NO proved to inhibit proliferation without causing cell death. On the other hand, larger, single-burst NO concentrations, inhibits proliferation, with concurrent significant cell death. Furthermore, our results indicate that endogenously produced NO inhibits MVSC differentiation to mesenchymal-like stem cells (MSCs) and subsequently to SMC as well.« less

Role of CD146 Enrichment in Purification of Stem Cells Derived from Dental Pulp Polyp.

PubMed

Tavangar, Maryam Sadat; Hosseini, Seyed-Mojtaba; Dehghani-Nazhvani, Ali; Monabati, Ahmad

2017-01-01

Hyperplastic pulpitis (pulp polyp) tissues contains cells with stem cell properties similar to that of the dental pulp stem cells (DPSCs). It has also been shown that CD146 enrichment can homogenize the cultures of DPSCs and enhance the colony forming potentials of their cultures. This study determines whether CD146 enrichment can help purifying the stem cells from heterogeneous cultures of the pulp polyp derived stem cells (PPSCs). Healthy dental pulps and pulp polyp tissues were enzymatically digested and the harvested single cells were sorted according to the presence of CD146 marker. The sorted cells were seeded directly for colony forming unit (CFU) assays of the negative and positive portions. Flowcytometric antigen panel and differentiation assays were used to see if these cells conform with mesenchymal stems cells (MSCs) definition. Differences between the between groups was assessed using independent t-test. The level of significance was set at 0.05. Normal pulp tissue derived cells formed higher colonies (42.5±16.8 per 10 4 cells) than the pulp polyp (17.75±8.9 per 10 4 cells) ( P =0.015). The CD146 positive portion of the polyp derived cells formed an average of 91.5±29.7 per 10 4 cells per CFU. On the other hand, CD146 negative portion did not show any colonies ( P <0.001). Both resources showed cells with flowcytometric antigen panel and differentiation potentials conforming to MSC definition. The entire CFU of PPSCs were formed within CD146 enriched portion. It seems that CD146 enrichment may reduce the number of possible fibroblasts of the pulp polyps and may further homogenize the culture of the PPSCs.

Quartz-Seq2: a high-throughput single-cell RNA-sequencing method that effectively uses limited sequence reads.

PubMed

Sasagawa, Yohei; Danno, Hiroki; Takada, Hitomi; Ebisawa, Masashi; Tanaka, Kaori; Hayashi, Tetsutaro; Kurisaki, Akira; Nikaido, Itoshi

2018-03-09

High-throughput single-cell RNA-seq methods assign limited unique molecular identifier (UMI) counts as gene expression values to single cells from shallow sequence reads and detect limited gene counts. We thus developed a high-throughput single-cell RNA-seq method, Quartz-Seq2, to overcome these issues. Our improvements in the reaction steps make it possible to effectively convert initial reads to UMI counts, at a rate of 30-50%, and detect more genes. To demonstrate the power of Quartz-Seq2, we analyzed approximately 10,000 transcriptomes from in vitro embryonic stem cells and an in vivo stromal vascular fraction with a limited number of reads.

Stem Cell Modeling of Core Binding Factor Acute Myeloid Leukemia

PubMed Central

Mosna, Federico

2016-01-01

Even though clonally originated from a single cell, acute leukemia loses its homogeneity soon and presents at clinical diagnosis as a hierarchy of cells endowed with different functions, of which only a minority possesses the ability to recapitulate the disease. Due to their analogy to hematopoietic stem cells, these cells have been named “leukemia stem cells,” and are thought to be chiefly responsible for disease relapse and ultimate survival after chemotherapy. Core Binding Factor (CBF) Acute Myeloid Leukemia (AML) is cytogenetically characterized by either the t(8;21) or the inv(16)/t(16;16) chromosomal abnormalities, which, although being pathognomonic, are not sufficient per se to induce overt leukemia but rather determine a preclinical phase of disease when preleukemic subclones compete until the acquisition of clonal dominance by one of them. In this review we summarize the concepts regarding the application of the “leukemia stem cell” theory to the development of CBF AML; we will analyze the studies investigating the leukemogenetic role of t(8;21) and inv(16)/t(16;16), the proposed theories of its clonal evolution, and the role played by the hematopoietic niches in preserving the disease. Finally, we will discuss the clinical implications of stem cell modeling of CBF AML for the therapy of the disease. PMID:26880987

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

PubMed

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

2010-12-06

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

Results of a Prospective Randomized, Open-Label, Noninferiority Study of Tbo-Filgrastim (Granix) versus Filgrastim (Neupogen) in Combination with Plerixafor for Autologous Stem Cell Mobilization in Patients with Multiple Myeloma and Non-Hodgkin Lymphoma.

PubMed

Bhamidipati, Pavan Kumar; Fiala, Mark A; Grossman, Brenda J; DiPersio, John F; Stockerl-Goldstein, Keith; Gao, Feng; Uy, Geoffrey L; Westervelt, Peter; Schroeder, Mark A; Cashen, Amanda F; Abboud, Camille N; Vij, Ravi

2017-12-01

Autologous hematopoietic stem cell transplantation (auto-HSCT) improves survival in patients with multiple myeloma (MM) and non-Hodgkin lymphoma (NHL). Traditionally, filgrastim (Neupogen; recombinant G-CSF) has been used in as a single agent or in combination with plerixafor for stem cell mobilization for auto-HSCT. In Europe, a biosimilar recombinant G-CSF (Tevagrastim) has been approved for various indications similar to those of reference filgrastim, including stem cell mobilization for auto-HSCT; however, in the United States, tbo-filgrastim (Granix) is registered under the original biological application and is not approved for stem cell mobilization. In retrospective studies, stem cell mobilization with tbo-filgrastim has shown similar efficacy and toxicity as filgrastim, but no prospective studies have been published to date. We have conducted the first prospective randomized trial comparing the safety and efficacy of tbo-filgrastim in combination with plerixafor with that of filgrastim in combination with plerixafor for stem cell mobilization in patients with MM and NHL. This is a phase 2 prospective randomized (1:1) open-label single-institution noninferiority study of tbo-filgrastim and filgrastim with plerixafor in patients with MM or NHL undergoing auto-HSCT. Here 10 µg/kg/day of tbo-filgrastim/filgrastim was administered s.c. for 5 days (days 1 to 5). On day 4 at approximately 1800 hours, 0.24 mg/kg of plerixafor was administered s.c. Apheresis was performed on day 5 with a target cumulative collection goal of at least 5.0 × 10 6 CD34 + cells/kg. The primary objective was to compare day 5 CD34 +  cells/kg collected. Secondary objectives included other mobilization endpoints, safety, engraftment outcomes, and hospital readmission rate. A total of 97 evaluable patients were enrolled (tbo-filgrastim, n = 46; filgrastim, n = 51). Tbo-filgrastim was not inferior to filgrastim in terms of day 5 CD34 +  cell collection (mean, 11.6 ± 6.7 CD34 + cells/kg versus 10.0  ± 6.8 CD34 + cells/kg. Multivariate analysis revealed a trend toward increased mobilization in the tbo-filgrastim arm, but this was not statistically significant. The tbo-filgrastim and filgrastim arms were similar in all secondary endpoints. Tbo-filgrastim is not inferior in efficacy and has similar safety compared to reference filgrastim when used for stem cell mobilization in patients with MM and NHL. Granix can be safely used instead of Neupogen for stem cell collection in patients undergoing auto-HSCT for MM or NHL. The study is registered at https://clinicaltrials.gov/ct2/show/NCT02098109. Copyright © 2017 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

Correction of the sickle cell disease mutation in human hematopoietic stem/progenitor cells.

PubMed

Hoban, Megan D; Cost, Gregory J; Mendel, Matthew C; Romero, Zulema; Kaufman, Michael L; Joglekar, Alok V; Ho, Michelle; Lumaquin, Dianne; Gray, David; Lill, Georgia R; Cooper, Aaron R; Urbinati, Fabrizia; Senadheera, Shantha; Zhu, Allen; Liu, Pei-Qi; Paschon, David E; Zhang, Lei; Rebar, Edward J; Wilber, Andrew; Wang, Xiaoyan; Gregory, Philip D; Holmes, Michael C; Reik, Andreas; Hollis, Roger P; Kohn, Donald B

2015-04-23

Sickle cell disease (SCD) is characterized by a single point mutation in the seventh codon of the β-globin gene. Site-specific correction of the sickle mutation in hematopoietic stem cells would allow for permanent production of normal red blood cells. Using zinc-finger nucleases (ZFNs) designed to flank the sickle mutation, we demonstrate efficient targeted cleavage at the β-globin locus with minimal off-target modification. By co-delivering a homologous donor template (either an integrase-defective lentiviral vector or a DNA oligonucleotide), high levels of gene modification were achieved in CD34(+) hematopoietic stem and progenitor cells. Modified cells maintained their ability to engraft NOD/SCID/IL2rγ(null) mice and to produce cells from multiple lineages, although with a reduction in the modification levels relative to the in vitro samples. Importantly, ZFN-driven gene correction in CD34(+) cells from the bone marrow of patients with SCD resulted in the production of wild-type hemoglobin tetramers. © 2015 by The American Society of Hematology.

Autologous hematopoietic stem cell transplantation in peripheral T-cell lymphoma using a uniform high-dose regimen.

PubMed

Smith, S D; Bolwell, B J; Rybicki, L A; Brown, S; Dean, R; Kalaycio, M; Sobecks, R; Andresen, S; Hsi, E D; Pohlman, B; Sweetenham, J W

2007-08-01

The role of high-dose therapy and autologous stem cell transplantation (ASCT) for patients with peripheral T-cell lymphoma (PTCL) is poorly defined. Comparisons of outcomes between PTCL and B-cell non-Hodgkin's lymphoma (NHL) have yielded conflicting results, in part due to the rarity and heterogeneity of PTCL. Some retrospective studies have found comparable survival rates for patients with T- and B-cell NHL. In this study, we report our single-center experience of ASCT over one decade using a uniform chemotherapy-only high-dose regimen. Thirty-two patients with PTCL-unspecified (PTCL-u; 11 patients) and anaplastic large-cell lymphoma (21 patients) underwent autologous stem cell transplant, mostly for relapsed or refractory disease. The preparative regimen consisted of busulfan, etoposide and cyclophosphamide. Kaplan-Meier 5-year overall survival (OS) and relapse-free survival (RFS) are 34 and 18%, respectively. These results suggest a poor outcome for patients with PTCL after ASCT, and new therapies for T-cell lymphoma are needed.

Explanation for excessive DNA single-strand breaks and endogenous repair foci in pluripotent mouse embryonic stem cells.

PubMed

Banáth, J P; Bañuelos, C A; Klokov, D; MacPhail, S M; Lansdorp, P M; Olive, P L

2009-05-01

Pluripotent mouse embryonic stem cells (mES cells) exhibit approximately 100 large gammaH2AX repair foci in the absence of measurable numbers of DNA double-strand breaks. Many of these cells also show excessive numbers of DNA single-strand breaks (>10,000 per cell) when analyzed using the alkaline comet assay. To understand the reasons for these unexpected observations, various methods for detecting DNA strand breaks were applied to wild-type mES cells and to mES cells lacking H2AX, ATM, or DNA-PKcs. H2AX phosphorylation and expression of other repair complexes were measured using flow and image analysis of antibody-stained cells. Results indicate that high numbers of endogenous gammaH2AX foci and single-strand breaks in pluripotent mES cells do not require ATM or DNA-PK kinase activity and appear to be associated with global chromatin decondensation rather than pre-existing DNA damage. This will limit applications of gammaH2AX foci analysis in mES cells to relatively high levels of initial or residual DNA damage. Excessive numbers of single-strand breaks in the alkaline comet assay can be explained by the vulnerability of replicating chromatin in mES cells to osmotic shock. This suggests that caution is needed in interpreting results with the alkaline comet assay when applied to certain cell types or after treatment with agents that make chromatin vulnerable to osmotic changes. Differentiation of mES cells caused a reduction in histone acetylation, gammaH2AX foci intensity, and DNA single-strand breakage, providing a link between chromatin structural organization, excessive gammaH2AX foci, and sensitivity of replicating mES cell chromatin to osmotic shock.

Multi-scale imaging and informatics pipeline for in situ pluripotent stem cell analysis.

PubMed

Gorman, Bryan R; Lu, Junjie; Baccei, Anna; Lowry, Nathan C; Purvis, Jeremy E; Mangoubi, Rami S; Lerou, Paul H

2014-01-01

Human pluripotent stem (hPS) cells are a potential source of cells for medical therapy and an ideal system to study fate decisions in early development. However, hPS cells cultured in vitro exhibit a high degree of heterogeneity, presenting an obstacle to clinical translation. hPS cells grow in spatially patterned colony structures, necessitating quantitative single-cell image analysis. We offer a tool for analyzing the spatial population context of hPS cells that integrates automated fluorescent microscopy with an analysis pipeline. It enables high-throughput detection of colonies at low resolution, with single-cellular and sub-cellular analysis at high resolutions, generating seamless in situ maps of single-cellular data organized by colony. We demonstrate the tool's utility by analyzing inter- and intra-colony heterogeneity of hPS cell cycle regulation and pluripotency marker expression. We measured the heterogeneity within individual colonies by analyzing cell cycle as a function of distance. Cells loosely associated with the outside of the colony are more likely to be in G1, reflecting a less pluripotent state, while cells within the first pluripotent layer are more likely to be in G2, possibly reflecting a G2/M block. Our multi-scale analysis tool groups colony regions into density classes, and cells belonging to those classes have distinct distributions of pluripotency markers and respond differently to DNA damage induction. Lastly, we demonstrate that our pipeline can robustly handle high-content, high-resolution single molecular mRNA FISH data by using novel image processing techniques. Overall, the imaging informatics pipeline presented offers a novel approach to the analysis of hPS cells that includes not only single cell features but also colony wide, and more generally, multi-scale spatial configuration.

Double versus single high-dose melphalan 200 mg/m2 and autologous stem cell transplantation for multiple myeloma: a region-based study in 484 patients from the Nordic area

PubMed Central

Björkstrand, Bo; Klausen, Tobias W.; Remes, Kari; Gruber, Astrid; Knudsen, Lene M.; Bergmann, Olav J.; Lenhoff, Stig; Johnsen, Hans E.

2009-01-01

Autologous stem cell transplantation is still considered the standard of care in young patients with multiple myeloma (MM). This disease is the most common indication for high-dose therapy (HDT) supported by hematopoietic stem cell transplantation and much data support the benefit of this procedure. Results of randomized studies are in favor of tandem autologous transplantation although the effect on overall survival is unclear. Based on sequential registration trials in the Nordic area, we aimed to evaluate the outcome of conventional single or double HDT. During 1994–2000 we registered a total of 484 previously untreated patients under the age of 60 years at diagnosis who on a regional basis initially were treated with single [Trial NMSG #5/94 and #7/98 (N=383)] or double [Trial Huddinge Karolinska Turku Herlev (N=101)] high-dose melphalan (200 mg/m2) therapy supported by autologous stem cell transplantation. A complete or very good partial response was achieved by 40% of patients in the single transplant group and 60% of patients in the double transplant group (p=0.0006). The probability of surviving progression free for five years after the diagnosis was 25% (95% CL 18–32%) in the singletransplant group and 46% (95% CL 33–55%) in the double transplant group (p=0.0014). The estimated overall five-year survival rate was 60% in the single transplant group and 64% in the doubletransplant (p=0.9). In a multivariate analysis of variables, including single versus double transplantation, β2 microglobulin level, age, sex and disease stage, only β2 microglobulin level was predictive for overall survival (p>0.0001) and progression free survival (p=0.001). In accordance with these results, a 1:1 case-control matched comparison between double and single transplantation did not identify significant differences in overall and progression free survival. In this retrospective analysis up front double transplantation with melphalan (200 mg/m2) as compared to single transplantation did not seem to improve the final outcome among patients in the Nordic area. These data are in accordance with recent publications from the Bologna 96 trial indicating that a second transplant should not be recommended up front as standard care.

Variation of mechanical property of single-walled carbon nanotubes-treated cells explored by atomic force microscopy.

PubMed

Dulińska-Molak, Ida; Mao, Hongli; Kawazoe, Naoki; Chen, Guoping

2014-04-01

With a range of biological properties, single-walled carbon nanotubes (SWCNTs) are a promising material for nanobiotechnology. Concerns about their potential effect on human health have led to the interest in understanding the interaction between SWCNTs and cells. There are many reports showing the potential cellular effects of SWCNTs but this issue is quite controversially discussed in the literature. In this study, we used conventional biological evaluation methods and atomic force microscopy (AFM) to compare the effects of SWCNTs on three different cell types: bovine articular chondrocytes, human bone marrow-derived mesenchymal stem cells and HeLa cells. No obvious effects of SWCNTs on cell morphology and viability were observed during 3 days in vitro culture. However, SWCNTs significantly increased the Young's modulus of all the three types of cells. The effect of SWCNTs on Young's modulus was in an increasing order of Hela cells < chondrocytes < mesenchymal stem cells. AFM was shown to be a useful tool for investigation of the effect of nanomaterials on mechanical property of cells.

Long-term monoclonal reconstitution of erythropoiesis in genetically anemic W/Wv mice by injection of 5-fluorouracil-treated bone marrow cells of Pgk-1b/Pgk-1a mice

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

Nakano, T.; Waki, N.; Asai, H.

The spleen colony-forming assay does not represent the number of hematopoietic stem cells with extensive self-maintaining capacity because five to 50 spleen colony-forming units (CFU-S) are necessary to rescue a genetically anemic (WB X C57BL/6)F1-W/Wv(WBB6F1-W/Wv) mouse. We investigated which is more important for the reconstitution of erythropoiesis, the transplantation of multiple CFU-S or that of a single stem cell with extensive self-maintaining potential. The electrophoretic pattern of hemoglobin was used as a marker of reconstitution and that of phosphoglycerate kinase (PGK), an X chromosome-linked enzyme, as a tool for estimating the number of stem cells. For this purpose, we developedmore » the C57BL/6 congeneic strain with the Pgk-1a gene. Bone marrow cells were harvested after injection of 5-fluorouracil from C57BL/6-Pgk-1b/Pgk-1a female mice in which each stem cell had either A-type PGK or B-type PGK due to the random inactivation of one or two X chromosomes. When a relatively small number of bone marrow cells (ie, 10(3) or 3 X 10(3) were injected into 200-rad-irradiated WBB6F1-W/Wv mice, the hemoglobin pattern changed from the recipient type (Hbbd/Hbbs) to the donor type (Hbbs/Hbbs) in seven of 150 mice for at least 8 weeks. Erythrocytes of all these WBB6F1-W/Wv mice showed either A-type PGK alone or B-type PGK alone during the time of reconstitution, which suggests that a single stem cell with extensive self-maintaining potential may sustain the whole erythropoiesis of a mouse for at least 8 weeks.« less

Single-cell metabolomics: analytical and biological perspectives.

PubMed

Zenobi, R

2013-12-06

There is currently much interest in broad molecular profiling of single cells; a cell's metabolome-its full complement of small-molecule metabolites-is a direct indicator of phenotypic diversity of single cells and a nearly immediate readout of how cells react to environmental influences. However, the metabolome is very difficult to measure at the single-cell level because of rapid metabolic dynamics, the structural diversity of the molecules, and the inability to amplify or tag small-molecule metabolites. Measurement techniques including mass spectrometry, capillary electrophoresis, and, to a lesser extent, optical spectroscopy and fluorescence detection have led to impressive advances in single-cell metabolomics. Even though none of these methodologies can currently measure the metabolome of a single cell completely, rapidly, and nondestructively, progress has been sufficient such that the field is witnessing a shift from feasibility studies to investigations that yield new biological insight. Particularly interesting fields of application are cancer biology, stem cell research, and monitoring of xenobiotics and drugs in tissue sections at the single-cell level.

Single cell analysis of normal and leukemic hematopoiesis.

PubMed

Povinelli, Benjamin J; Rodriguez-Meira, Alba; Mead, Adam J

2018-02-01

The hematopoietic system is well established as a paradigm for the study of cellular hierarchies, their disruption in disease and therapeutic use in regenerative medicine. Traditional approaches to study hematopoiesis involve purification of cell populations based on a small number of surface markers. However, such population-based analysis obscures underlying heterogeneity contained within any phenotypically defined cell population. This heterogeneity can only be resolved through single cell analysis. Recent advances in single cell techniques allow analysis of the genome, transcriptome, epigenome and proteome in single cells at an unprecedented scale. The application of these new single cell methods to investigate the hematopoietic system has led to paradigm shifts in our understanding of cellular heterogeneity in hematopoiesis and how this is disrupted in disease. In this review, we summarize how single cell techniques have been applied to the analysis of hematopoietic stem/progenitor cells in normal and malignant hematopoiesis, with a particular focus on recent advances in single-cell genomics, including how these might be utilized for clinical application. Copyright © 2017. Published by Elsevier Ltd.

Directed Differentiation of Human-Induced Pluripotent Stem Cells to Mesenchymal Stem Cells.

PubMed

Lian, Qizhou; Zhang, Yuelin; Liang, Xiaoting; Gao, Fei; Tse, Hung-Fat

2016-01-01

Multipotent stromal cells, also known as mesenchymal stem cells (MSCs), possess great potential to generate a wide range of cell types including endothelial cells, smooth muscle cells, bone, cartilage, and lipid cells. This protocol describes in detail how to perform highly efficient, lineage-specific differentiation of human-induced pluripotent stem cells (iPSCs) with an MSCs fate. The approach uses a clinically compliant protocol with chemically defined media, feeder-free conditions, and a CD105 positive and CD24 negative selection to achieve a single cell-based MSCs derivation from differentiating human pluripotent cells in approximately 20 days. Cells generated with this protocol express typical MSCs surface markers and undergo adipogenesis, osteogenesis, and chondrogenesis similar to adult bone marrow-derived MSCs (BM-MSCs). Nonetheless, compared with adult BM-MSCs, iPSC-MSCs display a higher proliferative capacity, up to 120 passages, without obvious loss of self-renewal potential and constitutively express MSCs surface antigens. MSCs generated with this protocol have numerous applications, including expansion to large scale cell numbers for tissue engineering and the development of cellular therapeutics. This approach has been used to rescue limb ischemia, allergic disorders, and cigarette smoke-induced lung damage and to model mesenchymal and vascular disorders of Hutchinson-Gilford progeria syndrome (HGPS).

High-density polymer microarrays: identifying synthetic polymers that control human embryonic stem cell growth.

PubMed

Hansen, Anne; Mjoseng, Heidi K; Zhang, Rong; Kalloudis, Michail; Koutsos, Vasileios; de Sousa, Paul A; Bradley, Mark

2014-06-01

The fabrication of high-density polymer microarray is described, allowing the simultaneous and efficient evaluation of more than 7000 different polymers in a single-cellular-based screen. These high-density polymer arrays are applied in the search for synthetic substrates for hESCs culture. Up-scaling of the identified hit polymers enables long-term cellular cultivation and promoted successful stem-cell maintenance. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A novel mode of enhancer evolution: The Tal1 stem cell enhancer recruited a MIR element to specifically boost its activity

PubMed Central

Smith, Aileen M.; Sanchez, Maria-Jose; Follows, George A.; Kinston, Sarah; Donaldson, Ian J.; Green, Anthony R.; Göttgens, Berthold

2008-01-01

Altered cis-regulation is thought to underpin much of metazoan evolution, yet the underlying mechanisms remain largely obscure. The stem cell leukemia TAL1 (also known as SCL) transcription factor is essential for the normal development of blood stem cells and we have previously shown that the Tal1 +19 enhancer directs expression to hematopoietic stem cells, hematopoietic progenitors, and to endothelium. Here we demonstrate that an adjacent region 1 kb upstream (+18 element) is in an open chromatin configuration and carries active histone marks but does not function as an enhancer in transgenic mice. Instead, it boosts activity of the +19 enhancer both in stable transfection assays and during differentiation of embryonic stem (ES) cells carrying single-copy reporter constructs targeted to the Hprt locus. The +18 element contains a mammalian interspersed repeat (MIR) which is essential for the +18 function and which was transposed to the Tal1 locus ∼160 million years ago at the time of the mammalian/marsupial branchpoint. Our data demonstrate a previously unrecognized mechanism whereby enhancer activity is modulated by a transposon exerting a “booster” function which would go undetected by conventional transgenic approaches. PMID:18687876

High risk of graft failure in patients with anti-HLA antibodies undergoing haploidentical stem-cell transplantation.

PubMed

Ciurea, Stefan O; de Lima, Marcos; Cano, Pedro; Korbling, Martin; Giralt, Sergio; Shpall, Elizabeth J; Wang, Xuemei; Thall, Peter F; Champlin, Richard E; Fernandez-Vina, Marcelo

2009-10-27

BACKGROUND.: Although donor-specific anti-human leukocyte antigen (HLA) antibodies (DSA) have been implicated in graft rejection in solid organ transplantation, their role in hematopoietic stem-cell transplantation remains unclear. METHODS.: To address the hypothesis that the presence of DSA contributes to the development graft failure, we tested 24 consecutive patients for the presence of anti-HLA antibodies determined by a sensitive and specific solid-phase/single-antigen assay. The study included a total of 28 haploidentical transplants, each with 2 to 5 HLA allele mismatches, at a single institution, from September 2005 to August 2008. RESULTS.: DSA were detected in five patients (21%). Three of four (75%) patients with DSA before the first transplant failed to engraft, compared with 1 of 20 (5%) without DSA (P=0.008). All four patients who experienced primary graft failure had second haploidentical transplants. One patient developed a second graft failure with persistent high DSA levels, whereas three engrafted, two of them in the absence of DSA. No other known factors that could negatively influence engraftment were associated with the development of graft failure in these patients. CONCLUSIONS.: These results suggest that donor-specific anti-HLA antibodies are associated with a high rate of graft rejection in patients undergoing haploidentical stem-cell transplantation. Anti-HLA sensitization should be evaluated routinely in hematopoietic stem-cell transplantation with HLA mismatched donors.

Single-cell transcriptome of early embryos and cultured embryonic stem cells of cynomolgus monkeys

PubMed Central

Nakamura, Tomonori; Yabuta, Yukihiro; Okamoto, Ikuhiro; Sasaki, Kotaro; Iwatani, Chizuru; Tsuchiya, Hideaki; Saitou, Mitinori

2017-01-01

In mammals, the development of pluripotency and specification of primordial germ cells (PGCs) have been studied predominantly using mice as a model organism. However, divergences among mammalian species for such processes have begun to be recognized. Between humans and mice, pre-implantation development appears relatively similar, but the manner and morphology of post-implantation development are significantly different. Nevertheless, the embryogenesis just after implantation in primates, including the specification of PGCs, has been unexplored due to the difficulties in analyzing the embryos at relevant developmental stages. Here, we present a comprehensive single-cell transcriptome dataset of pre- and early post-implantation embryo cells, PGCs and embryonic stem cells (ESCs) of cynomolgus monkeys as a model of higher primates. The identities of each transcriptome were also validated rigorously by other way such as immunofluorescent analysis. The information reported here will serve as a foundation for our understanding of a wide range of processes in the developmental biology of primates, including humans. PMID:28649393

Clonally expanded novel multipotent stem cells from human bone marrow regenerate myocardium after myocardial infarction

PubMed Central

Yoon, Young-sup; Wecker, Andrea; Heyd, Lindsay; Park, Jong-Seon; Tkebuchava, Tengiz; Kusano, Kengo; Hanley, Allison; Scadova, Heather; Qin, Gangjian; Cha, Dong-Hyun; Johnson, Kirby L.; Aikawa, Ryuichi; Asahara, Takayuki; Losordo, Douglas W.

2005-01-01

We have identified a subpopulation of stem cells within adult human BM, isolated at the single-cell level, that self-renew without loss of multipotency for more than 140 population doublings and exhibit the capacity for differentiation into cells of all 3 germ layers. Based on surface marker expression, these clonally expanded human BM-derived multipotent stem cells (hBMSCs) do not appear to belong to any previously described BM-derived stem cell population. Intramyocardial transplantation of hBMSCs after myocardial infarction resulted in robust engraftment of transplanted cells, which exhibited colocalization with markers of cardiomyocyte (CMC), EC, and smooth muscle cell (SMC) identity, consistent with differentiation of hBMSCs into multiple lineages in vivo. Furthermore, upregulation of paracrine factors including angiogenic cytokines and antiapoptotic factors, and proliferation of host ECs and CMCs, were observed in the hBMSC-transplanted hearts. Coculture of hBMSCs with CMCs, ECs, or SMCs revealed that phenotypic changes of hBMSCs result from both differentiation and fusion. Collectively, the favorable effect of hBMSC transplantation after myocardial infarction appears to be due to augmentation of proliferation and preservation of host myocardial tissues as well as differentiation of hBMSCs for tissue regeneration and repair. To our knowledge, this is the first demonstration that a specific population of multipotent human BM-derived stem cells can induce both therapeutic neovascularization and endogenous and exogenous cardiomyogenesis. PMID:15690083

Single-step generation of gene knockout-rescue system in pluripotent stem cells by promoter insertion with CRISPR/Cas9.

PubMed

Matsunaga, Taichi; Yamashita, Jun K

2014-02-07

Specific gene knockout and rescue experiments are powerful tools in developmental and stem cell biology. Nevertheless, the experiments require multiple steps of molecular manipulation for gene knockout and subsequent rescue procedures. Here we report an efficient and single step strategy to generate gene knockout-rescue system in pluripotent stem cells by promoter insertion with CRISPR/Cas9 genome editing technology. We inserted a tetracycline-regulated inducible gene promoter (tet-OFF/TRE-CMV) upstream of the endogenous promoter region of vascular endothelial growth factor receptor 2 (VEGFR2/Flk1) gene, an essential gene for endothelial cell (EC) differentiation, in mouse embryonic stem cells (ESCs) with homologous recombination. Both homo- and hetero-inserted clones were efficiently obtained through a simple selection with a drug-resistant gene. The insertion of TRE-CMV promoter disrupted endogenous Flk1 expression, resulting in null mutation in homo-inserted clones. When the inserted TRE-CMV promoter was activated with doxycycline (Dox) depletion, Flk1 expression was sufficiently recovered from the downstream genomic Flk1 gene. Whereas EC differentiation was almost completely perturbed in homo-inserted clones, Flk1 rescue with TRE-CMV promoter activation restored EC appearance, indicating that phenotypic changes in EC differentiation can be successfully reproduced with this knockout-rescue system. Thus, this promoter insertion strategy with CRISPR/Cas9 would be a novel attractive method for knockout-rescue experiments. Copyright © 2014 Elsevier Inc. All rights reserved.

Fever after peripheral blood stem cell infusion in haploidentical transplantation with post-transplant cyclophosphamide.

PubMed

Arango, Marcos; Combariza, Juan F

2017-06-01

Noninfection-related fever can occur after peripheral blood stem cell infusion in haploidentical hematopoietic stem cell transplantation with post-transplant cyclophosphamide. The objective of this study was to analyze the incidence of fever and characterize some clinical features of affected patients. A retrospective case-series study with 40 patients who received haploidentical hematopoietic stem cell transplantation was carried out. Thirty-three patients (82.5%) developed fever; no baseline characteristic was associated with its development. Median time to fever onset was 25.5h (range, 9.5-100h) and median peak temperature was 39.0°C (range, 38.1-40.5°C). Not a single patient developed hemodynamic or respiratory compromise that required admission to the intensive care unit. Fever was not explained by infection in any case. Ninety-one percent of the febrile episodes resolved within 96h of cyclophosphamide administration. No significant difference in overall survival, event-free survival, or graft versus host disease-free/relapse-free survival was found in the group of febrile individuals after peripheral blood stem cell infusion. Fever after peripheral blood stem cell infusion in this clinical setting was common; it usually subsides with cyclophosphamide administration. The development of fever was not associated with an adverse prognosis. Copyright © 2017 King Faisal Specialist Hospital & Research Centre. Published by Elsevier B.V. All rights reserved.

Six years' experience of tolerance induction in renal transplantation using stem cell therapy.

PubMed

Vanikar, Aruna V; Trivedi, Hargovind L; Thakkar, Umang G

2018-02-01

Tolerance induction (TI) has been attempted with chimerism/clonal deletion. We report results of TI protocol (TIP) using stem cell therapy (SCT) included adipose derived mesenchymal stem cells (AD-MSC) and hematopoietic stem cells (HSC) in 10 living-donor related renal transplantation (LDRT) patients under non-myeloablative conditioning with Bortezomib, Methylprednisone, rabbit-anti-thymoglobulin and Rituximab, without using conventional immunosuppression. Transplantation was performed following acceptable lymphocyte cross-match, flow cross-match, single antigen assay and negative mixed lymphocyte reaction (MLR). Monitoring included serum creatinine (SCr), donor specific antibodies (DSA) and MLR. Protocol biopsies were planned after 100days and yearly in willing patients. Rescue immunosuppression was planned for rejection/DSA/positive MLR. Over mean 6±0.37year follow-up patient survival was 80% and death-censored graft survival was 90%. Mean SCr was 1.44±0.41mg/dL. This is the first clinical report of sustained TI in LDRT for 6years using SCT. Copyright © 2017 Elsevier Inc. All rights reserved.

Non-senescent Hydra tolerates severe disturbances in the nuclear lamina.

PubMed

Klimovich, Alexander; Rehm, Arvid; Wittlieb, Jörg; Herbst, Eva-Maria; Benavente, Ricardo; Bosch, Thomas C G

2018-05-10

The cnidarian Hydra is known for its unlimited lifespan and non-senescence, due to the indefinite self-renewal capacity of its stem cells. While proteins of the Lamin family are recognized as critical factors affecting senescence and longevity in human and mice, their putative role in the extreme longevity and non-senescence in long-living animals remains unknown. Here we analyze the role of a single lamin protein in non-senescence of Hydra . We demonstrate that proliferation of stem cells in Hydra is robust against the disturbance of Lamin expression and localization. While Lamin is indispensable for Hydra , the stem cells tolerate overexpression, downregulation and mislocalization of Lamin, and disturbances in the nuclear envelope structure. This extraordinary robustness may underlie the indefinite self-renewal capacity of stem cells and the non-senescence of Hydra . A relatively low complexity of the nuclear envelope architecture in basal Metazoa might allow for their extreme lifespans, while an increasing complexity of the nuclear architecture in bilaterians resulted in restricted lifespans.

Non-senescent Hydra tolerates severe disturbances in the nuclear lamina

PubMed Central

Rehm, Arvid; Wittlieb, Jörg; Herbst, Eva-Maria; Benavente, Ricardo

2018-01-01

The cnidarian Hydra is known for its unlimited lifespan and non-senescence, due to the indefinite self-renewal capacity of its stem cells. While proteins of the Lamin family are recognized as critical factors affecting senescence and longevity in human and mice, their putative role in the extreme longevity and non-senescence in long-living animals remains unknown. Here we analyze the role of a single lamin protein in non-senescence of Hydra. We demonstrate that proliferation of stem cells in Hydra is robust against the disturbance of Lamin expression and localization. While Lamin is indispensable for Hydra, the stem cells tolerate overexpression, downregulation and mislocalization of Lamin, and disturbances in the nuclear envelope structure. This extraordinary robustness may underlie the indefinite self-renewal capacity of stem cells and the non-senescence of Hydra. A relatively low complexity of the nuclear envelope architecture in basal Metazoa might allow for their extreme lifespans, while an increasing complexity of the nuclear architecture in bilaterians resulted in restricted lifespans. PMID:29754147

[Isolation and identification of human periodontal ligament stem cells in vitro].

PubMed

Shen, Tao; Chang, Hui-jun; Jian, Cong-xiang; Yang, Yan-chun; Zhou, Ji-xiang

2011-02-01

To isolate and identify human periodontal ligament stem cells (PDLSC) by improved methods and assess the characteristics of PDLSC ex vivo. The periodontal ligament cells were obtained from the healthy impacted third molars and teeth extracted for orthodontic purposes and used to isolate PDLSC by limiting dilution assay. PDLSC were cultured and expanded in alpha-MEM supplemented with 10% FBS. Colony-forming assay, immunohistochemistry, flow cytometry, osteogenic and adipogenic induction were used to identify PDLSC. The obtained cells had high colony-forming efficiency and were positive staining for vimentin and negative for pancytokeratin. Flow cytometry revealed that the isolated cells were positive for STRO-1 and CD146 antibodies and most were in the G0/G1 phase of cell cycle. Under specific conditions, they could differentiate to the osteoblast and adipocyte lineages in vitro. Limiting dilution assay is an effective method to isolate PDLSC and the single-cell-derived colonies demonstrate the properties of stem cells in vitro.

Small-molecule-directed, efficient generation of retinal pigment epithelium from human pluripotent stem cells.

PubMed

Maruotti, Julien; Sripathi, Srinivas R; Bharti, Kapil; Fuller, John; Wahlin, Karl J; Ranganathan, Vinod; Sluch, Valentin M; Berlinicke, Cynthia A; Davis, Janine; Kim, Catherine; Zhao, Lijun; Wan, Jun; Qian, Jiang; Corneo, Barbara; Temple, Sally; Dubey, Ramin; Olenyuk, Bogdan Z; Bhutto, Imran; Lutty, Gerard A; Zack, Donald J

2015-09-01

Age-related macular degeneration (AMD) is associated with dysfunction and death of retinal pigment epithelial (RPE) cells. Cell-based approaches using RPE-like cells derived from human pluripotent stem cells (hPSCs) are being developed for AMD treatment. However, most efficient RPE differentiation protocols rely on complex, stepwise treatments and addition of growth factors, whereas small-molecule-only approaches developed to date display reduced yields. To identify new compounds that promote RPE differentiation, we developed and performed a high-throughput quantitative PCR screen complemented by a novel orthogonal human induced pluripotent stem cell (hiPSC)-based RPE reporter assay. Chetomin, an inhibitor of hypoxia-inducible factors, was found to strongly increase RPE differentiation; combination with nicotinamide resulted in conversion of over one-half of the differentiating cells into RPE. Single passage of the whole culture yielded a highly pure hPSC-RPE cell population that displayed many of the morphological, molecular, and functional characteristics of native RPE.

Small-molecule–directed, efficient generation of retinal pigment epithelium from human pluripotent stem cells

PubMed Central

Maruotti, Julien; Sripathi, Srinivas R.; Bharti, Kapil; Fuller, John; Wahlin, Karl J.; Ranganathan, Vinod; Sluch, Valentin M.; Berlinicke, Cynthia A.; Davis, Janine; Kim, Catherine; Zhao, Lijun; Wan, Jun; Qian, Jiang; Corneo, Barbara; Temple, Sally; Dubey, Ramin; Olenyuk, Bogdan Z.; Bhutto, Imran; Lutty, Gerard A.; Zack, Donald J.

2015-01-01

Age-related macular degeneration (AMD) is associated with dysfunction and death of retinal pigment epithelial (RPE) cells. Cell-based approaches using RPE-like cells derived from human pluripotent stem cells (hPSCs) are being developed for AMD treatment. However, most efficient RPE differentiation protocols rely on complex, stepwise treatments and addition of growth factors, whereas small-molecule–only approaches developed to date display reduced yields. To identify new compounds that promote RPE differentiation, we developed and performed a high-throughput quantitative PCR screen complemented by a novel orthogonal human induced pluripotent stem cell (hiPSC)-based RPE reporter assay. Chetomin, an inhibitor of hypoxia-inducible factors, was found to strongly increase RPE differentiation; combination with nicotinamide resulted in conversion of over one-half of the differentiating cells into RPE. Single passage of the whole culture yielded a highly pure hPSC-RPE cell population that displayed many of the morphological, molecular, and functional characteristics of native RPE. PMID:26269569

The dynamics of long-term transgene expression in engrafted neural stem cells.

PubMed

Lee, Jean-Pyo; Tsai, David J; In Park, Kook; Harvey, Alan R; Snyder, Evan Y

2009-07-01

To assess the dynamics and confounding variables that influence transgene expression in neural stem cells (NSCs), we generated distinct NSC clones from the same pool of cells, carrying the same reporter gene transcribed from the same promoter, transduced by the same retroviral vector, and transplanted similarly at the same differentiation state, at the same time and location, into the brains of newborn mouse littermates, and monitored in parallel for over a year in vivo (without immunosuppression). Therefore, the sole variables were transgene chromosomal insertion site and copy number. We then adapted and optimized a technique that tests, at the single cell level, persistence of stem cell-mediated transgene expression in vivo based on correlating the presence of the transgene in a given NSC's nucleus (by fluorescence in situ hybridization [FISH]) with the frequency of that transgene's product within the same cell (by combined immunohistochemistry [IHC]). Under the above-stated conditions, insertion site is likely the most contributory variable dictating transgene downregulation in an NSC after 3 months in vivo. We also observed that this obstacle could be effectively and safely counteracted by simple serial infections (as few as three) inserting redundant copies of the transgene into the prospective donor NSC. (The preservation of normal growth control mechanisms and an absence of tumorigenic potential can be readily screened and ensured ex vivo prior to transplantation.) The combined FISH/IHC strategy employed here for monitoring the dynamics of transgene expression at the single cell level in vivo may be used for other types of therapeutic and housekeeping genes in endogenous and exogenous stem cells of many organs and lineages. Copyright 2009 Wiley-Liss, Inc.

Generation of organized germ layers from a single mouse embryonic stem cell.

PubMed

Poh, Yeh-Chuin; Chen, Junwei; Hong, Ying; Yi, Haiying; Zhang, Shuang; Chen, Junjian; Wu, Douglas C; Wang, Lili; Jia, Qiong; Singh, Rishi; Yao, Wenting; Tan, Youhua; Tajik, Arash; Tanaka, Tetsuya S; Wang, Ning

2014-05-30

Mammalian inner cell mass cells undergo lineage-specific differentiation into germ layers of endoderm, mesoderm and ectoderm during gastrulation. It has been a long-standing challenge in developmental biology to replicate these organized germ layer patterns in culture. Here we present a method of generating organized germ layers from a single mouse embryonic stem cell cultured in a soft fibrin matrix. Spatial organization of germ layers is regulated by cortical tension of the colony, matrix dimensionality and softness, and cell-cell adhesion. Remarkably, anchorage of the embryoid colony from the 3D matrix to collagen-1-coated 2D substrates of ~1 kPa results in self-organization of all three germ layers: ectoderm on the outside layer, mesoderm in the middle and endoderm at the centre of the colony, reminiscent of generalized gastrulating chordate embryos. These results suggest that mechanical forces via cell-matrix and cell-cell interactions are crucial in spatial organization of germ layers during mammalian gastrulation. This new in vitro method could be used to gain insights on the mechanisms responsible for the regulation of germ layer formation.

Graphene oxide selectively targets cancer stem cells, across multiple tumor types: Implications for non-toxic cancer treatment, via “differentiation-based nano-therapy”

PubMed Central

Fiorillo, Marco; Verre, Andrea F.; Iliut, Maria; Peiris-Pagés, Maria; Ozsvari, Bela; Gandara, Ricardo; Cappello, Anna Rita; Sotgia, Federica; Vijayaraghavan, Aravind; Lisanti, Michael P.

2015-01-01

Tumor-initiating cells (TICs), a.k.a. cancer stem cells (CSCs), are difficult to eradicate with conventional approaches to cancer treatment, such as chemo-therapy and radiation. As a consequence, the survival of residual CSCs is thought to drive the onset of tumor recurrence, distant metastasis, and drug-resistance, which is a significant clinical problem for the effective treatment of cancer. Thus, novel approaches to cancer therapy are needed urgently, to address this clinical need. Towards this end, here we have investigated the therapeutic potential of graphene oxide to target cancer stem cells. Graphene and its derivatives are well-known, relatively inert and potentially non-toxic nano-materials that form stable dispersions in a variety of solvents. Here, we show that graphene oxide (of both big and small flake sizes) can be used to selectively inhibit the proliferative expansion of cancer stem cells, across multiple tumor types. For this purpose, we employed the tumor-sphere assay, which functionally measures the clonal expansion of single cancer stem cells under anchorage-independent conditions. More specifically, we show that graphene oxide effectively inhibits tumor-sphere formation in multiple cell lines, across 6 different cancer types, including breast, ovarian, prostate, lung and pancreatic cancers, as well as glioblastoma (brain). In striking contrast, graphene oxide is non-toxic for “bulk” cancer cells (non-stem) and normal fibroblasts. Mechanistically, we present evidence that GO exerts its striking effects on CSCs by inhibiting several key signal transduction pathways (WNT, Notch and STAT-signaling) and thereby inducing CSC differentiation. Thus, graphene oxide may be an effective non-toxic therapeutic strategy for the eradication of cancer stem cells, via differentiation-based nano-therapy. PMID:25708684

Imaging the distribution of individual platinum-based anticancer drug molecules attached to single-wall carbon nanotubes

PubMed Central

Bhirde, Ashwin A; Sousa, Alioscka A; Patel, Vyomesh; Azari, Afrouz A; Gutkind, J Silvio; Leapman, Richard D; Rusling, James F

2009-01-01

Aims To image the distribution of drug molecules attached to single-wall carbon nanotubes (SWNTs). Materials & methods Herein we report the use of scanning transmission electron microscopy (STEM) for atomic scale visualization and quantitation of single platinum-based drug molecules attached to SWNTs designed for targeted drug delivery. Fourier transform infrared spectroscopy and energy-dispersive x-ray spectroscopy were used for characterization of the SWNT drug conjugates. Results Z-contrast STEM imaging enabled visualization of the first-line anticancer drug cisplatin on the nanotubes at single molecule level. The identity and presence of cisplatin on the nanotubes was confirmed using energy-dispersive x-ray spectroscopy and Fourier transform infrared spectroscopy. STEM tomography was also used to provide additional insights concerning the nanotube conjugates. Finally, our observations provide a rationale for exploring the use of SWNT bioconjugates to selectively target and kill squamous cancer cells. Conclusion Z-contrast STEM imaging provides a means for direct visualization of heavy metal containing molecules (i.e., cisplatin) attached to surfaces of carbon SWNTs along with distribution and quantitation. PMID:19839812

Rotary orbital suspension culture of embryonic stem cell-derived neural stem/progenitor cells: impact of hydrodynamic culture on aggregate yield, morphology and cell phenotype.

PubMed

Laundos, Tiago L; Silva, Joana; Assunção, Marisa; Quelhas, Pedro; Monteiro, Cátia; Oliveira, Carla; Oliveira, Maria J; Pêgo, Ana P; Amaral, Isabel F

2017-08-01

Embryonic stem (ES)-derived neural stem/progenitor cells (ES-NSPCs) constitute a promising cell source for application in cell therapies for the treatment of central nervous system disorders. In this study, a rotary orbital hydrodynamic culture system was applied to single-cell suspensions of ES-NSPCs, to obtain homogeneously-sized ES-NSPC cellular aggregates (neurospheres). Hydrodynamic culture allowed the formation of ES-NSPC neurospheres with a narrower size distribution than statically cultured neurospheres, increasing orbital speeds leading to smaller-sized neurospheres and higher neurosphere yield. Neurospheres formed under hydrodynamic conditions (72 h at 55 rpm) showed higher cell compaction and comparable percentages of viable, dead, apoptotic and proliferative cells. Further characterization of cellular aggregates provided new insights into the effect of hydrodynamic shear on ES-NSPC behaviour. Rotary neurospheres exhibited reduced protein levels of N-cadherin and β-catenin, and higher deposition of laminin (without impacting fibronectin deposition), matrix metalloproteinase-2 (MMP-2) activity and percentage of neuronal cells. In line with the increased MMP-2 activity levels found, hydrodynamically-cultured neurospheres showed higher outward migration on laminin. Moreover, when cultured in a 3D fibrin hydrogel, rotary neurospheres generated an increased percentage of neuronal cells. In conclusion, the application of a constant orbital speed to single-cell suspensions of ES-NSPCs, besides allowing the formation of homogeneously-sized neurospheres, promoted ES-NSPC differentiation and outward migration, possibly by influencing the expression of cell-cell adhesion molecules and the secretion of proteases/extracellular matrix proteins. These findings are important when establishing the culture conditions needed to obtain uniformly-sized ES-NSPC aggregates, either for use in regenerative therapies or in in vitro platforms for biomaterial development or pharmacological screening. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Hematopoietic Stem Cell Transplantation in Thalassemia and Sickle Cell Anemia

PubMed Central

Lucarelli, Guido; Isgrò, Antonella; Sodani, Pietro; Gaziev, Javid

2012-01-01

The globally widespread single-gene disorders β-thalassemia and sickle cell anemia (SCA) can only be cured by allogeneic hematopoietic stem cell transplantation (HSCT). HSCT treatment of thalassemia has substantially improved over the last two decades, with advancements in preventive strategies, control of transplant-related complications, and preparative regimens. A risk class–based transplantation approach results in disease-free survival probabilities of 90%, 84%, and 78% for class 1, 2, and 3 thalassemia patients, respectively. Because of disease advancement, adult thalassemia patients have a higher risk for transplant-related toxicity and a 65% cure rate. Patients without matched donors could benefit from haploidentical mother-to-child transplantation. There is a high cure rate for children with SCA who receive HSCT following myeloablative conditioning protocols. Novel non-myeloablative transplantation protocols could make HSCT available to adult SCA patients who were previously excluded from allogeneic stem cell transplantation. PMID:22553502

The In Vitro Response of Tissue Stem Cells to Irradiation With Different Linear Energy Transfers

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

Nagle, Peter W.; Hosper, Nynke A.; Department of Radiation Oncology, University of Groningen, University Medical Center Groningen, Groningen

Purpose: A reduction in the dose, irradiated volume, and sensitivity of, in particular, normal tissue stem cells is needed to advance radiation therapy. This could be obtained with the use of particles for radiation therapy. However, the radiation response of normal tissue stem cells is still an enigma. Therefore, in the present study, we developed a model to investigate the in vitro response of stem cells to particle irradiation. Methods and Materials: We used the immortalized human salivary gland (HSG) cell line resembling salivary gland (SG) cells to translate the radiation response in 2-dimensional (2D) to 3-dimensional (3D) conditions. This responsemore » was subsequently translated to the response of SG stem cells (SGSCs). Dispersed single cells were irradiated with photons or carbon ions at different linear energy transfers (LETs; 48.76 ± 2.16, 149.9 ± 10.8, and 189 ± 15 keV/μm). Subsequently, 2D or 3D clonogenicity was determined by counting the colonies or secondary stem cell-derived spheres in Matrigel. γH2AX immunostaining was used to assess DNA double strand break repair. Results: The 2D response of HSG cells showed a similar increase in dose response to increasing higher LET irradiation as other cell lines. The 3D response of HSG cells to increasing LET irradiation was reduced compared with the 2D response. Finally, the response of mouse SGSCs to photons was similar to the 3D response of HSG cells. The response to higher LET irradiation was reduced in the stem cells. Conclusions: Mouse SGSC radiosensitivity seems reduced at higher LET radiation compared with transformed HSG cells. The developed model to assess the radiation response of SGSCs offers novel possibilities to study the radiation response of normal tissue in vitro.« less

Human organomics: a fresh approach to understanding human development using single-cell transcriptomics.

PubMed

Camp, J Gray; Treutlein, Barbara

2017-05-01

Innovative methods designed to recapitulate human organogenesis from pluripotent stem cells provide a means to explore human developmental biology. New technologies to sequence and analyze single-cell transcriptomes can deconstruct these 'organoids' into constituent parts, and reconstruct lineage trajectories during cell differentiation. In this Spotlight article we summarize the different approaches to performing single-cell transcriptomics on organoids, and discuss the opportunities and challenges of applying these techniques to generate organ-level, mechanistic models of human development and disease. Together, these technologies will move past characterization to the prediction of human developmental and disease-related phenomena. © 2017. Published by The Company of Biologists Ltd.

Single-cell epigenomics: techniques and emerging applications.

PubMed

Schwartzman, Omer; Tanay, Amos

2015-12-01

Epigenomics is the study of the physical modifications, associations and conformations of genomic DNA sequences, with the aim of linking these with epigenetic memory, cellular identity and tissue-specific functions. While current techniques in the field are characterizing the average epigenomic features across large cell ensembles, the increasing interest in the epigenetics within complex and heterogeneous tissues is driving the development of single-cell epigenomics. We review emerging single-cell methods for capturing DNA methylation, chromatin accessibility, histone modifications, chromosome conformation and replication dynamics. Together, these techniques are rapidly becoming a powerful tool in studies of cellular plasticity and diversity, as seen in stem cells and cancer.

Haematopoietic transplants combining a single unrelated cord blood unit and mobilized haematopoietic stem cells from an adult HLA-mismatched third party donor. Comparable results to transplants from HLA-identical related donors in adults with acute leukaemia and myelodysplastic syndromes.

PubMed

Sebrango, Ana; Vicuña, Isabel; de Laiglesia, Almudena; Millán, Isabel; Bautista, Guiomar; Martín-Donaire, Trinidad; Regidor, Carmen; Cabrera, Rafael; Fernandez, Manuel N

2010-06-01

We describe results of the strategy, developed by our group, of co-infusion of mobilized haematopoietic stem cells as a support for single-unit unrelated cord blood transplant (dual CB/TPD-MHSC transplants) for treatment of haematological malignancies in adults, and a comparative analysis of results obtained using this strategy and transplants performed with mobilized haematopoietic stem cells from related HLA-identical donors (RTD) for treatment of adults with acute leukaemia and myelodysplastic syndromes. Our data show that the dual CB/TPD-MHSC transplant strategy results in periods of post-transplant neutropenia, final rates of full donor chimerism and transplant-related mortality rates comparable to those of the RTD. Final survival outcomes are comparable in adults transplanted because of acute leukaemia, with different incidences of the complications that most influence these: a higher incidence of infections related to late recovery of protective immunity dependent on T cell functions, and a lower incidence of serious acute graft-versus-host disease and relapses. Recent advances in cord blood transplant techniques allow allogeneic haematopoietic stem cell transplantation (HSCT) to be a viable option for almost every patient who may benefit from this therapeutic approach. Development of innovative strategies to improve the post-transplant recovery of T cells function is currently the main challenge to further improving the possibilities of unrelated cord blood transplantation. Copyright © 2010 Elsevier Ltd. All rights reserved.

Single-Factor SOX2 Mediates Direct Neural Reprogramming of Human Mesenchymal Stem Cells via Transfection of In Vitro Transcribed mRNA.

PubMed

Kim, Bo-Eun; Choi, Soon Won; Shin, Ji-Hee; Kim, Jae-Jun; Kang, Insung; Lee, Byung-Chul; Lee, Jin Young; Kook, Myoung Geun; Kang, Kyung-Sun

2018-01-01

Neural stem cells (NSCs) are a prominent cell source for understanding neural pathogenesis and for developing therapeutic applications to treat neurodegenerative disease because of their regenerative capacity and multipotency. Recently, a variety of cellular reprogramming technologies have been developed to facilitate in vitro generation of NSCs, called induced NSCs (iNSCs). However, the genetic safety aspects of established virus-based reprogramming methods have been considered, and non-integrating reprogramming methods have been developed. Reprogramming with in vitro transcribed (IVT) mRNA is one of the genetically safe reprogramming methods because exogenous mRNA temporally exists in the cell and is not integrated into the chromosome. Here, we successfully generated expandable iNSCs from human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) via transfection with IVT mRNA encoding SOX2 (SOX2 mRNA) with properly optimized conditions. We confirmed that generated human UCB-MSC-derived iNSCs (UM-iNSCs) possess characteristics of NSCs, including multipotency and self-renewal capacity. Additionally, we transfected human dermal fibroblasts (HDFs) with SOX2 mRNA. Compared with human embryonic stem cell-derived NSCs, HDFs transfected with SOX2 mRNA exhibited neural reprogramming with similar morphologies and NSC-enriched mRNA levels, but they showed limited proliferation ability. Our results demonstrated that human UCB-MSCs can be used for direct reprogramming into NSCs through transfection with IVT mRNA encoding a single factor, which provides an integration-free reprogramming tool for future therapeutic application.

Ssb1 and Ssb2 cooperate to regulate mouse hematopoietic stem and progenitor cells by resolving replicative stress.

PubMed

Shi, Wei; Vu, Therese; Boucher, Didier; Biernacka, Anna; Nde, Jules; Pandita, Raj K; Straube, Jasmin; Boyle, Glen M; Al-Ejeh, Fares; Nag, Purba; Jeffery, Jessie; Harris, Janelle L; Bain, Amanda L; Grzelak, Marta; Skrzypczak, Magdalena; Mitra, Abhishek; Dojer, Norbert; Crosetto, Nicola; Cloonan, Nicole; Becherel, Olivier J; Finnie, John; Skaar, Jeffrey R; Walkley, Carl R; Pandita, Tej K; Rowicka, Maga; Ginalski, Krzysztof; Lane, Steven W; Khanna, Kum Kum

2017-05-04

Hematopoietic stem and progenitor cells (HSPCs) are vulnerable to endogenous damage and defects in DNA repair can limit their function. The 2 single-stranded DNA (ssDNA) binding proteins SSB1 and SSB2 are crucial regulators of the DNA damage response; however, their overlapping roles during normal physiology are incompletely understood. We generated mice in which both Ssb1 and Ssb2 were constitutively or conditionally deleted. Constitutive Ssb1/Ssb2 double knockout (DKO) caused early embryonic lethality, whereas conditional Ssb1/Ssb2 double knockout (cDKO) in adult mice resulted in acute lethality due to bone marrow failure and intestinal atrophy featuring stem and progenitor cell depletion, a phenotype unexpected from the previously reported single knockout models of Ssb1 or Ssb2 Mechanistically, cDKO HSPCs showed altered replication fork dynamics, massive accumulation of DNA damage, genome-wide double-strand breaks enriched at Ssb-binding regions and CpG islands, together with the accumulation of R -loops and cytosolic ssDNA. Transcriptional profiling of cDKO HSPCs revealed the activation of p53 and interferon (IFN) pathways, which enforced cell cycling in quiescent HSPCs, resulting in their apoptotic death. The rapid cell death phenotype was reproducible in in vitro cultured cDKO-hematopoietic stem cells, which were significantly rescued by nucleotide supplementation or after depletion of p53. Collectively, Ssb1 and Ssb2 control crucial aspects of HSPC function, including proliferation and survival in vivo by resolving replicative stress to maintain genomic stability. © 2017 by The American Society of Hematology.

Ssb1 and Ssb2 cooperate to regulate mouse hematopoietic stem and progenitor cells by resolving replicative stress

PubMed Central

Vu, Therese; Boucher, Didier; Biernacka, Anna; Nde, Jules; Pandita, Raj K.; Straube, Jasmin; Boyle, Glen M.; Al-Ejeh, Fares; Jeffery, Jessie; Harris, Janelle L.; Bain, Amanda L.; Grzelak, Marta; Skrzypczak, Magdalena; Mitra, Abhishek; Dojer, Norbert; Crosetto, Nicola; Cloonan, Nicole; Becherel, Olivier J.; Finnie, John; Skaar, Jeffrey R.; Walkley, Carl R.; Pandita, Tej K.; Rowicka, Maga; Ginalski, Krzysztof

2017-01-01

Hematopoietic stem and progenitor cells (HSPCs) are vulnerable to endogenous damage and defects in DNA repair can limit their function. The 2 single-stranded DNA (ssDNA) binding proteins SSB1 and SSB2 are crucial regulators of the DNA damage response; however, their overlapping roles during normal physiology are incompletely understood. We generated mice in which both Ssb1 and Ssb2 were constitutively or conditionally deleted. Constitutive Ssb1/Ssb2 double knockout (DKO) caused early embryonic lethality, whereas conditional Ssb1/Ssb2 double knockout (cDKO) in adult mice resulted in acute lethality due to bone marrow failure and intestinal atrophy featuring stem and progenitor cell depletion, a phenotype unexpected from the previously reported single knockout models of Ssb1 or Ssb2. Mechanistically, cDKO HSPCs showed altered replication fork dynamics, massive accumulation of DNA damage, genome-wide double-strand breaks enriched at Ssb-binding regions and CpG islands, together with the accumulation of R-loops and cytosolic ssDNA. Transcriptional profiling of cDKO HSPCs revealed the activation of p53 and interferon (IFN) pathways, which enforced cell cycling in quiescent HSPCs, resulting in their apoptotic death. The rapid cell death phenotype was reproducible in in vitro cultured cDKO-hematopoietic stem cells, which were significantly rescued by nucleotide supplementation or after depletion of p53. Collectively, Ssb1 and Ssb2 control crucial aspects of HSPC function, including proliferation and survival in vivo by resolving replicative stress to maintain genomic stability. PMID:28270450

Concise review: programming human pluripotent stem cells into blood.

PubMed

Easterbrook, Jennifer; Fidanza, Antonella; Forrester, Lesley M

2016-06-01

Blood disorders are treated with cell therapies including haematopoietic stem cell (HSC) transplantation as well as platelet and red blood cell transfusions. However the source of cells is entirely dependent on donors, procedures are susceptible to transfusion-transmitted infections and serious complications can arise in recipients due to immunological incompatibility. These problems could be alleviated if it was possible to produce haematopoietic cells in vitro from an autologous and renewable cell source. The production of haematopoietic cells in the laboratory from human induced pluripotent stem cells (iPSCs) may provide a route to realize this goal but it has proven challenging to generate long-term reconstituting HSCs. To date, the optimization of differentiation protocols has mostly relied on the manipulation of extrinsic signals to mimic the in vivo environment. We review studies that have taken an alternative approach to modulate intrinsic signals by enforced expression of transcription factors. Single and combinations of multiple transcription factors have been used in a variety of contexts to enhance the production of haematopoietic cells from human pluripotent stem cells. This programming approach, together with the recent advances in the production and use of synthetic transcription factors, holds great promise for the production of fully functional HSCs in the future. © 2016 The Authors. British Journal of Haematology published by John Wiley & Sons Ltd.

Human Finger-Prick Induced Pluripotent Stem Cells Facilitate the Development of Stem Cell Banking

PubMed Central

Tan, Hong-Kee; Toh, Cheng-Xu Delon; Ma, Dongrui; Yang, Binxia; Liu, Tong Ming; Lu, Jun; Wong, Chee-Wai; Tan, Tze-Kai; Li, Hu; Syn, Christopher; Tan, Eng-Lee; Lim, Bing; Lim, Yoon-Pin; Cook, Stuart A.

2014-01-01

Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients can be a good model for studying human diseases and for future therapeutic regenerative medicine. Current initiatives to establish human iPSC (hiPSC) banking face challenges in recruiting large numbers of donors with diverse diseased, genetic, and phenotypic representations. In this study, we describe the efficient derivation of transgene-free hiPSCs from human finger-prick blood. Finger-prick sample collection can be performed on a “do-it-yourself” basis by donors and sent to the hiPSC facility for reprogramming. We show that single-drop volumes of finger-prick samples are sufficient for performing cellular reprogramming, DNA sequencing, and blood serotyping in parallel. Our novel strategy has the potential to facilitate the development of large-scale hiPSC banking worldwide. PMID:24646489

Adult human mesenchymal stem cells delivered via intra-articular injection to the knee following partial medial meniscectomy: a randomized, double-blind, controlled study.

PubMed

Vangsness, C Thomas; Farr, Jack; Boyd, Joel; Dellaero, David T; Mills, C Randal; LeRoux-Williams, Michelle

2014-01-15

There are limited treatment options for tissue restoration and the prevention of degenerative changes in the knee. Stem cells have been a focus of intense preclinical research into tissue regeneration but limited clinical investigation. In a randomized, double-blind, controlled study, the safety of the intra-articular injection of human mesenchymal stem cells into the knee, the ability of mesenchymal stem cells to promote meniscus regeneration following partial meniscectomy, and the effects of mesenchymal stem cells on osteoarthritic changes in the knee were investigated. A total of fifty-five patients at seven institutions underwent a partial medial meniscectomy. A single superolateral knee injection was given within seven to ten days after the meniscectomy. Patients were randomized to one of three treatment groups: Group A, in which patients received an injection of 50 × 10⁶ allogeneic mesenchymal stem cells; Group B, 150 × 10⁶ allogeneic mesenchymal stem cells; and the control group, a sodium hyaluronate (hyaluronic acid/hyaluronan) vehicle control. Patients were followed to evaluate safety, meniscus regeneration, the overall condition of the knee joint, and clinical outcomes at intervals through two years. Evaluations included sequential magnetic resonance imaging (MRI). No ectopic tissue formation or clinically important safety issues were identified. There was significantly increased meniscal volume (defined a priori as a 15% threshold) determined by quantitative MRI in 24% of patients in Group A and 6% in Group B at twelve months post meniscectomy (p = 0.022). No patients in the control group met the 15% threshold for increased meniscal volume. Patients with osteoarthritic changes who received mesenchymal stem cells experienced a significant reduction in pain compared with those who received the control, on the basis of visual analog scale assessments. There was evidence of meniscus regeneration and improvement in knee pain following treatment with allogeneic human mesenchymal stem cells. These results support the study of human mesenchymal stem cells for the apparent knee-tissue regeneration and protective effects.

Polycistronic lentiviral vector for "hit and run" reprogramming of adult skin fibroblasts to induced pluripotent stem cells.

PubMed

Chang, Chia-Wei; Lai, Yi-Shin; Pawlik, Kevin M; Liu, Kaimao; Sun, Chiao-Wang; Li, Chao; Schoeb, Trenton R; Townes, Tim M

2009-05-01

We report the derivation of induced pluripotent stem (iPS) cells from adult skin fibroblasts using a single, polycistronic lentiviral vector encoding the reprogramming factors Oct4, Sox2, and Klf4. Porcine teschovirus-1 2A sequences that trigger ribosome skipping were inserted between human cDNAs for these factors, and the polycistron was subcloned downstream of the elongation factor 1 alpha promoter in a self-inactivating (SIN) lentiviral vector containing a loxP site in the truncated 3' long terminal repeat (LTR). Adult skin fibroblasts from a humanized mouse model of sickle cell disease were transduced with this single lentiviral vector, and iPS cell colonies were picked within 30 days. These cells expressed endogenous Oct4, Sox2, Nanog, alkaline phosphatase, stage-specific embryonic antigen-1, and other markers of pluripotency. The iPS cells produced teratomas containing tissue derived from all three germ layers after injection into immunocompromised mice and formed high-level chimeras after injection into murine blastocysts. iPS cell lines with as few as three lentiviral insertions were obtained. Expression of Cre recombinase in these iPS cells resulted in deletion of the lentiviral vector, and sequencing of insertion sites demonstrated that remnant 291-bp SIN LTRs containing a single loxP site did not interrupt coding sequences, promoters, or known regulatory elements. These results suggest that a single, polycistronic "hit and run" vector can safely and effectively reprogram adult dermal fibroblasts into iPS cells.

Effects of Hypoglycemia on Circulating Stem and Progenitor Cells in Diabetic Patients.

PubMed

Fadini, Gian Paolo; Boscari, Federico; Cappellari, Roberta; Galasso, Silvia; Rigato, Mauro; Bonora, Benedetta Maria; D'Anna, Marianna; Bruttomesso, Daniela; Avogaro, Angelo

2018-03-01

Iatrogenic hypoglycemia is the most common acute diabetic complication, and it significantly increases morbidity. In people with diabetes, reduction in the levels of circulating stem and progenitor cells predicts adverse outcomes. To evaluate whether hypoglycemia in diabetes affects circulating stem cells and endothelial progenitor cells (EPCs). We performed an experimental hypoglycemia study (Study 1) and a case-control study (Study 2). Tertiary referral inpatient clinic. Type 1 diabetic patients (Study 1, n = 19); diabetic patients hospitalized for severe iatrogenic hypoglycemia, matched inpatient and outpatient controls (Study 2, n = 22/group). Type 1 diabetic patients underwent two in-hospital sessions of glucose monitoring during a breakfast meal with or without induction of hypoglycemia in random order. In Study 2, patients hospitalized for hypoglycemia and matched controls were compared. Circulating stem cells and EPCs were measured by flow cytometry based on the expression of CD34 and kinase insert domain receptor (KDR). In Study 1, the physiologic decline of CD34+KDR+ EPCs from 8 am to 2 pm was abolished by insulin-induced hypoglycemia in type 1 diabetic patients. In Study 2, diabetic patients hospitalized for severe iatrogenic hypoglycemia had significantly lower levels of CD34+ stem cells and CD34+KDR+ EPCs compared with diabetic inpatients or outpatient controls. In diabetic patients, a single mild hypoglycemic episode can compromise the physiologic EPC fluctuation, whereas severe hypoglycemia is associated with a marked reduction in stem cells and EPCs. These data provide a possible link between hypoglycemia and adverse outcomes of diabetes.

The effects of restricted glycolysis on stem-cell like characteristics of breast cancer cells

PubMed Central

Banerjee, Arindam; Arvinrad, Pardis; Darley, Matthew; Laversin, Stéphanie A.; Parker, Rachel; Rose-Zerilli, Matthew J.J.; Townsend, Paul A.; Cutress, Ramsey I.; Beers, Stephen A.; Houghton, Franchesca D.; Birts, Charles N.; Blaydes, Jeremy P.

2018-01-01

Altered glycolysis is a characteristic of many cancers, and can also be associated with changes in stem cell-like cancer (SCLC) cell populations. We therefore set out to directly examine the effect of glycolysis on SCLC cell phenotype, using a model where glycolysis is stably reduced by adapting the cells to a sugar source other than glucose. Restricting glycolysis using this approach consistently resulted in cells with increased oncogenic potential; including an increase in SCLC cells, proliferation in 3D matrigel, invasiveness, chemoresistance, and altered global gene expression. Tumorigenicity in vivo was also markedly increased. SCLC cells exhibited increased dependence upon alternate metabolic pathways. They also became c-KIT dependent, indicating that their apparent state of maturation is regulated by glycolysis. Single-cell mRNA sequencing identified altered networks of metabolic-, stem- and signaling- gene expression within SCLC-enriched populations in response to glycolytic restriction. Therefore, reduced glycolysis, which may occur in niches within tumors where glucose availability is limiting, can promote tumor aggressiveness by increasing SCLC cell populations, but can also introduce novel, potentially exploitable, vulnerabilities in SCLC cells. PMID:29796188

Serum replacement with a growth factor-free synthetic substance in culture medium contributes to effective establishment of mouse embryonic stem cells of various origins.

PubMed

Lee, Seung Tae; Oh, Se Woong; Kim, Dae Yong; Han, Jae Yong; Moon, Shin Yong; Lim, Jeong Mook

2006-10-01

To evaluate whether serum replacement with growth factor-free synthetic substances contributed to the effective establishment of embryonic stem (ES) cells. Randomized, prospective model study. Gamete and stem cell biotechnology laboratory at Seoul National University in Korea. F1 (C57BL6 x DBA2) mice. Blastocysts of different origins were cultured in serum-replaced media. Embryonic stem cell establishment. Eight batches of ES cells were established from colony-forming inner cell mass cells after the replacement of fetal bovine serum (FBS) with synthetic knockout serum replacement (KSR) in mkDMEM. The established cells were positive for ES cell markers and formed both embryoid bodies in vitro and teratomas in vivo, but the established cell batches and control (transformed) ES cells responded differently to the culture media. Higher levels of cell viability were detected after the replacement with the 75:25 FBS-KSR mixture than with any other mixtures, and a gradual decrease in viability was detected as the KSR volume ratio was increased. The 75:25 FBS-KSR mixture-containing medium supported ES cell establishment of outbred ICR, F1, and F2 of C57BL6/DBA2; F1 parthenogenetic and ES cell-complemented tetraploid blastocysts; and single ES-cell cultures. A serum-replaced medium could be used for effective ES-cell establishment of various origins.

Sox2 acts in a dose-dependent fashion to regulate proliferation of cortical progenitors.

PubMed

Hagey, Daniel W; Muhr, Jonas

2014-12-11

Organ formation and maintenance depends on slowly self-renewing stem cells that supply an intermediate population of rapidly dividing progenitors, but how this proliferative hierarchy is regulated is unknown. By performing genome-wide single-cell and functional analyses in the cortex, we demonstrate that reduced Sox2 expression is a key regulatory signature of the transition between stem cells and rapidly dividing progenitors. In stem cells, Sox2 is expressed at high levels, which enables its repression of proproliferative genes, of which Cyclin D1 is the most potent target. Sox2 confers this function through binding to low-affinity motifs, which facilitate the recruitment of Gro/Tle corepressors in synergy with Tcf/Lef proteins. Upon differentiation, proneural factors reduce Sox2 expression, which derepresses Cyclin D1 and promotes proliferation. Our results show how concentration-dependent Sox2 occupancy of DNA motifs of varying affinities translates into recruitment of repressive complexes, which regulate the proliferative dynamics of neural stem and progenitor cells. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Multipotential differentiation of human urine-derived stem cells: potential for therapeutic applications in urology.

PubMed

Bharadwaj, Shantaram; Liu, Guihua; Shi, Yingai; Wu, Rongpei; Yang, Bin; He, Tongchuan; Fan, Yuxin; Lu, Xinyan; Zhou, Xiaobo; Liu, Hong; Atala, Anthony; Rohozinski, Jan; Zhang, Yuanyuan

2013-09-01

We sought to biologically characterize and identify a subpopulation of urine-derived stem cells (USCs) with the capacity for multipotent differentiation. We demonstrated that single USCs can expand to a large population with 60-70 population doublings. Nine of 15 individual USC clones expressed detectable levels of telomerase and have long telomeres. These cells expressed pericyte and mesenchymal stem cell markers. Upon induction with appropriate media in vitro, USCs differentiated into bladder-associated cell types, including functional urothelial and smooth muscle cell lineages. When the differentiated USCs were seeded onto a scaffold and subcutaneously implanted into nude mice, multilayered tissue-like structures formed consisting of urothelium and smooth muscle. Additionally, USCs were able to differentiate into endothelial, osteogenic, chondrogenic, adipogenic, skeletal myogenic, and neurogenic lineages but did not form teratomas during the 1-month study despite telomerase activity. USCs may be useful in cell-based therapies and tissue engineering applications, including urogenital reconstruction. © AlphaMed Press.

High-Content Analysis of CRISPR-Cas9 Gene-Edited Human Embryonic Stem Cells.

PubMed

Carlson-Stevermer, Jared; Goedland, Madelyn; Steyer, Benjamin; Movaghar, Arezoo; Lou, Meng; Kohlenberg, Lucille; Prestil, Ryan; Saha, Krishanu

2016-01-12

CRISPR-Cas9 gene editing of human cells and tissues holds much promise to advance medicine and biology, but standard editing methods require weeks to months of reagent preparation and selection where much or all of the initial edited samples are destroyed during analysis. ArrayEdit, a simple approach utilizing surface-modified multiwell plates containing one-pot transcribed single-guide RNAs, separates thousands of edited cell populations for automated, live, high-content imaging and analysis. The approach lowers the time and cost of gene editing and produces edited human embryonic stem cells at high efficiencies. Edited genes can be expressed in both pluripotent stem cells and differentiated cells. This preclinical platform adds important capabilities to observe editing and selection in situ within complex structures generated by human cells, ultimately enabling optical and other molecular perturbations in the editing workflow that could refine the specificity and versatility of gene editing. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Fluorescent nanodiamonds enable quantitative tracking of human mesenchymal stem cells in miniature pigs

NASA Astrophysics Data System (ADS)

Su, Long-Jyun; Wu, Meng-Shiue; Hui, Yuen Yung; Chang, Be-Ming; Pan, Lei; Hsu, Pei-Chen; Chen, Yit-Tsong; Ho, Hong-Nerng; Huang, Yen-Hua; Ling, Thai-Yen; Hsu, Hsao-Hsun; Chang, Huan-Cheng

2017-03-01

Cell therapy is a promising strategy for the treatment of human diseases. While the first use of cells for therapeutic purposes can be traced to the 19th century, there has been a lack of general and reliable methods to study the biodistribution and associated pharmacokinetics of transplanted cells in various animal models for preclinical evaluation. Here, we present a new platform using albumin-conjugated fluorescent nanodiamonds (FNDs) as biocompatible and photostable labels for quantitative tracking of human placenta choriodecidual membrane-derived mesenchymal stem cells (pcMSCs) in miniature pigs by magnetic modulation. With this background-free detection technique and time-gated fluorescence imaging, we have been able to precisely determine the numbers as well as positions of the transplanted FND-labeled pcMSCs in organs and tissues of the miniature pigs after intravenous administration. The method is applicable to single-cell imaging and quantitative tracking of human stem/progenitor cells in rodents and other animal models as well.

Composite cell sheet for periodontal regeneration: crosstalk between different types of MSCs in cell sheet facilitates complex periodontal-like tissue regeneration.

PubMed

Zhang, Hao; Liu, Shiyu; Zhu, Bin; Xu, Qiu; Ding, Yin; Jin, Yan

2016-11-14

Tissue-engineering strategies based on mesenchymal stem cells (MSCs) and cell sheets have been widely used for periodontal tissue regeneration. However, given the complexity in periodontal structure, the regeneration methods using a single species of MSC could not fulfill the requirement for periodontal regeneration. We researched the interaction between the periodontal ligament stem cells (PDLSCs) and jaw bone marrow-derived mesenchymal stem cells (JBMMSCs), and constructed a composite cell sheet comprising both of the above MSCs to regenerate complex periodontium-like structures in nude mice. Our results show that by co-culturing PDLSCs and JBMMSCs, the expressions of bone and extracellular matrix (ECM)-related genes and proteins were significantly improved in both MSCs. Further investigations showed that, compared to the cell sheet using PDLSCs or JBMMSCs, the composite stem cell sheet (CSCS), which comprises these two MSCs, expressed higher levels of bone- and ECM-related genes and proteins, and generated a composite structure more similar to the native periodontal tissue physiologically in vivo. In conclusion, our results demonstrate that the crosstalk between PDLSCs and JBMMSCs in cell sheets facilitate regeneration of complex periodontium-like structures, providing a promising new strategy for physiological and functional regeneration of periodontal tissue.

Monolayer culturing and cloning of human pluripotent stem cells on laminin-521-based matrices under xeno-free and chemically defined conditions.

PubMed

Rodin, Sergey; Antonsson, Liselotte; Hovatta, Outi; Tryggvason, Karl

2014-10-01

A robust method for culturing human pluripotent stem (hPS) cells under chemically defined and xeno-free conditions is an important tool for stem cell research and for the development of regenerative medicine. Here, we describe a protocol for monolayer culturing of Oct-4-positive hPS cells on a specific laminin-521 (LN-521) isoform, under xeno-free and chemically defined conditions. The cells are dispersed into single-cell suspension and then plated on LN-521 isoform at densities higher than 5,000 cells per cm², where they attach, migrate and survive by forming small monolayer cell groups. The cells avidly divide and expand horizontally until the entire dish is covered by a confluent monolayer. LN-521, in combination with E-cadherin, allows cloning of individual hPS cells in separate wells of 96-well plates without the presence of rho-associated protein kinase (ROCK) inhibitors or any other inhibitors of anoikis. Characterization of cells maintained for several months in culture reveals pluripotency with a minimal degree of genetic abnormalities.

Zika Virus Disrupts Phospho-TBK1 Localization and Mitosis in Human Neuroepithelial Stem Cells and Radial Glia.

PubMed

Onorati, Marco; Li, Zhen; Liu, Fuchen; Sousa, André M M; Nakagawa, Naoki; Li, Mingfeng; Dell'Anno, Maria Teresa; Gulden, Forrest O; Pochareddy, Sirisha; Tebbenkamp, Andrew T N; Han, Wenqi; Pletikos, Mihovil; Gao, Tianliuyun; Zhu, Ying; Bichsel, Candace; Varela, Luis; Szigeti-Buck, Klara; Lisgo, Steven; Zhang, Yalan; Testen, Anze; Gao, Xiao-Bing; Mlakar, Jernej; Popovic, Mara; Flamand, Marie; Strittmatter, Stephen M; Kaczmarek, Leonard K; Anton, E S; Horvath, Tamas L; Lindenbach, Brett D; Sestan, Nenad

2016-09-06

The mechanisms underlying Zika virus (ZIKV)-related microcephaly and other neurodevelopment defects remain poorly understood. Here, we describe the derivation and characterization, including single-cell RNA-seq, of neocortical and spinal cord neuroepithelial stem (NES) cells to model early human neurodevelopment and ZIKV-related neuropathogenesis. By analyzing human NES cells, organotypic fetal brain slices, and a ZIKV-infected micrencephalic brain, we show that ZIKV infects both neocortical and spinal NES cells as well as their fetal homolog, radial glial cells (RGCs), causing disrupted mitoses, supernumerary centrosomes, structural disorganization, and cell death. ZIKV infection of NES cells and RGCs causes centrosomal depletion and mitochondrial sequestration of phospho-TBK1 during mitosis. We also found that nucleoside analogs inhibit ZIKV replication in NES cells, protecting them from ZIKV-induced pTBK1 relocalization and cell death. We established a model system of human neural stem cells to reveal cellular and molecular mechanisms underlying neurodevelopmental defects associated with ZIKV infection and its potential treatment. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Self-targeted salinomycin-loaded DSPE-PEG-methotrexate nanomicelles for targeting both head and neck squamous cell carcinoma cancer cells and cancer stem cells.

PubMed

Zhu, Minhui; Chen, Shicai; Hua, Libo; Zhang, Caiyun; Chen, Mengjie; Chen, Donghui; Dong, Yinmei; Zhang, Yingying; Li, Meng; Song, Xianmin; Chen, Huaiwen; Zheng, Hongliang

2017-02-01

To target both head and neck squamous cell carcinoma (HNSCC) cells and cancer stem cells (CSCs) by salinomycin-loaded DSPE-PEG-MTX (synthesized using DSPE-PEG2000-NH2 and methotrexate) nanomicelles (M-SAL-MTX). The characterization, antitumor activity and mechanism of M-SAL-MTX were evaluated. M-SAL-MTX showed enhanced inhibitory effect toward both HNSCC CSCs and non-CSCs compared with a single treatment of methotrexate and salinomycin. In nude mice-bearing HNSCC xenografts, M-SAL-MTX suppressed tumor growth more effectively than other controls including combination of methotrexate and salinomycin. Therefore, M-SAL-MTX may provide a strategy for treating HNSCC by targeting both HNSCC CSCs and HNSCC cells.

Salinomycin Hydroxamic Acids: Synthesis, Structure, and Biological Activity of Polyether Ionophore Hybrids.

PubMed

Borgström, Björn; Huang, Xiaoli; Chygorin, Eduard; Oredsson, Stina; Strand, Daniel

2016-06-09

The polyether ionophore salinomycin has recently gained attention due to its exceptional ability to selectively reduce the proportion of cancer stem cells within a number of cancer cell lines. Efficient single step strategies for the preparation of hydroxamic acid hybrids of this compound varying in N- and O-alkylation are presented. The parent hydroxamic acid, salinomycin-NHOH, forms both inclusion complexes and well-defined electroneutral complexes with potassium and sodium cations via 1,3-coordination by the hydroxamic acid moiety to the metal ion. A crystal structure of an cationic sodium complex with a noncoordinating anion corroborates this finding and, moreover, reveals a novel type of hydrogen bond network that stabilizes the head-to-tail conformation that encapsulates the cation analogously to the native structure. The hydroxamic acid derivatives display down to single digit micromolar activity against cancer cells but unlike salinomycin selective reduction of ALDH(+) cells, a phenotype associated with cancer stem cells was not observed. Mechanistic implications are discussed.

Salinomycin Hydroxamic Acids: Synthesis, Structure, and Biological Activity of Polyether Ionophore Hybrids

PubMed Central

2016-01-01

The polyether ionophore salinomycin has recently gained attention due to its exceptional ability to selectively reduce the proportion of cancer stem cells within a number of cancer cell lines. Efficient single step strategies for the preparation of hydroxamic acid hybrids of this compound varying in N- and O-alkylation are presented. The parent hydroxamic acid, salinomycin-NHOH, forms both inclusion complexes and well-defined electroneutral complexes with potassium and sodium cations via 1,3-coordination by the hydroxamic acid moiety to the metal ion. A crystal structure of an cationic sodium complex with a noncoordinating anion corroborates this finding and, moreover, reveals a novel type of hydrogen bond network that stabilizes the head-to-tail conformation that encapsulates the cation analogously to the native structure. The hydroxamic acid derivatives display down to single digit micromolar activity against cancer cells but unlike salinomycin selective reduction of ALDH+ cells, a phenotype associated with cancer stem cells was not observed. Mechanistic implications are discussed. PMID:27326340

[Investigation of neural stem cell-derived donor contribution in the inner ear following blastocyst injection].

PubMed

Volkenstein, S; Brors, D; Hansen, S; Mlynski, R; Dinger, T C; Müller, A M; Dazert, S

2008-03-01

Utilising the enormous proliferation and multi-lineage differentiation potentials of somatic stem cells represents a possible therapeutical strategy for diseases of non-regenerative tissues like the inner ear. In the current study, the possibility of murine neural stem cells to contribute to the developing inner ear following blastocyst injection was investigated. Fetal brain-derived neural stem cells from the embryonic day 14 cortex of male mice were isolated and expanded for four weeks in neurobasal media supplemented with bFGF and EGF. Neural stem cells of male animals were harvested, injected into blastocysts and the blastocysts were transferred into pseudo-pregnant foster animals. Each blastocyst was injected with 5-15 microspheres growing from single cell suspension from neurospheres dissociated the day before. The resulting mice were investigated six months POST PARTUM for the presence of donor cells. Brainstem evoked response audiometry (BERA) was performed in six animals. To visualize donor cells Lac-Z staining was performed on sliced cochleas of two animals. In addition, the cochleas of four female animals were isolated and genomic DNA of the entire cochlea was analyzed for donor contribution by Y-chromosome-specific PCR. All animals had normal thresholds in brainstem evoked response audiometry. The male-specific PCR product indicating the presence of male donor cells were detected in the cochleas of three of the four female animals investigated. In two animals, male donor cells were detected unilateral, in one animal bilateral. The results suggest that descendants of neural stem cells are detectable in the inner ear after injection into blastocysts and possess the ability to integrate into the developing inner ear without obvious loss in hearing function.

Multiphoton autofluorescence lifetime imaging of induced pluripotent stem cells

NASA Astrophysics Data System (ADS)

Uchugonova, Aisada

2017-06-01

The multiphoton fluorescence lifetime imaging tomograph MPTflex with its flexible 360-deg scan head, articulated arm, and tunable femtosecond laser source was employed to study induced pluripotent stem cell (iPS) cultures. Autofluorescence (AF) lifetime imaging was performed with 250-ps temporal resolution and submicron spatial resolution using time-correlated single-photon counting. The two-photon excited AF was based on the metabolic coenzymes NAD(P)H and flavin adenine dinucleotide/flavoproteins. iPS cells generated from mouse embryonic fibroblasts (MEFs) and cocultured with growth-arrested MEFs as feeder cells have been studied. Significant differences on AF lifetime signatures were identified between iPS and feeder cells as well as between their differentiating counterparts.

Maintenance and induction of murine embryonic stem cell differentiation using E-cadherin-Fc substrata without colony formation

NASA Astrophysics Data System (ADS)

Meng, Qing-Yuan; Akaike, Toshihiro

2013-03-01

Induced embryonic stem (ES) cells are expected to be promising cell resources for the observation of the cell behaviors in developmental biology as well as the implantation in cell treatments in human diseases. A recombinant E-cadherin substratum was developed as a cell recognizable substratum to maintain the ES cells' self-renewal and pluripotency at single cell level. Furthermore, the generation of various cell lineages in different germ layers, including hepatic or neural cells, was achieved on the chimeric protein layer precisely and effectively. The induction and isolation of specific cell population was carried out with the enhancing effect of other artificial extracellular matrices (ECMs) in enzyme-free process. The murine ES cell-derived cells showed highly morphological similarities and functional expressions to matured hepatocytes or neural progenitor cells.

Chemical Proteomic Approaches Targeting Cancer Stem Cells: A Review of Current Literature.

PubMed

Jung, Hye Jin

2017-01-01

Cancer stem cells (CSCs) have been proposed as central drivers of tumor initiation, progression, recurrence, and therapeutic resistance. Therefore, identifying stem-like cells within cancers and understanding their properties is crucial for the development of effective anticancer therapies. Recently, chemical proteomics has become a powerful tool to efficiently determine protein networks responsible for CSC pathophysiology and comprehensively elucidate molecular mechanisms of drug action against CSCs. This review provides an overview of major methodologies utilized in chemical proteomic approaches. In addition, recent successful chemical proteomic applications targeting CSCs are highlighted. Future direction of potential CSC research by integrating chemical genomic and proteomic data obtained from a single biological sample of CSCs are also suggested in this review. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

In situ single cell detection via microfluidic magnetic bead assay.

PubMed

Liu, Fan; Kc, Pawan; Zhang, Ge; Zhe, Jiang

2017-01-01

We present a single cell detection device based on magnetic bead assay and micro Coulter counters. This device consists of two successive micro Coulter counters, coupled with a high gradient magnetic field generated by an external magnet. The device can identify single cells in terms of the transit time difference of the cell through the two micro Coulter counters. Target cells are conjugated with magnetic beads via specific antibody and antigen binding. A target cell traveling through the two Coulter counters interacts with the magnetic field, and have a longer transit time at the 1st counter than that at the 2nd counter. In comparison, a non-target cell has no interaction with the magnetic field, and hence has nearly the same transit times through the two counters. Each cell passing through the two counters generates two consecutive voltage pulses one after the other; the pulse widths and magnitudes indicating the cell's transit times through the counters and the cell's size respectively. Thus, by measuring the pulse widths (transit times) of each cell through the two counters, each single target cell can be differentiated from non-target cells even if they have similar sizes. We experimentally proved that the target human umbilical vein endothelial cells (HUVECs) and non-target rat adipose-derived stem cells (rASCs) have significant different transit time distribution, from which we can determine the recognition regions for both cell groups quantitatively. We further demonstrated that within a mixed cell population of rASCs and HUVECs, HUVECs can be detected in situ and the measured HUVECs ratios agree well with the pre-set ratios. With the simple device structure and easy sample preparation, this method is expected to enable single cell detection in a continuous flow and can be applied to facilitate general cell detection applications such as stem cell identification and enumeration.

Single-cell DNA methylome sequencing and bioinformatic inference of epigenomic cell-state dynamics.

PubMed

Farlik, Matthias; Sheffield, Nathan C; Nuzzo, Angelo; Datlinger, Paul; Schönegger, Andreas; Klughammer, Johanna; Bock, Christoph

2015-03-03

Methods for single-cell genome and transcriptome sequencing have contributed to our understanding of cellular heterogeneity, whereas methods for single-cell epigenomics are much less established. Here, we describe a whole-genome bisulfite sequencing (WGBS) assay that enables DNA methylation mapping in very small cell populations (μWGBS) and single cells (scWGBS). Our assay is optimized for profiling many samples at low coverage, and we describe a bioinformatic method that analyzes collections of single-cell methylomes to infer cell-state dynamics. Using these technological advances, we studied epigenomic cell-state dynamics in three in vitro models of cellular differentiation and pluripotency, where we observed characteristic patterns of epigenome remodeling and cell-to-cell heterogeneity. The described method enables single-cell analysis of DNA methylation in a broad range of biological systems, including embryonic development, stem cell differentiation, and cancer. It can also be used to establish composite methylomes that account for cell-to-cell heterogeneity in complex tissue samples. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Biocompatability of carbon nanotubes with stem cells to treat CNS injuries.

PubMed

Bokara, Kiran Kumar; Kim, Jong Youl; Lee, Young Il; Yun, Kyungeun; Webster, Tom J; Lee, Jong Eun

2013-06-01

Cases reporting traumatic injuries to the brain and spinal cord are extended range of disorders that affect a large percentage of the world's population. But, there are only few effective treatments available for central nervous system (CNS) injuries because the CNS is refractory to axonal regeneration and relatively inaccessible to many pharmacological treatments. The use of stem cell therapy in regenerative medicine has been extensively examined to replace lost cells during CNS injuries. But, given the complexity of CNS injuries oxidative stress, toxic byproducts, which prevails in the microenvironment during the diseased condition, may limit the survival of the transplanted stem cells affecting tissue regeneration and even longevity. Carbon nanotubes (CNT) are a new class of nanomaterials, which have been shown to be promising in different areas of nanomedicine for the prevention, diagnosis and therapy of certain diseases, including CNS diseases. In particular, the use of CNTs as substrates/scaffolds for supporting the stem cell differentiation has been an area of active research. Single-walled and multi-walled CNT's have been increasingly used as scaffolds for neuronal growth and more recently for neural stem cell growth and differentiation. This review summarizes recent research on the application of CNT-based materials to direct the differentiation of progenitor and stem cells toward specific neurons and to enhance axon regeneration and synaptogenesis for the effective treatment of CNS injuries. Nonetheless, accumulating data support the use of CNTs as a biocompatible and permissive substrate/scaffold for neural cells and such application holds great potential in neurological research.

Biocompatability of carbon nanotubes with stem cells to treat CNS injuries

PubMed Central

Bokara, Kiran Kumar; Kim, Jong Youl; Lee, Young Il; Yun, Kyungeun; Webster, Tom J

2013-01-01

Cases reporting traumatic injuries to the brain and spinal cord are extended range of disorders that affect a large percentage of the world's population. But, there are only few effective treatments available for central nervous system (CNS) injuries because the CNS is refractory to axonal regeneration and relatively inaccessible to many pharmacological treatments. The use of stem cell therapy in regenerative medicine has been extensively examined to replace lost cells during CNS injuries. But, given the complexity of CNS injuries oxidative stress, toxic byproducts, which prevails in the microenvironment during the diseased condition, may limit the survival of the transplanted stem cells affecting tissue regeneration and even longevity. Carbon nanotubes (CNT) are a new class of nanomaterials, which have been shown to be promising in different areas of nanomedicine for the prevention, diagnosis and therapy of certain diseases, including CNS diseases. In particular, the use of CNTs as substrates/scaffolds for supporting the stem cell differentiation has been an area of active research. Single-walled and multi-walled CNT's have been increasingly used as scaffolds for neuronal growth and more recently for neural stem cell growth and differentiation. This review summarizes recent research on the application of CNT-based materials to direct the differentiation of progenitor and stem cells toward specific neurons and to enhance axon regeneration and synaptogenesis for the effective treatment of CNS injuries. Nonetheless, accumulating data support the use of CNTs as a biocompatible and permissive substrate/scaffold for neural cells and such application holds great potential in neurological research. PMID:23869255

A Common Origin for B-1a and B-2 Lymphocytes in Clonal Pre- Hematopoietic Stem Cells.

PubMed

Hadland, Brandon K; Varnum-Finney, Barbara; Mandal, Pankaj K; Rossi, Derrick J; Poulos, Michael G; Butler, Jason M; Rafii, Shahin; Yoder, Mervin C; Yoshimoto, Momoko; Bernstein, Irwin D

2017-06-06

Recent evidence points to the embryonic emergence of some tissue-resident innate immune cells, such as B-1a lymphocytes, prior to and independently of hematopoietic stem cells (HSCs). However, whether the full hematopoietic repertoire of embryonic HSCs initially includes these unique lineages of innate immune cells has been difficult to assess due to lack of clonal assays that identify and assess HSC precursor (pre-HSC) potential. Here, by combining index sorting of single embryonic hemogenic precursors with in vitro HSC maturation and transplantation assays, we analyze emerging pre-HSCs at the single-cell level, revealing their unique stage-specific properties and clonal lineage potential. Remarkably, clonal pre-HSCs detected between E9.5 and E11.5 contribute to the complete B cell repertoire, including B-1a lymphocytes, revealing a previously unappreciated common precursor for all B cell lineages at the pre-HSC stage and a second embryonic origin for B-1a lymphocytes. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Hematopoietic stem cell fate through metabolic control.

PubMed

Ito, Kyoko; Ito, Keisuke

2018-05-25

Hematopoietic stem cells (HSCs) maintain a quiescent state in the bone marrow to preserve their self-renewal capacity, but also undergo cell divisions as required. Organelles such as the mitochondria sustain cumulative damage during these cell divisions, and this damage may eventually compromise the cells' self-renewal capacity. HSC divisions result in either self-renewal or differentiation, with the balance between the two directly impacting hematopoietic homeostasis; but the heterogeneity of available HSC-enriched fractions, together with the technical challenges of observing HSC behavior, has long hindered the analysis of individual HSCs, and prevented the elucidation of this process. However, recent advances in genetic models, metabolomics analyses and single-cell approaches have revealed the contributions made to HSC self-renewal by metabolic cues, mitochondrial biogenesis, and autophagy/mitophagy, which have highlighted mitochondrial quality as a key control factor in the equilibrium of HSCs. A deeper understanding of precisely how specific modes of metabolism control HSC fate at the single cell level is therefore not only of great biological interest, but will have clear clinical implications for the development of therapies for hematological disease. Copyright © 2018. Published by Elsevier Inc.

Generation of Functional Blood Vessels from a Single c-kit+ Adult Vascular Endothelial Stem Cell

PubMed Central

Fang, Shentong; Wei, Jing; Pentinmikko, Nalle; Leinonen, Hannele; Salven, Petri

2012-01-01

In adults, the growth of blood vessels, a process known as angiogenesis, is essential for organ growth and repair. In many disorders including cancer, angiogenesis becomes excessive. The cellular origin of new vascular endothelial cells (ECs) during blood vessel growth in angiogenic situations has remained unknown. Here, we provide evidence for adult vascular endothelial stem cells (VESCs) that reside in the blood vessel wall endothelium. VESCs constitute a small subpopulation within CD117+ (c-kit+) ECs capable of undergoing clonal expansion while other ECs have a very limited proliferative capacity. Isolated VESCs can produce tens of millions of endothelial daughter cells in vitro. A single transplanted c-kit-expressing VESC by the phenotype lin−CD31+CD105+Sca1+CD117+ can generate in vivo functional blood vessels that connect to host circulation. VESCs also have long-term self-renewal capacity, a defining functional property of adult stem cells. To provide functional verification on the role of c-kit in VESCs, we show that a genetic deficit in endothelial c-kit expression markedly decreases total colony-forming VESCs. In vivo, c-kit expression deficit resulted in impaired EC proliferation and angiogenesis and retardation of tumor growth. Isolated VESCs could be used in cell-based therapies for cardiovascular repair to restore tissue vascularization after ischemic events. VESCs also provide a novel cellular target to block pathological angiogenesis and cancer growth. PMID:23091420

Identification of multipotent mesenchymal stromal cells in the reactive stroma of a prostate cancer xenograft by side population analysis

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

Santamaria-Martinez, Albert; Universitat de Barcelona, Barcelona; Barquinero, Jordi

2009-10-15

Cancer stem cells are a distinct cellular population that is believed to be responsible for tumor initiation and maintenance. Recent data suggest that solid tumors also contain another type of stem cells, the mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs), which contribute to the formation of tumor-associated stroma. The Hoechst 33342 efflux assay has proved useful to identify a rare cellular fraction, named Side Population (SP), enriched in cells with stem-like properties. Using this assay, we identified SP cells in a prostate cancer xenograft containing human prostate cancer cells and mouse stromal cells. The SP isolation, subculture andmore » sequential sorting allowed the generation of single-cell-derived clones of murine origin that were recognized as MSC by their morphology, plastic adherence, proliferative potential, adipogenic and osteogenic differentiation ability and immunophenotype (CD45{sup -}, CD81{sup +} and Sca-1{sup +}). We also demonstrated that SP clonal cells secrete transforming growth factor {beta}1 (TGF-{beta}1) and that their inhibition reduces proliferation and accelerates differentiation. These results reveal the existence of SP cells in the stroma of a cancer xenograft, and provide evidence supporting their MSC nature and the role of TGF-{beta}1 in maintaining their proliferation and undifferentiated status. Our data also reveal the usefulness of the SP assay to identify and isolate MSC cells from carcinomas.« less

Identification of multipotent mesenchymal stromal cells in the reactive stroma of a prostate cancer xenograft by side population analysis.

PubMed

Santamaria-Martínez, Albert; Barquinero, Jordi; Barbosa-Desongles, Anna; Hurtado, Antoni; Pinós, Tomàs; Seoane, Joan; Poupon, Marie-France; Morote, Joan; Reventós, Jaume; Munell, Francina

2009-10-15

Cancer stem cells are a distinct cellular population that is believed to be responsible for tumor initiation and maintenance. Recent data suggest that solid tumors also contain another type of stem cells, the mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs), which contribute to the formation of tumor-associated stroma. The Hoechst 33342 efflux assay has proved useful to identify a rare cellular fraction, named Side Population (SP), enriched in cells with stem-like properties. Using this assay, we identified SP cells in a prostate cancer xenograft containing human prostate cancer cells and mouse stromal cells. The SP isolation, subculture and sequential sorting allowed the generation of single-cell-derived clones of murine origin that were recognized as MSC by their morphology, plastic adherence, proliferative potential, adipogenic and osteogenic differentiation ability and immunophenotype (CD45(-), CD81(+) and Sca-1(+)). We also demonstrated that SP clonal cells secrete transforming growth factor beta1 (TGF-beta1) and that their inhibition reduces proliferation and accelerates differentiation. These results reveal the existence of SP cells in the stroma of a cancer xenograft, and provide evidence supporting their MSC nature and the role of TGF-beta1 in maintaining their proliferation and undifferentiated status. Our data also reveal the usefulness of the SP assay to identify and isolate MSC cells from carcinomas.

TOOTH (The Open study Of dental pulp stem cell Therapy in Humans): Study protocol for evaluating safety and feasibility of autologous human adult dental pulp stem cell therapy in patients with chronic disability after stroke.

PubMed

Nagpal, Anjali; Kremer, Karlea L; Hamilton-Bruce, Monica A; Kaidonis, Xenia; Milton, Austin G; Levi, Christopher; Shi, Songtao; Carey, Leeanne; Hillier, Susan; Rose, Miranda; Zacest, Andrew; Takhar, Parabjit; Koblar, Simon A

2016-07-01

Stroke represents a significant global disease burden. As of 2015, there is no chemical or biological therapy proven to actively enhance neurological recovery during the chronic phase post-stroke. Globally, cell-based therapy in stroke is at the stage of clinical translation and may improve neurological function through various mechanisms such as neural replacement, neuroprotection, angiogenesis, immuno-modulation, and neuroplasticity. Preclinical evidence in a rodent model of middle cerebral artery ischemic stroke as reported in four independent studies indicates improvement in neurobehavioral function with adult human dental pulp stem cell therapy. Human adult dental pulp stem cells present an exciting potential therapeutic option for improving post-stroke disability. TOOTH (The Open study Of dental pulp stem cell Therapy in Humans) will investigate the use of autologous stem cell therapy for stroke survivors with chronic disability, with the following objectives: (a) determine the maximum tolerable dose of autologous dental pulp stem cell therapy; (b) define that dental pulp stem cell therapy at the maximum tolerable dose is safe and feasible in chronic stroke; and (c) estimate the parameters of efficacy required to design a future Phase 2/3 clinical trial. TOOTH is a Phase 1, open-label, single-blinded clinical trial with a pragmatic design that comprises three stages: Stage 1 will involve the selection of 27 participants with middle cerebral artery ischemic stroke and the commencement of autologous dental pulp stem cell isolation, growth, and testing in sequential cohorts (n = 3). Stage 2 will involve the transplantation of dental pulp stem cell in each cohort of participants with an ascending dose and subsequent observation for a 6-month period for any dental pulp stem cell-related adverse events. Stage 3 will investigate the neurosurgical intervention of the maximum tolerable dose of autologous dental pulp stem cell followed by 9 weeks of intensive task-specific rehabilitation. Advanced magnetic resonance and positron emission tomography neuro-imaging, and clinical assessment will be employed to probe any change afforded by stem cell therapy in combination with rehabilitation. Nine participants will step-wise progress in Stage 2 to a dose of up to 10 million dental pulp stem cell, employing a cumulative 3 + 3 statistical design with low starting stem cell dose and subsequent dose escalation, assuming that an acceptable probability of dose-limiting complications is between 1 in 6 (17%) and 1 in 3 (33%) of patients. In Stage 3, another 18 participants will receive an intracranial injection with the maximum tolerable dose of dental pulp stem cell. The primary outcomes to be measured are safety and feasibility of intracranial administration of autologous human adult DPSC in patients with chronic stroke and determination of the maximum tolerable dose in human subjects. Secondary outcomes include estimation of the measures of effectiveness required to design a future Phase 2/3 clinical trial. © 2016 World Stroke Organization.

Ovarian cancer stem cells.

PubMed

Zeimet, A G; Reimer, D; Sopper, S; Boesch, M; Martowicz, A; Roessler, J; Wiedemair, A M; Rumpold, H; Untergasser, G; Concin, N; Hofstetter, G; Muller-Holzner, E; Fiegl, H; Marth, C; Wolf, D; Pesta, M; Hatina, J

2012-01-01

Because of its semi-solid character in dissemination and growth, advanced ovarian cancer with its hundreds of peritoneal tumor nodules and plaques appears to be an excellent in vivo model for studying the cancer stem cell hypothesis. The most important obstacle, however, is to adequately define and isolate these tumor-initiating cells endowed with the properties of anoikis-resistance and unlimited self-renewal. Until now, no universal single marker or marker constellation has been found to faithfully isolate (ovarian) cancer stem cells. As these multipotent cells are known to possess highly elaborated efflux systems for cytotoxic agents, these pump systems have been exploited to outline putative stem cells as a side-population (SP) via dye exclusion analysis. Furthermore, the cells in question have been isolated via flow cytometry on the basis of cell surface markers thought to be characteristic for stem cells.In the Vienna variant of the ovarian cancer cell line A2780 a proof-of-principle model with both a stable SP and a stable ALDH1A1+ cell population was established. Double staining clearly revealed that both cell fractions were not identical. Of note, A2780V cells were negative for expression of surface markers CD44 and CD117 (c-kit). When cultured on monolayers of healthy human mesothelial cells, green-fluorescence-protein (GFP)-transfected SP of A2780V exhibited spheroid-formation, whereas non-side-population (NSP) developed a spare monolayer growing over the healthy mesothelium. Furthermore, A2780V SP was found to be partially resistant to platinum. However, this resistance could not be explained by over-expression of the "excision repair cross-complementation group 1" (ERCC1) gene, which is essentially involved in the repair of platinated DNA damage. ERCC1 was, nonetheless, over-expressed in A2780V cells grown as spheres under stem cell-selective conditions as compared to adherent monolayers cultured under differentiating conditions. The same was true for the primary ovarian cancer cells B-57.In summary our investigations indicate that even in multi-passaged cancer cell lines hierarchic government of growth and differentiation is conserved and that the key cancer stem cell population may be composed of small overlapping cell fractions defined by various arbitrary markers.

Anti-Inflammatory Effects of Adult Stem Cells in Sustained Lung Injury: A Comparative Study

PubMed Central

Moodley, Yuben; Vaghjiani, Vijesh; Chan, James; Baltic, Svetlana; Ryan, Marisa; Tchongue, Jorge; Samuel, Chrishan S.; Murthi, Padma; Parolini, Ornella; Manuelpillai, Ursula

2013-01-01

Lung diseases are a major cause of global morbidity and mortality that are treated with limited efficacy. Recently stem cell therapies have been shown to effectively treat animal models of lung disease. However, there are limitations to the translation of these cell therapies to clinical disease. Studies have shown that delayed treatment of animal models does not improve outcomes and that the models do not reflect the repeated injury that is present in most lung diseases. We tested the efficacy of amnion mesenchymal stem cells (AM-MSC), bone marrow MSC (BM-MSC) and human amniotic epithelial cells (hAEC) in C57BL/6 mice using a repeat dose bleomycin-induced model of lung injury that better reflects the repeat injury seen in lung diseases. The dual bleomycin dose led to significantly higher levels of inflammation and fibrosis in the mouse lung compared to a single bleomycin dose. Intravenously infused stem cells were present in the lung in similar numbers at days 7 and 21 post cell injection. In addition, stem cell injection resulted in a significant decrease in inflammatory cell infiltrate and a reduction in IL-1 (AM-MSC), IL-6 (AM-MSC, BM-MSC, hAEC) and TNF-α (AM-MSC). The only trophic factor tested that increased following stem cell injection was IL-1RA (AM-MSC). IL-1RA levels may be modulated by GM-CSF produced by AM-MSC. Furthermore, only AM-MSC reduced collagen deposition and increased MMP-9 activity in the lung although there was a reduction of the pro-fibrogenic cytokine TGF-β following BM-MSC, AM-MSC and hAEC treatment. Therefore, AM-MSC may be more effective in reducing injury following delayed injection in the setting of repeated lung injury. PMID:23936322

In situ single cell detection via microfluidic magnetic bead assay

PubMed Central

KC, Pawan; Zhang, Ge; Zhe, Jiang

2017-01-01

We present a single cell detection device based on magnetic bead assay and micro Coulter counters. This device consists of two successive micro Coulter counters, coupled with a high gradient magnetic field generated by an external magnet. The device can identify single cells in terms of the transit time difference of the cell through the two micro Coulter counters. Target cells are conjugated with magnetic beads via specific antibody and antigen binding. A target cell traveling through the two Coulter counters interacts with the magnetic field, and have a longer transit time at the 1st counter than that at the 2nd counter. In comparison, a non-target cell has no interaction with the magnetic field, and hence has nearly the same transit times through the two counters. Each cell passing through the two counters generates two consecutive voltage pulses one after the other; the pulse widths and magnitudes indicating the cell’s transit times through the counters and the cell’s size respectively. Thus, by measuring the pulse widths (transit times) of each cell through the two counters, each single target cell can be differentiated from non-target cells even if they have similar sizes. We experimentally proved that the target human umbilical vein endothelial cells (HUVECs) and non-target rat adipose-derived stem cells (rASCs) have significant different transit time distribution, from which we can determine the recognition regions for both cell groups quantitatively. We further demonstrated that within a mixed cell population of rASCs and HUVECs, HUVECs can be detected in situ and the measured HUVECs ratios agree well with the pre-set ratios. With the simple device structure and easy sample preparation, this method is expected to enable single cell detection in a continuous flow and can be applied to facilitate general cell detection applications such as stem cell identification and enumeration. PMID:28222140

Establishment of a Novel Lingual Organoid Culture System: Generation of Organoids Having Mature Keratinized Epithelium from Adult Epithelial Stem Cells

NASA Astrophysics Data System (ADS)

Hisha, Hiroko; Tanaka, Toshihiro; Kanno, Shohei; Tokuyama, Yoko; Komai, Yoshihiro; Ohe, Shuichi; Yanai, Hirotsugu; Omachi, Taichi; Ueno, Hiroo

2013-11-01

Despite the strong need for the establishment of a lingual epithelial cell culture system, a simple and convenient culture method has not yet been established. Here, we report the establishment of a novel lingual epithelium organoid culture system using a three-dimensional matrix and growth factors. Histological analyses showed that the generated organoids had both a stratified squamous epithelial cell layer and a stratum corneum. Very recently, we showed via a multicolor lineage tracing method that Bmi1-positive stem cells exist at the base of the epithelial basal layer in the interpapillary pit. Using our new culture system, we found that organoids could be generated by single Bmi1-positive stem cells and that in the established organoids, multiple Bmi1-positive stem cells were generated at the outermost layer. Moreover, we observed that organoids harvested at an early point in culture could be engrafted and maturate in the tongue of recipient mice and that the organoids generated from carcinogen-treated mice had an abnormal morphology. Thus, this culture system presents valuable settings for studying not only the regulatory mechanisms of lingual epithelium but also lingual regeneration and carcinogenesis.

Granulocyte-colony stimulating factor administration among hemoglobin S trait donors: A single center experience from the Eastern Mediterranean region.

PubMed

Gereklioglu, Cigdem; Asma, Suheyl; Korur, Aslı; Tepebaşı, Songul; Aytan, Pelin; Yeral, Mahmut; Kozanoglu, Ilknur; Boga, Can; Ozdogu, Hakan

2018-02-01

Assessment of Hemoglobin S trait donors has gained importance together with the increased allogeneic peripheral stem cell transplant activity for sickle cell disease in the regions where the disease is prevalent. Outcomes of Granulocyte-Colony Stimulating Factor (G-CSF) administration are obscure for hemoglobin S trait donors. This study aims at investigating the incidence of hemoglobin S carrier status and outcomes of G-CSF administration among donors who live in Eastern Mediterranean region. The cross-sectional, single-center cohort study was performed with 147 donors between January 2013 and March 2017. Prevalence of hemoglobin S trait was estimated and subjects with or without Hemogobin S trait were compared with regard to stem cell characteristics, early and late clinical outcomes after G-CSF administration. Eleven out of 147 donors (7.48%) were found as hemoglobin S trait. G-CSF administration was successfully completed and yielded good harvesting results in hemoglobin S trait donors. No statistically significant difference was found between groups with regard to early and late side effects, stem cell characteristics. Blood pressures and QTc values were within normal ranges in both groups. Groups were similar with regard to CD34 values. G-CSF seems safe in hemoglobin S trait donors. Their being eligible as donors would increase the chance of the patients for allogeneic stem cell transplantation in high prevalence regions. Further studies are required to reveal the safety profile of G-SCF in hemoglobin S carriers in different regions. © 2017 Wiley Periodicals, Inc.

Spectroscopic signature of mouse embryonic stem cell-derived hepatocytes using synchrotron Fourier transform infrared microspectroscopy

NASA Astrophysics Data System (ADS)

Thumanu, Kanjana; Tanthanuch, Waraporn; Ye, Danna; Sangmalee, Anawat; Lorthongpanich, Chanchao; Parnpai, Rangsun; Heraud, Philip

2011-05-01

Stem cell-based therapy for liver regeneration has been proposed to overcome the persistent shortage in the supply of suitable donor organs. A requirement for this to succeed is to find a rapid method to detect functional hepatocytes, differentiated from embryonic stem cells. We propose Fourier transform infrared (FTIR) microspectroscopy as a versatile method to identify the early and last stages of the differentiation process leading to the formation of hepatocytes. Using synchrotron-FTIR microspectroscopy, the means of identifying hepatocytes at the single-cell level is possible and explored. Principal component analysis and subsequent partial least-squares (PLS) discriminant analysis is applied to distinguish endoderm induction from hepatic progenitor cells and matured hepatocyte-like cells. The data are well modeled by PLS with endoderm induction, hepatic progenitor cells, and mature hepatocyte-like cells able to be discriminated with very high sensitivity and specificity. This method provides a practical tool to monitor endoderm induction and has the potential to be applied for quality control of cell differentiation leading to hepatocyte formation.

High-dose therapy and autologous hematopoietic stem cell transplant in T-cell lymphoma: a single center experience.

PubMed

Cairoli, Anne; Ketterer, Nicolas; Barelli, Stefano; Duchosal, Michel A

2014-08-01

We report here the long-term outcome of autologous stem cell transplant in peripheral T-cell lymphoma (PTCL). Forty-three consecutive patients with PTCL diagnosed between 2000 and 2011 were treated with high-dose chemotherapy (HDCT) and autologous stem cell transplant (ASCT) in our center. Diagnoses included PTCL-not otherwise specified (n = 19), anaplastic large cell lymphoma (n = 11), angioimmunoblastic T-cell lymphoma (n = 5), enteropathy-associated T-cell lymphoma (n = 5) and other rare subtypes (n = 3). Thirty-six patients with a median age of 50 years (range 22-65) were transplanted in first response and seven after relapse. After a median follow-up of 63 months, estimated overall survival at 12 years was 40%, progression-free survival at 12 years was 34% and event-free survival at 12 years was 30%. On univariate analysis, age less than 50 years and no B symptoms at diagnosis were significantly associated with prolonged overall and progression-free-survival. HDCT/ASCT for peripheral T-cell lymphoma can lead to long-term survival for patients responding to induction chemotherapy.

Temperature variation and distribution of living cells within tree stems: implications for stem respiration modeling and scale-up.

PubMed

Stockfors, J

2000-09-01

Few studies have examined variation in respiration rates within trees, and even fewer studies have focused on variation caused by within-stem temperature differences. In this study, stem temperatures at 40 positions in the stem of one 30-year-old Norway spruce (Picea abies (L.) Karst.) were measured during 40 days between July 1994 and June 1995. The temperature data were used to simulate variations in respiration rate within the stem. The simulations assumed that the temperature-respiration relationship was constant (Q10 = 2) for all days and all stem positions. Total respiration for the whole stem was calculated by interpolating the temperature between the thermocouples and integrating the respiration rates in three dimensions. Total respiration rate of the stem was then compared to respiration rate scaled up from horizontal planes at the thermocouple heights (40, 140, 240 and 340 cm) on a surface area and on a sapwood volume basis. Simulations were made for three distributions of living cells in the stems: one with a constant 5% fraction of living cells, disregarding depth into the stem; one with a living cell fraction decreasing linearly with depth into the stem; and one with an exponentially decreasing fraction of living cells. Mean temperature variation within the stem was 3.7 degrees C, and was more than 10 degrees C for 8% of the time. The maximum measured temperature difference was 21.5 degrees C. The corresponding mean variation in respiration was 35% and was more than 50% for 24% of the time. Scaling up respiration rates from different heights between 40 and 240 cm to the whole stem produced an error of 2 to 58% for the whole year. For a single sunny day, the error was between 2 and 72%. Thus, within-stem variations in temperature may significantly affect the accuracy of scaling respiration data obtained from small samples to whole trees. A careful choice of chamber position and basis for scaling is necessary to minimize errors from variation in temperature.

A Randomized Phase IV Control Trial of Single High Dose Oral Vitamin D3 in Pediatric Patients Undergoing HSCT

ClinicalTrials.gov

2017-06-01

Vitamin D Deficiency; Stem Cell Transplant Complications; Pediatric Cancer; Blood Disorder; Pediatric Acute Myeloid Leukemia; Pediatric Acute Lymphoid Leukemia; Myelodysplastic Syndromes; Sickle Cell Anemia in Children; Aplastic Anemia; Thalassemia in Children

FANCA knockout in human embryonic stem cells causes a severe growth disadvantage.

PubMed

Vanuytsel, Kim; Cai, Qing; Nair, Nisha; Khurana, Satish; Shetty, Swati; Vermeesch, Joris R; Ordovas, Laura; Verfaillie, Catherine M

2014-09-01

Fanconi anemia (FA) is an autosomal recessive disorder characterized by progressive bone marrow failure (BMF) during childhood, aside from numerous congenital abnormalities. FA mouse models have been generated; however, they do not fully mimic the hematopoietic phenotype. As there is mounting evidence that the hematopoietic impairment starts already in utero, a human pluripotent stem cell model would constitute a more appropriate system to investigate the mechanisms underlying BMF in FA and its developmental basis. Using zinc finger nuclease (ZFN) technology, we have created a knockout of FANCA in human embryonic stem cells (hESC). We introduced a selection cassette into exon 2 thereby disrupting the FANCA coding sequence and found that whereas mono-allelically targeted cells retain an unaltered proliferation potential, disruption of the second allele causes a severe growth disadvantage. As a result, heterogeneous cultures arise due to the presence of cells still carrying an unaffected FANCA allele, quickly outgrowing the knockout cells. When pure cultures of FANCA knockout hESC are pursued either through selection or single cell cloning, this rapidly results in growth arrest and such cultures cannot be maintained. These data highlight the importance of a functional FA pathway at the pluripotent stem cell stage. Copyright © 2014. Published by Elsevier B.V.

Imaging mesenchymal stem cells containing single wall nanotube nanoprobes in a 3D scaffold using photo-thermal optical coherence tomography

NASA Astrophysics Data System (ADS)

Connolly, Emma; Subhash, Hrebesh M.; Leahy, Martin; Rooney, Niall; Barry, Frank; Murphy, Mary; Barron, Valerie

2014-02-01

Despite the fact, that a range of clinically viable imaging modalities, such as magnetic resonance imaging (MRI), computed tomography (CT), photo emission tomography (PET), ultrasound and bioluminescence imaging are being optimised to track cells in vivo, many of these techniques are subject to limitations such as the levels of contrast agent required, toxic effects of radiotracers, photo attenuation of tissue and backscatter. With the advent of nanotechnology, nanoprobes are leading the charge to overcome these limitations. In particular, single wall nanotubes (SWNT) have been shown to be taken up by cells and as such are effective nanoprobes for cell imaging. Consequently, the main aim of this research is to employ mesenchymal stem cells (MSC) containing SWNT nanoprobes to image cell distribution in a 3D scaffold for cartilage repair. To this end, MSC were cultured in the presence of 32μg/ml SWNT in cell culture medium (αMEM, 10% FBS, 1% penicillin/streptomycin) for 24 hours. Upon confirmation of cell viability, the MSC containing SWNT were encapsulated in hyaluronic acid gels and loaded on polylactic acid polycaprolactone scaffolds. After 28 days in complete chondrogenic medium, with medium changes every 2 days, chondrogenesis was confirmed by the presence of glycosaminoglycan. Moreover, using photothermal optical coherence tomography (PT-OCT), the cells were seen to be distributed through the scaffold with high resolution. In summary, these data reveal that MSC containing SWNT nanoprobes in combination with PT-OCT offer an exciting opportunity for stem cell tracking in vitro for assessing seeding scaffolds and in vivo for determining biodistribution.

Hydrogels and Cell Based Therapies in Spinal Cord Injury Regeneration

PubMed Central

Assunção-Silva, Rita C.; Gomes, Eduardo D.; Silva, Nuno A.; Salgado, António J.

2015-01-01

Spinal cord injury (SCI) is a central nervous system- (CNS-) related disorder for which there is yet no successful treatment. Within the past several years, cell-based therapies have been explored for SCI repair, including the use of pluripotent human stem cells, and a number of adult-derived stem and mature cells such as mesenchymal stem cells, olfactory ensheathing cells, and Schwann cells. Although promising, cell transplantation is often overturned by the poor cell survival in the treatment of spinal cord injuries. Alternatively, the therapeutic role of different cells has been used in tissue engineering approaches by engrafting cells with biomaterials. The latter have the advantages of physically mimicking the CNS tissue, while promoting a more permissive environment for cell survival, growth, and differentiation. The roles of both cell- and biomaterial-based therapies as single therapeutic approaches for SCI repair will be discussed in this review. Moreover, as the multifactorial inhibitory environment of a SCI suggests that combinatorial approaches would be more effective, the importance of using biomaterials as cell carriers will be herein highlighted, as well as the recent advances and achievements of these promising tools for neural tissue regeneration. PMID:26124844

Eph receptor interclass cooperation is required for the regulation of cell proliferation

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

Jurek, Aleksandra; Genander, Maria; Kundu, Parag

2016-10-15

Cancer often arises by the constitutive activation of mitogenic pathways by mutations in stem cells. Eph receptors are unusual in that although they regulate the proliferation of stem/progenitor cells in many adult organs, they typically fail to transform cells. Multiple ephrins and Eph receptors are often co-expressed and are thought to be redundant, but we here describe an unexpected dichotomy with two homologous ligands, ephrin-B1 and ephrin-B2, regulating specifically migration or proliferation in the intestinal stem cell niche. We demonstrate that the combined activity of two different coexpressed Eph receptors of the A and B class assembled into common signalingmore » clusters in response to ephrin-B2 is required for mitogenic signaling. The requirement of two different Eph receptors to convey mitogenic signals identifies a new type of cooperation within this receptor family and helps explain why constitutive activation of a single receptor fails to transform cells. - Highlights: • We demonstrate that ephrin-B1 and ephrin-B2 have largely non-overlapping functions in the intestinal stem cell niche. • Ephrin-B1 regulates cell positioning and ephrin-B2 regulates cell proliferation in the intestinal stem cell niche. • EphA4/B2 receptor cooperation in response to ephrin-B2 binding is obligatory to convey mitogenic signals in the intestine. • EphA4 facilitates EphB2 phosphorylation in response to ephrin-B2 in SW480 adenocarcinoma cells. • Ephrin-B1 and ephrin-B2 induce phosphorylation and degradation of the EphB2 receptor with different kinetics.« less

NSC30049 inhibits Chk1 pathway in 5-FU-resistant CRC bulk and stem cell populations.

PubMed

Narayan, Satya; Jaiswal, Aruna S; Sharma, Ritika; Nawab, Akbar; Duckworth, Lizette Vila; Law, Brian K; Zajac-Kaye, Maria; George, Thomas J; Sharma, Jay; Sharma, Arun K; Hromas, Robert A

2017-08-22

The 5-fluorouracil (5-FU) treatment induces DNA damage and stalling of DNA replication forks. These stalled replication forks then collapse to form one sided double-strand breaks, leading to apoptosis. However, colorectal cancer (CRC) stem cells rapidly repair the stalled/collapsed replication forks and overcome treatment effects. Recent evidence suggests a critical role of checkpoint kinase 1 (Chk1) in preventing the replicative stress. Therefore, Chk1 kinase has been a target for developing mono or combination therapeutic agents. In the present study, we have identified a novel orphan molecule NSC30049 (NSC49L) that is effective alone, and in combination potentiates 5-FU-mediated growth inhibition of CRC heterogeneous bulk and FOLFOX-resistant cell lines in culture with minimal effect on normal colonic epithelial cells. It also inhibits the sphere forming activity of CRC stem cells, and decreases the expression levels of mRNAs of CRC stem cell marker genes. Results showed that NSC49L induces 5-FU-mediated S-phase cell cycle arrest due to increased load of DNA damage and increased γ-H2AX staining as a mechanism of cytotoxicity. The pharmacokinetic analysis showed a higher bioavailability of this compound, however, with a short plasma half-life. The drug is highly tolerated by animals with no pathological aberrations. Furthermore, NSC49L showed very potent activity in a HDTX model of CRC stem cell tumors either alone or in combination with 5-FU. Thus, NSC49L as a single agent or combined with 5-FU can be developed as a therapeutic agent by targeting the Chk1 pathway in 5-FU-resistant CRC heterogeneous bulk and CRC stem cell populations.

Modulation of Ocular Inflammation by Mesenchymal Stem Cells

DTIC Science & Technology

2017-03-01

mature myeloid cells in 64 host defense and resolution of inflammation, excessive innate immune response can have 65 deleterious effects on tissue...that MSCs can regulate 69 functions of mature innate immune cells , including polarization of inflammatory macrophages 70 into an anti-inflammatory... cells 191 As immune cells are primarily developed in lymphoid organs, single cell suspensions from bone 192 marrow, spleen, and submandibular lymph

Nonpermissiveness for mouse embryonic stem (ES) cell derivation circumvented by a single backcross to 129/Sv strain: establishment of ES cell lines bearing the Omd conditional lethal mutation.

PubMed

Kress, C; Vandormael-Pournin, S; Baldacci, P; Cohen-Tannoudji, M; Babinet, C

1998-12-01

The inbred mouse strain DDK carries a conditional early embryonic lethal mutation that is manifested when DDK females are crossed to males of other inbred strains but not in the corresponding reciprocal crosses. It has been shown that embryonic lethality could be assigned to a single genetic locus called Ovum mutant (Om), on Chromosome (Chr) 11 near Syca 1. In the course of our study of the molecular mechanisms underlying the embryonic lethality, we were interested in deriving an embryonic stem cell bearing the Om mutation in the homozygous state (Omd/Omd). However, it turned out that DDK is nonpermissive for ES cell establishment, with a standard protocol. Here we show that permissiveness could be obtained using Omd/Omd blastocysts with a 75% 129/Sv and 25% DDK genetic background. Several germline-competent Omd/Omd ES cell lines have been derived from blastocysts of this genotype. Such a scenario could be extended to the generation of ES cell lines bearing any mutation present in an otherwise nonpermissive mouse strain.

Establishing a human pancreatic stem cell line and transplanting induced pancreatic islets to reverse experimental diabetes in rats.

PubMed

Xiao, Mei; An, LiLong; Yang, XueYi; Ge, Xin; Qiao, Hai; Zhao, Ting; Ma, XiaoFei; Fan, JingZhuang; Zhu, MengYang; Dou, ZhongYing

2008-09-01

The major obstacle in using pancreatic islet transplantation to cure type I and some type II diabetes is the shortage of the donors. One of ways to overcome such obstacle is to isolate and clone pancreatic stem cells as "seed cells" and induce their differentiation into functional islets as an abundant transplantation source. In this study, a monoclonal human pancreatic stem cell (mhPSC) line was obtained from abortive fetal pancreatic tissues. Pancreatic tissues were taken from abortive fetus by sterile procedures, and digested into single cells and cell clusters with 0.1% type IV collagenase. Cultured in modified glucose-low DMEM with 10% fetal bovine serum (FBS), these single cells and cell clusters adhered to culture dishes, and then primary epidermal-like pancreatic stem cells started to clone. After digesting with 0.25% trypsin and 0.04% EDTA, fibroblasts and other cells were gradually eliminated and epithelioid pancreatic stem cells were gradually purified during generations. Using clone-ring selection, the mhPSCs were obtained. After addition of 10 ng/mL epidermal growth factor (EGF) in cell culture medium, the mhPSCs quickly grew and formed a gravelstone-like monolayer. Continuously proliferated, a mhPSC line, which was derived from a male abortive fetus of 4 months old, has been passed through 50 generations. More than 1 x 10(9) mhPSCs were cryo-preserved in liquid nitrogen. Karyotype analysis showed that the chromosome set of the mhPSC line was normal diploid. Immunocytochemistry results demonstrated that the mhPSC line was positive for the pdx1, glucagon, nestin and CK19, and negative for the insulin, CD34, CD44 and CD45 protein expression. RT-PCR revealed further that the mhPSCs expressed transcription factors of the pdx1, glucagon, nestin and CK19. Also, in vitro induced with beta-mercaptoethanol, the mhPSCs differentiated into nerve cells that expressed the NF protein. Induced with nicotinamide, the mhPSCs differentiated into functional islet-like clusters, as identified by dithizone staining, which expressed the transcription factor of the insulin and secreted the insulin and C-peptide. Furthermore, the transplantation of mhPSCs-induced pancreatic islets into the subcapsular region of the kidney in streptozotocin-induced diabetic rats could reduce blood glucose levels and prolong the life time.

Acute and Fractionated Exposure to High-LET 56Fe HZE-Particle Radiation Both Result in Similar Long-Term Deficits in Adult Hippocampal Neurogenesis

PubMed Central

Rivera, Phillip D.; Shih, Hung-Ying; LeBlanc, Junie A.; Cole, Mara G.; Amaral, Wellington Z.; Mukherjee, Shibani; Zhang, Shichuan; Lucero, Melanie J.; DeCarolis, Nathan A.; Chen, Benjamin P. C.; Eisch, Amelia J.

2014-01-01

Astronauts on multi-year interplanetary missions will be exposed to a low, chronic dose of high-energy, high-charge particles. Studies in rodents show acute, nonfractionated exposure to these particles causes brain changes such as fewer adult-generated hippocampal neurons and stem cells that may be detrimental to cognition and mood regulation and thus compromise mission success. However, the influence of a low, chronic dose of these particles on neurogenesis and stem cells is unknown. To examine the influence of galactic cosmic radiation on neurogenesis, adult-generated stem and progenitor cells in Nestin-CreERT2/R26R-YFP transgenic mice were inducibly labeled to allow fate tracking. Mice were then sham exposed or given one acute 100 cGy 56Fe-particle exposure or five fractionated 20 cGy 56Fe-particle exposures. Adult-generated hippocampal neurons and stem cells were quantified 24 h or 3 months later. Both acute and fractionated exposure decreased the amount of proliferating cells and immature neurons relative to sham exposure. Unexpectedly, neither acute nor fractionated exposure decreased the number of adult neural stem cells relative to sham expsoure. Our findings show that single and fractionated exposures of 56Fe-particle irradiation are similarly detrimental to adult-generated neurons. Implications for future missions and ground-based studies in space radiation are discussed. PMID:24320054

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

PubMed

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

2018-06-07

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

Self-assembled adult adipose-derived stem cell spheroids combined with biomaterials promote wound healing in a rat skin repair model.

PubMed

Hsu, Shan-Hui; Hsieh, Pai-Shan

2015-01-01

Adult adipose-derived stem cells (ASCs) are a type of multipotent mesenchymal stem cells (MSCs) with easy availability and serve as a potential cell source for cell-based therapy. Three-dimensional MSC spheroids may be derived from the self-assembly of individual MSCs grown on certain polymer membranes. In this study, we demonstrated that the self-assembled ASC spheroids on chitosan-hyaluronan membranes expressed more cytokine genes (fibroblast growth factor 1, vascular endothelial growth factor, and chemokine [C-C motif] ligand 2) as well as migration-associated genes (chemokine [C-X-C motif] receptor type 4 and matrix metalloprotease 1) compared with ASC dispersed single cells grown on culture dish. To evaluate the in vivo effects of these spheroids, we applied ASC single cells and ASC spheroids in a designed rat skin repair model. Wounds of 15 × 15 mm were created on rat dorsal skin, where ASCs were administered and covered with hyaluronan gel/chitosan sponge to maintain a moist environment. Results showed that skin wounds treated with ASC spheroids had faster wound closure and a significantly higher ratio of angiogenesis. Tracking of fluorescently labeled ASCs showed close localization of ASC spheroids to microvessels, suggesting enhanced angiogenesis through paracrine effects. Based on the in vitro and in vivo results, the self-assembled ASC spheroids may be a promising cellular source for skin tissue engineering and wound regeneration. © 2014 by the Wound Healing Society.

Specificity and Heterogeneity of Terahertz Radiation Effect on Gene Expression in Mouse Mesenchymal Stem Cells

DOE PAGES

Alexandrov, Boian S.; Phipps, M. Lisa; Alexandrov, Ludmil B.; ...

2013-01-31

In this paper, we report that terahertz (THz) irradiation of mouse mesenchymal stem cells (mMSCs) with a single-frequency (SF) 2.52 THz laser or pulsed broadband (centered at 10 THz) source results in irradiation specific heterogenic changes in gene expression. The THz effect depends on irradiation parameters such as the duration and type of THz source, and on the degree of stem cell differentiation. Our microarray survey and RT-PCR experiments demonstrate that prolonged broadband THz irradiation drives mMSCs toward differentiation, while 2-hour irradiation (regardless of THz sources) affects genes transcriptionally active in pluripotent stem cells. The strictly controlled experimental environment indicatesmore » minimal temperature changes and the absence of any discernable response to heat shock and cellular stress genes imply a non-thermal response. Computer simulations of the core promoters of two pluripotency markers reveal association between gene upregulation and propensity for DNA breathing. Finally, we propose that THz radiation has potential for non-contact control of cellular gene expression.« less

Combining Single Strand Oligodeoxynucleotides and CRISPR/Cas9 to Correct Gene Mutations in β-Thalassemia-induced Pluripotent Stem Cells.

PubMed

Niu, Xiaohua; He, Wenyin; Song, Bing; Ou, Zhanhui; Fan, Di; Chen, Yuchang; Fan, Yong; Sun, Xiaofang

2016-08-05

β-Thalassemia (β-Thal) is one of the most common genetic diseases in the world. The generation of patient-specific β-Thal-induced pluripotent stem cells (iPSCs), correction of the disease-causing mutations in those cells, and then differentiation into hematopoietic stem cells offers a new therapeutic strategy for this disease. Here, we designed a CRISPR/Cas9 to specifically target the Homo sapiens hemoglobin β (HBB) gene CD41/42(-CTTT) mutation. We demonstrated that the combination of single strand oligodeoxynucleotides with CRISPR/Cas9 was capable of correcting the HBB gene CD41/42 mutation in β-Thal iPSCs. After applying a correction-specific PCR assay to purify the corrected clones followed by sequencing to confirm mutation correction, we verified that the purified clones retained full pluripotency and exhibited normal karyotyping. Additionally, whole-exome sequencing showed that the mutation load to the exomes was minimal after CRISPR/Cas9 targeting. Furthermore, the corrected iPSCs were selected for erythroblast differentiation and restored the expression of HBB protein compared with the parental iPSCs. This method provides an efficient and safe strategy to correct the HBB gene mutation in β-Thal iPSCs. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Large-scale culture of a megakaryocytic progenitor cell line with a single-use bioreactor system.

PubMed

Nurhayati, Retno Wahyu; Ojima, Yoshihiro; Dohda, Takeaki; Kino-Oka, Masahiro

2018-03-01

The increasing application of regenerative medicine has generated a growing demand for stem cells and their derivatives. Single-use bioreactors offer an attractive platform for stem cell expansion owing to their scalability for large-scale production and feasibility of meeting clinical-grade standards. The current work evaluated the capacity of a single-use bioreactor system (1 L working volume) for expanding Meg01 cells, a megakaryocytic (MK) progenitor cell line. Oxygen supply was provided by surface aeration to minimize foaming and orbital shaking was used to promote oxygen transfer. Oxygen transfer rates (k L a) of shaking speeds 50, 100, and 125 rpm were estimated to be 0.39, 1.12, and 10.45 h -1 , respectively. Shaking speed was a critical factor for optimizing cell growth. At 50 rpm, Meg01 cells exhibited restricted growth due to insufficient mixing. A negative effect occurred when the shaking speed was increased to 125 rpm, likely caused by high hydrodynamic shear stress. The bioreactor culture achieved the highest growth profile when shaken at 100 rpm, achieving a total expansion rate up to 5.7-fold with a total cell number of 1.2 ± 0.2 × 10 9 cells L -1 . In addition, cells expanded using the bioreactor system could maintain their potency to differentiate following the MK lineage, as analyzed from specific surface protein and morphological similarity with the cells grown in the conventional culturing system. Our study reports the impact of operational variables such as shaking speed for growth profile and MK differentiation potential of a progenitor cell line in a single-use bioreactor. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:362-369, 2018. © 2017 American Institute of Chemical Engineers.

Single-Cell Functional Analysis of Stem-Cell Derived Cardiomyocytes on Micropatterned Flexible Substrates.

PubMed

Kijlstra, Jan David; Hu, Dongjian; van der Meer, Peter; Domian, Ibrahim J

2017-11-15

Human pluripotent stem-cell derived cardiomyocytes (hPSC-CMs) hold great promise for applications in human disease modeling, drug discovery, cardiotoxicity screening, and, ultimately, regenerative medicine. The ability to study multiple parameters of hPSC-CM function, such as contractile and electrical activity, calcium cycling, and force generation, is therefore of paramount importance. hPSC-CMs cultured on stiff substrates like glass or polystyrene do not have the ability to shorten during contraction, making them less suitable for the study of hPSC-CM contractile function. Other approaches require highly specialized hardware and are difficult to reproduce. Here we describe a protocol for the preparation of hPSC-CMs on soft substrates that enable shortening, and subsequently the simultaneous quantitative analysis of their contractile and electrical activity, calcium cycling, and force generation at single-cell resolution. This protocol requires only affordable and readily available materials and works with standard imaging hardware. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Single-Cell Transcriptomics Reveals a Population of Dormant Neural Stem Cells that Become Activated upon Brain Injury.

PubMed

Llorens-Bobadilla, Enric; Zhao, Sheng; Baser, Avni; Saiz-Castro, Gonzalo; Zwadlo, Klara; Martin-Villalba, Ana

2015-09-03

Heterogeneous pools of adult neural stem cells (NSCs) contribute to brain maintenance and regeneration after injury. The balance of NSC activation and quiescence, as well as the induction of lineage-specific transcription factors, may contribute to diversity of neuronal and glial fates. To identify molecular hallmarks governing these characteristics, we performed single-cell sequencing of an unbiased pool of adult subventricular zone NSCs. This analysis identified a discrete, dormant NSC subpopulation that already expresses distinct combinations of lineage-specific transcription factors during homeostasis. Dormant NSCs enter a primed-quiescent state before activation, which is accompanied by downregulation of glycolytic metabolism, Notch, and BMP signaling and a concomitant upregulation of lineage-specific transcription factors and protein synthesis. In response to brain ischemia, interferon gamma signaling induces dormant NSC subpopulations to enter the primed-quiescent state. This study unveils general principles underlying NSC activation and lineage priming and opens potential avenues for regenerative medicine in the brain. Copyright © 2015 Elsevier Inc. All rights reserved.

siRNA Nanoparticle Functionalization of Nanostructured Scaffolds Enables Controlled Multilineage Differentiation of Stem Cells

PubMed Central

Andersen, Morten Ø; Nygaard, Jens V; Burns, Jorge S; Raarup, Merete K; Nyengaard, Jens R; Bünger, Cody; Besenbacher, Flemming; Howard, Kenneth A; Kassem, Moustapha; Kjems, Jørgen

2010-01-01

The creation of complex tissues and organs is the ultimate goal in tissue engineering. Engineered morphogenesis necessitates spatially controlled development of multiple cell types within a scaffold implant. We present a novel method to achieve this by adhering nanoparticles containing different small-interfering RNAs (siRNAs) into nanostructured scaffolds. This allows spatial retention of the RNAs within nanopores until their cellular delivery. The released siRNAs were capable of gene silencing BCL2L2 and TRIB2, in mesenchymal stem cells (MSCs), enhancing osteogenic and adipogenic differentiation, respectively. This approach for enhancing a single type of differentiation is immediately applicable to all areas of tissue engineering. Different nanoparticles localized to spatially distinct locations within a single implant allowed two different tissue types to develop in controllable areas of an implant. As a consequence of this, we predict that complex tissues and organs can be engineered by the in situ development of multiple cell types guided by spatially restricted nanoparticles. PMID:20808289

Production of cloned and transgenic embryos using buffalo (Bubalus bubalis) embryonic stem cell-like cells isolated from in vitro fertilized and cloned blastocysts.

PubMed

George, Aman; Sharma, Ruchi; Singh, Karn P; Panda, Sudeepta K; Singla, Suresh K; Palta, Prabhat; Manik, Radhaysham; Chauhan, Manmohan S

2011-06-01

Here, we report the isolation and characterization of embryonic stem (ES) cell-like cells from cloned blastocysts, generated using fibroblasts derived from an adult buffalo (BAF). These nuclear transfer embryonic stem cell-like cells (NT-ES) grew in well-defined and dome-shaped colonies. The expression pattern of pluripotency marker genes was similar in both NT-ES and in vitro fertilization (IVF) embryo-derived embryonic stem cell-like cells (F-ES). Upon spontaneous differentiation via embryoid body formation, cells of different morphology were observed, among which predominant were endodermal-like and epithelial-like cell types. The ES cell-like cells could be passaged only mechanically and did not form colonies when plated as single cell suspension at different concentrations. When F-ES cell-like, NT-ES cell-like, and BAF cells of same genotype were used for hand-made cloning (HMC), no significant difference (p > 0.05) was observed in cleavage and blastocyst rate. Following transfer of HMC embryos to synchronized recipients, pregnancies were established only with F-ES cell-like and BAF cell-derived embryos, and one live calf was born from F-ES cell-like cells. Further, when transfected NT-ES cell-like cells and BAF were used for HMC, no significant difference (p > 0.05) was observed between cleavage and blastocyst rate. In conclusion, here we report for the first time the derivation of ES cell-like cells from an adult buffalo, and its genetic modification. We also report the birth of a live cloned calf from buffalo ES cell-like cells.

CD34+CD38-CD123+ Cells Are Present in Virtually All Acute Myeloid Leukaemia Blasts: A Promising Single Unique Phenotype for Minimal Residual Disease Detection.

PubMed

Al-Mawali, Adhra; Pinto, Avinash Daniel; Al-Zadjali, Shoaib

In CD34-positive acute myeloid leukaemia (AML), the leukaemia-initiating event likely takes place in the CD34+CD38- cell compartment. CD123 has been shown to be a unique marker of leukaemic stem cells within the CD34+CD38- compartment. The aim of this study was to identify the percentage of CD34+CD38-CD123+ cells in AML blasts, AML CD34+CD38- stem cells, and normal and regenerating bone marrow CD34+CD38- stem cells from non-myeloid malignancies. Thirty-eight adult de novo AML patients with intention to treat were enrolled after the application of inclusion criteria from February 2012 to February 2017. The percentage of the CD34+CD38-CD123+ phenotype in the blast population at diagnosis was determined using a CD45-gating strategy and CD34+ backgating by flow cytometry. We studied the CD34+CD38-CD123+ fraction in AML blasts at diagnosis, and its utility as a unique phenotype for minimal residual disease (MRD) of AML patients. CD123+ cells were present in 97% of AML blasts in patients at diagnosis (median 90%; range 21-99%). CD123+ cells were also present in 97% of the CD34+CD38- compartment (median 0.8164%, range 0.0262-39.7%). Interestingly, CD123 was not present in normal and regenerating CD34+CD38- bone marrow stem cells (range 0.002- 0.067 and 0.004-0.086, respectively). The CD34+CD38-CD123+ phenotype is present in virtually all AML blasts and it may be used as a unique single phenotype for MRD detection in AML patients. © 2017 The Author(s) Published by S. Karger AG, Basel.

Characterization of bone marrow derived mesenchymal stem cells in suspension

PubMed Central

2012-01-01

Introduction Bone marrow mesenchymal stem cells (BMMSCs) are a heterogeneous population of postnatal precursor cells with the capacity of adhering to culture dishes generating colony-forming unit-fibroblasts (CFU-F). Here we identify a new subset of BMMSCs that fail to adhere to plastic culture dishes and remain in culture suspension (S-BMMSCs). Methods To catch S-BMMSCs, we used BMMSCs-produced extracellular cell matrix (ECM)-coated dishes. Isolated S-BMMSCs were analyzed by in vitro stem cell analysis approaches, including flow cytometry, inductive multiple differentiation, western blot and in vivo implantation to assess the bone regeneration ability of S-BMMSCs. Furthermore, we performed systemic S-BMMSCs transplantation to treat systemic lupus erythematosus (SLE)-like MRL/lpr mice. Results S-BMMSCs are capable of adhering to ECM-coated dishes and showing mesenchymal stem cell characteristics with distinction from hematopoietic cells as evidenced by co-expression of CD73 or Oct-4 with CD34, forming a single colony cluster on ECM, and failure to differentiate into hematopoietic cell lineage. Moreover, we found that culture-expanded S-BMMSCs exhibited significantly increased immunomodulatory capacities in vitro and an efficacious treatment for SLE-like MRL/lpr mice by rebalancing regulatory T cells (Tregs) and T helper 17 cells (Th17) through high NO production. Conclusions These data suggest that it is feasible to improve immunotherapy by identifying a new subset BMMSCs. PMID:23083975

Addition of High-Dose Cytarabine to Fludarabine-Based Conditioning for Hematopoietic Stem Cell Transplantation for Treating Fanconi Anemia Patients with Advanced Myeloid Malignancy: A Single-Center Experience and Literature Review.

PubMed

Aoki, Takahiro; Koh, Katsuyoshi; Ikeda, Yuhachi; Sekinaka, Yujin; Akiyama, Kosuke; Mori, Makiko; Arakawa, Yuki; Hanada, Ryoji

2016-09-01

The complication of Fanconi anemia (FA) with acute leukemia is rare and challenging to treat because of high relapse rates, despite the improved outcome of hematopoietic stem cell transplantation with fludarabine-based conditioning for treating FA patients with hematological abnormalities. We added high-dose cytarabine to fludarabine-based conditioning to promote an enhanced antitumor effect and successfully subjected 4 patients with FA, including 3 with acute leukemia, to hematopoietic stem cell transplantation. All patients remain alive without treatment-related mortality or evidence of disease. Adding high-dose cytarabine to fludarabine-based conditioning may be tolerable and effective for treating FA patients with acute leukemia. Copyright © 2016 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

Design and Validation of CRISPR/Cas9 Systems for Targeted Gene Modification in Induced Pluripotent Stem Cells.

PubMed

Lee, Ciaran M; Zhu, Haibao; Davis, Timothy H; Deshmukh, Harshahardhan; Bao, Gang

2017-01-01

The CRISPR/Cas9 system is a powerful tool for precision genome editing. The ability to accurately modify genomic DNA in situ with single nucleotide precision opens up new possibilities for not only basic research but also biotechnology applications and clinical translation. In this chapter, we outline the procedures for design, screening, and validation of CRISPR/Cas9 systems for targeted modification of coding sequences in the human genome and how to perform genome editing in induced pluripotent stem cells with high efficiency and specificity.

Mesenchymal Stem Cells in Cardiology

PubMed Central

White, Ian A.; Sanina, Cristina; Balkan, Wayne; Hare, Joshua M.

2017-01-01

Cardiovascular disease (CVD) accounts for more deaths globally than any other single disease. There are on average 1.5 million episodes of myocardial infarction (heart attack) each year in the United States alone with roughly one third resulting in death. There is therefore a major need for developing new and effective strategies to promote cardiac repair. Intramyocardial transplantation of mesenchymal stem cells (MSCs) has emerged as a leading contender in the pursuit of clinical intervention and therapy. MSCs are potent mediators of cardiac repair and are therefore an attractive tool in the development of pre-clinical and clinical trials. MSCs are capable of secreting a large array of soluble factors, which have had demonstrated effects on pathogenic cardiac remolding, fibrosis, immune activation and cardiac stem cell proliferation within the damaged heart. MSCs are also capable of differentiation into cardiomyocytes, endothelial cells and vascular smooth muscle cells, although the relative contribution of trilineage differentiation and paracrine effectors on cardiac repair remains the subject of active investigation. PMID:27236666

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

PubMed

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

2009-03-01

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

Mouse Regenerating Myofibers Detected as False-Positive Donor Myofibers with Anti-Human Spectrin

PubMed Central

Rozkalne, Anete; Adkin, Carl; Meng, Jinhong; Lapan, Ariya; Morgan, Jennifer E.

2014-01-01

Abstract Stem cell transplantation is being tested as a potential therapy for a number of diseases. Stem cells isolated directly from tissue specimens or generated via reprogramming of differentiated cells require rigorous testing for both safety and efficacy in preclinical models. The availability of mice with immune-deficient background that carry additional mutations in specific genes facilitates testing the efficacy of cell transplantation in disease models. The muscular dystrophies are a heterogeneous group of disorders, of which Duchenne muscular dystrophy is the most severe and common type. Cell-based therapy for muscular dystrophy has been under investigation for several decades, with a wide selection of cell types being studied, including tissue-specific stem cells and reprogrammed stem cells. Several immune-deficient mouse models of muscular dystrophy have been generated, in which human cells obtained from various sources are injected to assess their preclinical potential. After transplantation, the presence of engrafted human cells is detected via immunofluorescence staining, using antibodies that recognize human, but not mouse, proteins. Here we show that one antibody specific to human spectrin, which is commonly used to evaluate the efficacy of transplanted human cells in mouse muscle, detects myofibers in muscles of NOD/Rag1nullmdx5cv, NOD/LtSz-scid IL2Rγnull mice, or mdx nude mice, irrespective of whether they were injected with human cells. These “reactive” clusters are regenerating myofibers, which are normally present in dystrophic tissue and the spectrin antibody is likely recognizing utrophin, which contains spectrin-like repeats. Therefore, caution should be used in interpreting data based on detection of single human-specific proteins, and evaluation of human stem cell engraftment should be performed using multiple human-specific labeling strategies. PMID:24152287

The selection of light emitting diode irradiation parameters for stimulation of human mesenchymal stem cells proliferation

NASA Astrophysics Data System (ADS)

Lewandowski, Rafał; Trafny, ElŻbieta A.; Stepińska, Małgorzata; Gietka, Andrzej; Kotowski, Paweł; Dobrzyńska, Monika; Łapiński, Mariusz P.

2016-12-01

Human mesenchymal stem cells (hMSCs) with their vast differentiation potential are very useful for cell-based regenerative medicine. To achieve sufficient numbers of cells for tissue engineering, many different methods have been used to reach the effective increase of cell proliferation. Low-energy red light provided by light emitting diodes (LEDs) have been recently introduced as a method that promoted biomodulation and proliferation of hMSCs in vitro. The purpose of this study was to find the optimum stimulatory dosimetric parameters of LED (630 nm) irradiation on the hMSCs proliferation. The energy density was 2, 3, 4, 10, 20 J/cm2 and the power density used was 7, 17 or 30 mW/cm2. Human MSCs were irradiated with single or triple exposures daily at room temperature and the cell proliferation rate was evaluated during nine days after irradiation. The results showed that after irradiation 4 J/cm2 and 17 mW/cm2 at a single dose the proliferation rate of hMSCs increased on day 5 and 9 (13% and 7%, respectively) when compared to nonirradiated cells. However, triple LED irradiation under the same parameters resulted in the decline in the cell proliferation rate on day 5, but the proliferation rate was at the same level on day 9, when compared with the cell proliferation after irradiation with a single dose. The effect of a single dose irradiation with 4 J/cm2 and 17 mW/cm2 on the proliferation of cells was the highest when the cells were irradiated in phosphate-buffered saline (PBS) instead of MSCGM culture medium.

Cell type transcriptome atlas for the planarian Schmidtea mediterranea.

PubMed

Fincher, Christopher T; Wurtzel, Omri; de Hoog, Thom; Kravarik, Kellie M; Reddien, Peter W

2018-05-25

The transcriptome of a cell dictates its unique cell type biology. We used single-cell RNA sequencing to determine the transcriptomes for essentially every cell type of a complete animal: the regenerative planarian Schmidtea mediterranea. Planarians contain a diverse array of cell types, possess lineage progenitors for differentiated cells (including pluripotent stem cells), and constitutively express positional information, making them ideal for this undertaking. We generated data for 66,783 cells, defining transcriptomes for known and many previously unknown planarian cell types and for putative transition states between stem and differentiated cells. We also uncovered regionally expressed genes in muscle, which harbors positional information. Identifying the transcriptomes for potentially all cell types for many organisms should be readily attainable and represents a powerful approach to metazoan biology. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Converging roads: evidence for an adult hemangioblast.

PubMed

Bailey, Alexis S; Fleming, William H

2003-11-01

Classical studies of the developing embryo first suggested the existence of the hemangioblast, a precursor cell with the potential to differentiate into both blood and blood vessels. Several lines of investigation demonstrated that many of the genes activated during early hematopoietic development are also expressed in the vascular endothelium. Gene-targeting studies using embryonic stem cells have identified Flk-1, SCL, and Runx-1 as important regulatory molecules that specify both hematopoietic and vascular outcomes. Although it was anticipated that the hemangioblast would be present only during the earliest stages of vascular development in the yolk sac, accumulating evidence now indicates that hematopoietic cells with hemangioblast activity persist into adulthood. In the adult, bone marrow-derived, circulating endothelial progenitors contribute to postnatal neovascularization and enhance vascular repair following ischemic injury. Highly purified populations of hematopoietic stem cells from humans and mice can differentiate into both blood cells and vascular tissue at the single cell level. These recent findings suggest that bone marrow-derived hematopoietic stem cells or their progeny may contribute to the maintenance and repair of both the hematopoietic and the vascular systems during adult life.

Mex3a Marks a Slowly Dividing Subpopulation of Lgr5+ Intestinal Stem Cells.

PubMed

Barriga, Francisco M; Montagni, Elisa; Mana, Miyeko; Mendez-Lago, Maria; Hernando-Momblona, Xavier; Sevillano, Marta; Guillaumet-Adkins, Amy; Rodriguez-Esteban, Gustavo; Buczacki, Simon J A; Gut, Marta; Heyn, Holger; Winton, Douglas J; Yilmaz, Omer H; Attolini, Camille Stephan-Otto; Gut, Ivo; Batlle, Eduard

2017-06-01

Highly proliferative Lgr5+ stem cells maintain the intestinal epithelium and are thought to be largely homogeneous. Although quiescent intestinal stem cell (ISC) populations have been described, the identity and features of such a population remain controversial. Here we report unanticipated heterogeneity within the Lgr5+ ISC pool. We found that expression of the RNA-binding protein Mex3a labels a slowly cycling subpopulation of Lgr5+ ISCs that contribute to all intestinal lineages with distinct kinetics. Single-cell transcriptome profiling revealed that Lgr5+ cells adopt two discrete states, one of which is defined by a Mex3a expression program and relatively low levels of proliferation genes. During homeostasis, Mex3a+ cells continually shift into the rapidly dividing, self-renewing ISC pool. Chemotherapy and radiation preferentially target rapidly dividing Lgr5+ cells but spare the Mex3a-high/Lgr5+ population, helping to promote regeneration of the intestinal epithelium following toxic insults. Thus, Mex3a defines a reserve-like ISC population within the Lgr5+ compartment. Copyright © 2017 Elsevier Inc. All rights reserved.

The carbocyanine dye DiD labels in vitro and in vivo neural stem cells of the subventricular zone as well as myelinated structures following in vivo injection in the lateral ventricle.

PubMed

Carradori, Dario; Barreau, Kristell; Eyer, Joël

2016-02-01

Carbocyanines are fluorescent lipophilic cationic dyes used since the early 1980s as neuronal tracers. Several applications of these compounds have been developed thanks to their low cell toxicity, lateral diffusion within the cellular membranes, and good photostability. 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine 4-chlorobenzenesulfonate (DiD) is an interesting component of this family because, in addition to the classic carbocyanine properties, it has a longer wavelength compared with its analogues. That makes DiD an excellent carbocyanine for labeling cells and tissues with significant intrinsic fluorescence. Drug encapsulation, drug delivery, and cellular transplantation are also fields using DiD-based systems where having detailed knowledge about its behavior as a single entity is important. Recently, promising studies concerned neural stem cells from the subventricular zone of the lateral ventricle in the brain (their natural niche) and their potential therapeutic use. Here, we show that DiD is able to label these stem cells in vitro and present basilar information concerning its pharmacokinetics, concentrations, and microscope protocols. Moreover, when DiD is injected in vivo in the cerebrospinal fluid present in the lateral ventricle of rat, it also labels stem cells as well as myelinated structures of the caudoputamen. This analysis provides a database to consult when planning experiments concerning DiD and neural stem cells from the subventricular zone. © 2015 Wiley Periodicals, Inc.

Engineering spheroids potentiating cell-cell and cell-ECM interactions by self-assembly of stem cell microlayer.

PubMed

Lee, Yu Bin; Kim, Eun Mi; Byun, Hayeon; Chang, Hyung-Kwan; Jeong, Kwanghee; Aman, Zachary M; Choi, Yu Suk; Park, Jungyul; Shin, Heungsoo

2018-05-01

Numerous methods have been reported for the fabrication of 3D multi-cellular spheroids and their use in stem cell culture. Current methods typically relying on the self-assembly of trypsinized, suspended stem cells, however, show limitations with respect to cell viability, throughput, and accurate recapitulation of the natural microenvironment. In this study, we developed a new system for engineering cell spheroids by self-assembly of micro-scale monolayer of stem cells. We prepared synthetic hydrogels with the surface of chemically formed micropatterns (squares/circles with width/diameter of 200 μm) on which mesenchymal stem cells isolated from human nasal turbinate tissue (hTMSCs) were selectively attached and formed a monolayer. The hydrogel is capable of thermally controlled expansion. As the temperature was decreased from 37 to 4 °C, the cell layer detached rapidly (<10 min) and assembled to form spheroids with consistent size (∼100 μm) and high viability (>90%). Spheroidization was significantly delayed and occurred with reduced efficiency on circle patterns compared to square patterns. Multi-physics mapping supported that delamination of the micro-scale monolayer may be affected by stress concentrated at the corners of the square pattern. In contrast, stress was distributed symmetrically along the boundary of the circle pattern. In addition, treatment of the micro-scale monolayer with a ROCK inhibitor significantly retarded spheroidization, highlighting the importance of contraction mediated by actin stress fibers for the stable generation of spheroidal stem cell structures. Spheroids prepared from the assembly of monolayers showed higher expression, both on the mRNA and protein levels, of ECM proteins (fibronectin and laminin) and stemness markers (Oct4, Sox2, and Nanog) compared to spheroids prepared from low-attachment plates, in which trypsinized single cells are assembled. The hTMSC spheroids also presented enhanced expression levels of markers related to tri-lineage (osteogenic, chondrogenic and adipogenic) differentiation. The changes in microcellular environments and functionalities were double-confirmed by using adipose derived mesenchymal stem cells (ADSCs). This spheroid engineering technique may have versatile applications in regenerative medicine for functionally improved 3D culture and therapeutic cell delivery. Copyright © 2018 Elsevier Ltd. All rights reserved.

Human embryonic and induced pluripotent stem cells maintain phenotype but alter their metabolism after exposure to ROCK inhibitor.

PubMed

Vernardis, Spyros I; Terzoudis, Konstantinos; Panoskaltsis, Nicki; Mantalaris, Athanasios

2017-02-06

Human pluripotent stem cells (hPSCs) are adhesion-dependent cells that require cultivation in colonies to maintain growth and pluripotency. Robust differentiation protocols necessitate single cell cultures that are achieved by use of ROCK (Rho kinase) inhibitors. ROCK inhibition enables maintenance of stem cell phenotype; its effects on metabolism are unknown. hPSCs were exposed to 10 μM ROCK inhibitor for varying exposure times. Pluripotency (TRA-1-81, SSEA3, OCT4, NANOG, SOX2) remained unaffected, until after prolonged exposure (96 hrs). Gas chromatography-mass spectrometry metabolomics analysis identified differences between ROCK-treated and untreated cells as early as 12 hrs. Exposure for 48 hours resulted in reduction in glycolysis, glutaminolysis, the citric acid (TCA) cycle as well as the amino acids pools, suggesting the adaptation of the cells to the new culture conditions, which was also reflected by the expression of the metabolic regulators, mTORC1 and tp53 and correlated with cellular proliferation status. While gene expression and protein levels did not reveal any changes in the physiology of the cells, metabolomics revealed the fluctuating state of the metabolism. The above highlight the usefulness of metabolomics in providing accurate and sensitive information on cellular physiological status, which could lead to the development of robust and optimal stem cell bioprocesses.

Single-cell mRNA profiling reveals transcriptional heterogeneity among pancreatic circulating tumour cells.

PubMed

Lapin, Morten; Tjensvoll, Kjersti; Oltedal, Satu; Javle, Milind; Smaaland, Rune; Gilje, Bjørnar; Nordgård, Oddmund

2017-05-31

Single-cell mRNA profiling of circulating tumour cells may contribute to a better understanding of the biology of these cells and their role in the metastatic process. In addition, such analyses may reveal new knowledge about the mechanisms underlying chemotherapy resistance and tumour progression in patients with cancer. Single circulating tumour cells were isolated from patients with locally advanced or metastatic pancreatic cancer with immuno-magnetic depletion and immuno-fluorescence microscopy. mRNA expression was analysed with single-cell multiplex RT-qPCR. Hierarchical clustering and principal component analysis were performed to identify expression patterns. Circulating tumour cells were detected in 33 of 56 (59%) examined blood samples. Single-cell mRNA profiling of intact isolated circulating tumour cells revealed both epithelial-like and mesenchymal-like subpopulations, which were distinct from leucocytes. The profiled circulating tumour cells also expressed elevated levels of stem cell markers, and the extracellular matrix protein, SPARC. The expression of SPARC might correspond to an epithelial-mesenchymal transition in pancreatic circulating tumour cells. The analysis of single pancreatic circulating tumour cells identified distinct subpopulations and revealed elevated expression of transcripts relevant to the dissemination of circulating tumour cells to distant organ sites.

Positron emission tomography response at the time of autologous stem cell transplantation predicts outcome of patients with relapsed and/or refractory Hodgkin’s lymphoma responding to prior salvage therapy

PubMed Central

Devillier, Raynier; Coso, Diane; Castagna, Luca; Brenot Rossi, Isabelle; Anastasia, Antonella; Chiti, Arturo; Ivanov, Vadim; Schiano, Jean Marc; Santoro, Armando; Chabannon, Christian; Balzarotti, Monica; Blaise, Didier; Bouabdallah, Reda

2012-01-01

Background High-dose chemotherapy followed by autologous stem cell transplantation is the standard treatment for relapsed and/or refractory Hodgkin’s lymphoma although half of patients relapse after transplantation. Predictive factors, such as relapse within 12 months, Ann-Arbor stage at relapse, and relapse in previously irradiated fields are classically used to identify patients with poor outcome. Recently, 18-fluorodeoxyglucose positron emission tomography has emerged as a new method for providing information to predict outcome. The aim of this study was to confirm the predictive value of positron emission tomography status after salvage therapy and to compare single versus tandem autologous stem cell transplantation in patients with relapsed and/or refractory Hodgkin’s lymphoma. Design and Methods We report a series of 111 consecutive patients with treatment-sensitive relapsed and/or treatment-refractory Hodgkin’s lymphoma who achieved complete (positron emission tomography-negative group) or partial remission (positron emission tomography-positive group) at positron emission tomography evaluation after salvage chemotherapy and who underwent single or tandem autologous stem cell transplantation. Results Five-year overall and progression-free survival rates were 81% and 64%, respectively. There were significant differences in 5-year progression-free survival (79% versus 23%; P<0.001) and 5-year overall survival (90% versus 55%, P=0.001) between the positron emission tomography-negative and -positive groups, respectively. A complete response, as determined by positron emission tomography evaluation, after salvage therapy predicted significantly better 5-year overall survival rates in both intermediate (91% versus 50%; P=0.029) and unfavorable (89% versus 58%; P=0.026) risk subgroup analyses. In the positron emission tomography-positive subgroup, tandem transplantation improved 5-year progression-free survival from 0% (in the single transplantation group) to 43% (P=0.034). Multivariate analysis showed that positron emission tomography status (hazard ratio: 5.26 [2.57–10.73]) and tandem transplantation (hazard ratio: 0.39 [0.19–0.78]) but not risk factors at relapse (hazard ratio: 1.77 [0.80–3.92]) significantly influenced progression-free survival, while only tomography status significantly influenced overall survival (hazard ratio: 4.03 [1.38–11.75]). Conclusions In patients with relapsed/refractory Hodgkin’s lymphoma responding to prior salvage therapy, positron emission tomography response at time of autologous stem cell transplantation favorably influences outcome and enables identification of patients requiring single or tandem transplantation. PMID:22271893

Concentration dependent survival and neural differentiation of murine embryonic stem cells cultured on polyethylene glycol dimethacrylate hydrogels possessing a continuous concentration gradient of n-cadherin derived peptide His-Ala-Val-Asp-Lle.

PubMed

Lim, Hyun Ju; Mosley, Matthew C; Kurosu, Yuki; Smith Callahan, Laura A

2017-07-01

N-cadherin cell-cell signaling plays a key role in the structure and function of the nervous system. However, few studies have incorporated bioactive signaling from n-cadherin into tissue engineering matrices. The present study uses a continuous gradient approach in polyethylene glycol dimethacrylate hydrogels to identify concentration dependent effects of n-cadherin peptide, His-Ala-Val-Asp-Lle (HAVDI), on murine embryonic stem cell survival and neural differentiation. The n-cadherin peptide was found to affect the expression of pluripotency marker, alkaline phosphatase, in murine embryonic stem cells cultured on n-cadherin peptide containing hydrogels in a concentration dependent manner. Increasing n-cadherin peptide concentrations in the hydrogels elicited a biphasic response in neurite extension length and mRNA expression of neural differentiation marker, neuron-specific class III β-tubulin, in murine embryonic stem cells cultured on the hydrogels. High concentrations of n-cadherin peptide in the hydrogels were found to increase the expression of apoptotic marker, caspase 3/7, in murine embryonic stem cells compared to that of murine embryonic stem cell cultures on hydrogels containing lower concentrations of n-cadherin peptide. Increasing the n-cadherin peptide concentration in the hydrogels facilitated greater survival of murine embryonic stem cells exposed to increasing oxidative stress caused by hydrogen peroxide exposure. The combinatorial approach presented in this work demonstrates concentration dependent effects of n-cadherin signaling on mouse embryonic stem cell behavior, underscoring the need for the greater use of systematic approaches in tissue engineering matrix design in order to understand and optimize bioactive signaling in the matrix for tissue formation. Single cell encapsulation is common in tissue engineering matrices. This eliminates cellular access to cell-cell signaling. N-cadherin, a cell-cell signaling molecule, plays a vital role in the development of neural tissues, but has not been well studied as a bioactive signaling element in neural tissue engineering matrices. The present study uses a systematic continuous gradient approach to identify concentration dependent effects of n-cadherin derived peptide, HAVDI, on the survival and neural differentiation of murine embryonic stem cells. This work underscores the need for greater use to combinatorial strategies to understand the effect complex bioactive signaling, such as n-cadherin, and the need to optimize the concentration of such bioactive signaling within tissue engineering matrices for maximal cellular response. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Bone Repair Cells for Craniofacial Regeneration

PubMed Central

Pagni, G; Kaigler, D; Rasperini, G; Avila-Ortiz, G; Bartel, R; Giannobile, WV

2012-01-01

Reconstruction of complex craniofacial deformities is a clinical challenge in situations of injury, congenital defects or disease. The use of cell-based therapies represents one of the most advanced methods for enhancing the regenerative response for craniofacial wound healing. Both Somatic and Stem Cells have been adopted in the treatment of complex osseous defects and advances have been made in finding the most adequate scaffold for the delivery of cell therapies in human regenerative medicine. As an example of such approaches for clinical application for craniofacial regeneration, Ixmyelocel-T or bone repair cells are a source of bone marrow derived stem and progenitor cells. They are produced through the use of single pass perfusion bioreactors for CD90+ mesenchymal stem cells and CD14+ monocyte/macrophage progenitor cells. The application of ixmyelocel-T has shown potential in the regeneration of muscular, vascular, nervous and osseous tissue. The purpose of this manuscript is to highlight cell therapies used to repair bony and soft tissue defects in the oral and craniofacial complex. The field at this point remains at an early stage, however this review will provide insights into the progress being made using cell therapies for eventual development into clinical practice. PMID:22433781

Arabidopsis Myosins XI1, XI2, and XIK Are Crucial for Gravity-Induced Bending of Inflorescence Stems

PubMed Central

Talts, Kristiina; Ilau, Birger; Ojangu, Eve-Ly; Tanner, Krista; Peremyslov, Valera V.; Dolja, Valerian V.; Truve, Erkki; Paves, Heiti

2016-01-01

Myosins and actin filaments in the actomyosin system act in concert in regulating cell structure and dynamics and are also assumed to contribute to plant gravitropic response. To investigate the role of the actomyosin system in the inflorescence stem gravitropism, we used single and multiple mutants affecting each of the 17 Arabidopsis myosins of class VIII and XI. We show that class XI but not class VIII myosins are required for stem gravitropism. Simultaneous loss of function of myosins XI1, XI2, and XIK leads to impaired gravitropic bending that is correlated with altered growth, stiffness, and insufficient sedimentation of gravity sensing amyloplasts in stem endodermal cells. The gravitropic defect of the corresponding triple mutant xi1 xi2 xik could be rescued by stable expression of the functional XIK:YFP in the mutant background, indicating a role of class XI myosins in this process. Altogether, our results emphasize the critical contributions of myosins XI in stem gravitropism of Arabidopsis. PMID:28066484

How to select the best available related or unrelated donor of hematopoietic stem cells?

PubMed

Tiercy, Jean-Marie

2016-06-01

Recognition of HLA incompatibilities by the immune system represents a major barrier to allogeneic hematopoietic stem cell transplantation. HLA genotypically identical sibling donors are, therefore, the gold standard for transplantation purposes, but only 30% patients have such a donor. For the remaining 70% patients alternative sources of stem cells are a matched unrelated adult volunteer donor, a haploidentical donor or a cord blood unit. The definition of 'HLA matching' depends on the level of resolution and on which loci are tested. The development of HLA molecular typing technologies and the availability of more than 27 million donors in the international database has greatly facilitated unrelated donor searches. The gold standard is high resolution typing at the HLA-A, -B, -C, -DRB1, and -DQB1 loci (10/10 match). Single disparities for HLA-A, -B, - C, or -DRB1 are associated with increased risk of post-transplant complications, but less so in patients with advanced disease, and in those undergoing T-cell-depleted allografting. HLA-DQB1 mismatches seem to be better tolerated and some HLA-C, -DRB1 and -DPB1 disparities are potentially less immunogenic. HLA typing by next-generation sequencing methods is likely to change matching algorithms by providing full sequence information on all HLA loci in a single step. In most European populations a 10/10 matched donor can be found for at least 50% of patients and an additional 20-30% patients may have a 9/10 matched donor. Genetic factors that help in identifying donors with less immunogenic mismatches are discussed. Haploidentical donors are increasingly used as an alternative source of stem cells for those patients lacking a matched unrelated donor. Copyright© Ferrata Storti Foundation.

Zscan4 restores the developmental potency of embryonic stem cells

PubMed Central

Amano, Tomokazu; Hirata, Tetsuya; Falco, Geppino; Monti, Manuela; Sharova, Lioudmila V.; Amano, Misa; Sheer, Sarah; Hoang, Hien G.; Piao, Yulan; Stagg, Carole A.; Yamamizu, Kohei; Akiyama, Tomohiko; Ko, Minoru S.H.

2013-01-01

The developmental potency of mouse embryonic stem (ES) cells, which is the ability to contribute to a whole embryo is known to deteriorate during long-term cell culture. Previously we have shown that ES cells oscillate between Zscan4- and Zscan4+ states, and the transient activation of Zscan4 is required for the maintenance of telomeres and genome stability of ES cells. Here we show that increasing the frequency of Zscan4 activation in mouse ES cells restores and maintains their developmental potency in long-term cell culture. Injection of a single ES cell with such increased potency into a tetraploid blastocyst gives rise to an entire embryo with a higher success rate. These results not only provide a means to rejuvenate ES cells by manipulating Zscan4 expression, but also indicate the active roles of Zscan4 in the long-term maintenance of ES cell potency. PMID:23739662

Tenascin-C mimetic Peptide nanofibers direct stem cell differentiation to osteogenic lineage.

PubMed

Sever, Melike; Mammadov, Busra; Guler, Mustafa O; Tekinay, Ayse B

2014-12-08

Extracellular matrix contains various signals for cell surface receptors that regulate cell fate through modulation of cellular activities such as proliferation and differentiation. Cues from extracellular matrix components can be used for development of new materials to control the stem cell fate. In this study, we achieved control of stem cell fate toward osteogenic commitment by using a single extracellular matrix element despite the contradictory effect of mechanical stiffness. For this purpose, we mimicked bone extracellular matrix by incorporating functional sequence of fibronectin type III domain from native tenascin-C on self-assembled peptide nanofibers. When rat mesenchymal stem cells (rMSCs) were cultured on these peptide nanofibers, alkaline phosphatase (ALP) activity and alizarin red staining indicated osteogenic differentiation even in the absence of osteogenic supplements. Moreover, expression levels of osteogenic marker genes were significantly enhanced revealed by quantitative real-time polymerase chain reaction (qRT-PCR), which showed the remarkable bioactive role of this nanofiber system on osteogenic differentiation. Overall, these results showed that tenascin-C mimetic peptides significantly enhanced the attachment, proliferation, and osteogenic differentiation of rMSCs even in the absence of any external bioactive factors and regardless of the suitable stiff mechanical properties normally required for osteogenic differentiation. Thus, these peptide nanofibers provide a promising new platform for bone regeneration.

Functional melanocytes derived from human pluripotent stem cells engraft into pluristratified epidermis.

PubMed

Nissan, Xavier; Larribere, Lionel; Saidani, Manoubia; Hurbain, Ilse; Delevoye, Cédric; Feteira, Jessica; Lemaitre, Gilles; Peschanski, Marc; Baldeschi, Christine

2011-09-06

Melanocytes are essential for skin homeostasis and protection, and their defects in humans lead to a wide array of diseases that are potentially extremely severe. To date, the analysis of molecular mechanisms and the function of human melanocytes have been limited because of the difficulties in accessing large numbers of cells with the specific phenotypes. This issue can now be addressed via a differentiation protocol that allows melanocytes to be obtained from pluripotent stem cell lines, either induced or of embryonic origin, based on the use of moderate concentrations of a single cytokine, bone morphogenic protein 4. Human melanocytes derived from pluripotent stem cells exhibit all the characteristic features of their adult counterparts. This includes the enzymatic machinery required for the production and functional delivery of melanin to keratinocytes. Melanocytes also integrate appropriately into organotypic epidermis reconstructed in vitro. The availability of human cells committed to the melanocytic lineage in vitro will enable the investigation of those mechanisms that guide the developmental processes and will facilitate analysis of the molecular mechanisms responsible for genetic diseases. Access to an unlimited resource may also prove a vital tool for the treatment of hypopigmentation disorders when donors with matching haplotypes become available in clinically relevant banks of pluripotent stem cell lines.

Gene targeting and cloning in pigs using fetal liver derived cells.

PubMed

Waghmare, Sanjeev K; Estrada, Jose; Reyes, Luz; Li, Ping; Ivary, Bess; Sidner, Richard A; Burlak, Chris; Tector, A Joseph

2011-12-01

Since there are no pig embryonic stem cells, pig genetic engineering is done in fetal fibroblasts that remain totipotent for only 3 to 5 wk. Nuclear donor cells that remain totipotent for longer periods of time would facilitate complicated genetic engineering in pigs. The goal of this study was to test the feasibility of using fetal liver-derived cells (FLDC) to perform gene targeting, and create a genetic knockout pig. FLDC were isolated and processed using a human liver stem cell protocol. Single copy α-1,3-galactosyl transferase knockout (GTKO) FLDCs were created using electroporation and neomycin resistant colonies were screened using PCR. Homozygous GTKO cells were created through loss of heterozygosity mutations in single GTKO FLDCs. Double GTKO FLDCs were used in somatic cell nuclear transfer (SCNT) to create GTKO pigs. FLDCs grew for more than 80 population doublings, maintaining normal karyotype. Gene targeting and loss of heterozygosity mutations produced homozygous GTKO FLDCs. FLDCs used in SCNT gave rise to homozygous GTKO pigs. FDLCs can be used in gene targeting and SCNT to produce genetically modified pigs. The increased life span in culture compared to fetal fibroblasts may facilitate genetic engineering in the pig. Copyright © 2011 Elsevier Inc. All rights reserved.

Evidence of In Vitro Preservation of Human Nephrogenesis at the Single-Cell Level.

PubMed

Pode-Shakked, Naomi; Gershon, Rotem; Tam, Gal; Omer, Dorit; Gnatek, Yehudit; Kanter, Itamar; Oriel, Sarit; Katz, Guy; Harari-Steinberg, Orit; Kalisky, Tomer; Dekel, Benjamin

2017-07-11

During nephrogenesis, stem/progenitor cells differentiate and give rise to early nephron structures that segment to proximal and distal nephron cell types. Previously, we prospectively isolated progenitors from human fetal kidney (hFK) utilizing a combination of surface markers. However, upon culture nephron progenitors differentiated and could not be robustly maintained in vitro. Here, by culturing hFK in a modified medium used for in vitro growth of mouse nephron progenitors, and by dissection of NCAM + /CD133 - progenitor cells according to EpCAM expression (NCAM + /CD133 - /EpCAM - , NCAM + /CD133 - /EpCAM dim , NCAM + /CD133 - /EpCAM bright ), we show at single-cell resolution a preservation of uninduced and induced cap mesenchyme as well as a transitioning mesenchymal-epithelial state. Concomitantly, differentiating and differentiated epithelial lineages are also maintained. In vitro expansion of discrete stages of early human nephrogenesis in nephron stem cell cultures may be used for drug screening on a full repertoire of developing kidney cells and for prospective isolation of mesenchymal or epithelial renal lineages for regenerative medicine. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Isolating LacZ-expressing cells from mouse inner ear tissues using flow cytometry.

PubMed

Jan, Taha A; Chai, Renjie; Sayyid, Zahra N; Cheng, Alan G

2011-12-23

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae. The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown. To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs.

PubMed

Hayashi, Tetsutaro; Ozaki, Haruka; Sasagawa, Yohei; Umeda, Mana; Danno, Hiroki; Nikaido, Itoshi

2018-02-12

Total RNA sequencing has been used to reveal poly(A) and non-poly(A) RNA expression, RNA processing and enhancer activity. To date, no method for full-length total RNA sequencing of single cells has been developed despite the potential of this technology for single-cell biology. Here we describe random displacement amplification sequencing (RamDA-seq), the first full-length total RNA-sequencing method for single cells. Compared with other methods, RamDA-seq shows high sensitivity to non-poly(A) RNA and near-complete full-length transcript coverage. Using RamDA-seq with differentiation time course samples of mouse embryonic stem cells, we reveal hundreds of dynamically regulated non-poly(A) transcripts, including histone transcripts and long noncoding RNA Neat1. Moreover, RamDA-seq profiles recursive splicing in >300-kb introns. RamDA-seq also detects enhancer RNAs and their cell type-specific activity in single cells. Taken together, we demonstrate that RamDA-seq could help investigate the dynamics of gene expression, RNA-processing events and transcriptional regulation in single cells.

Transfer of minimally manipulated CMV-specific T cells from stem cell or third-party donors to treat CMV infection after allo-HSCT.

PubMed

Neuenhahn, M; Albrecht, J; Odendahl, M; Schlott, F; Dössinger, G; Schiemann, M; Lakshmipathi, S; Martin, K; Bunjes, D; Harsdorf, S; Weissinger, E M; Menzel, H; Verbeek, M; Uharek, L; Kröger, N; Wagner, E; Kobbe, G; Schroeder, T; Schmitt, M; Held, G; Herr, W; Germeroth, L; Bonig, H; Tonn, T; Einsele, H; Busch, D H; Grigoleit, G U

2017-10-01

Cytomegalovirus (CMV) infection is a common, potentially life-threatening complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT). We assessed prospectively the safety and efficacy of stem cell-donor- or third-party-donor-derived CMV-specific T cells for the treatment of persistent CMV infections after allo-HSCT in a phase I/IIa trial. Allo-HSCT patients with drug-refractory CMV infection and lacking virus-specific T cells were treated with a single dose of ex vivo major histocompatibility complex-Streptamer-isolated CMV epitope-specific donor T cells. Forty-four allo-HSCT patients receiving a T-cell-replete (D + repl; n=28) or T-cell-depleted (D + depl; n=16) graft from a CMV-seropositive donor were screened for CMV-specific T-cell immunity. Eight D + depl recipients received adoptive T-cell therapy from their stem cell donor. CMV epitope-specific T cells were well supported and became detectable in all treated patients. Complete and partial virological response rates were 62.5% and 25%, respectively. Owing to longsome third-party donor (TPD) identification, only 8 of the 57 CMV patients transplanted from CMV-seronegative donors (D - ) received antigen-specific T cells from partially human leukocyte antigen (HLA)-matched TPDs. In all but one, TPD-derived CMV-specific T cells remained undetectable. In summary, adoptive transfer correlated with functional virus-specific T-cell reconstitution in D + depl patients. Suboptimal HLA match may counteract expansion of TPD-derived virus-specific T cells in D - patients.

uPAR-controlled oncolytic adenoviruses eliminate cancer stem cells in human pancreatic tumors.

PubMed

Sobrevals, Luciano; Mato-Berciano, Ana; Urtasun, Nerea; Mazo, Adela; Fillat, Cristina

2014-01-01

Pancreatic tumors contain cancer stem cells highly resistant to chemotherapy. The identification of therapies that can eliminate this population of cells might provide with more effective treatments. In the current work we evaluated the potential of oncolytic adenoviruses to act against pancreatic cancer stem cells (PCSC). PCSC from two patient-derived xenograft models were isolated from orthotopic pancreatic tumors treated with saline, or with the chemotherapeutic agent gemcitabine. An enrichment in the number of PCSC expressing the cell surface marker CD133 and a marked enhancement on tumorsphere formation was observed in gemcitabine treated tumors. No significant increase in the CD44, CD24, and epithelial-specific antigen (ESA) positive cells was observed. Neoplastic sphere-forming cells were susceptible to adenoviral infection and exposure to oncolytic adenoviruses resulted in elevated cytotoxicity with both Adwt and the tumor specific AduPARE1A adenovirus. In vivo, intravenous administration of a single dose of AduPARE1A in human-derived pancreatic xenografts led to a remarkable anti-tumor effect. In contrast to gemcitabine AduPARE1A treatment did not result in PCSC enrichment. No enrichment on tumorspheres neither on the CD133(+) population was detected. Therefore our data provide evidences of the relevance of uPAR-controlled oncolytic adenoviruses for the elimination of pancreatic cancer stem cells. © 2013.

Skeletal stem cell isolation: A review on the state-of-the-art microfluidic label-free sorting techniques.

PubMed

Xavier, Miguel; Oreffo, Richard O C; Morgan, Hywel

2016-01-01

Skeletal stem cells (SSC) are a sub-population of bone marrow stromal cells that reside in postnatal bone marrow with osteogenic, chondrogenic and adipogenic differentiation potential. SSCs reside only in the bone marrow and have organisational and regulatory functions in the bone marrow microenvironment and give rise to the haematopoiesis-supportive stroma. Their differentiation capacity is restricted to skeletal lineages and therefore the term SSC should be clearly distinguished from mesenchymal stem cells which are reported to exist in extra-skeletal tissues and, critically, do not contribute to skeletal development. SSCs are responsible for the unique regeneration capacity of bone and offer unlimited potential for application in bone regenerative therapies. A current unmet challenge is the isolation of homogeneous populations of SSCs, in vitro, with homogeneous regeneration and differentiation capacities. Challenges that limit SSC isolation include a) the scarcity of SSCs in bone marrow aspirates, estimated at between 1 in 10-100,000 mononuclear cells; b) the absence of specific markers and thus the phenotypic ambiguity of the SSC and c) the complexity of bone marrow tissue. Microfluidics provides innovative approaches for cell separation based on bio-physical features of single cells. Here we review the physical principles underlying label-free microfluidic sorting techniques and review their capacity for stem cell selection/sorting from complex (heterogeneous) samples. Copyright © 2016 Elsevier Inc. All rights reserved.

Single-cell Hi-C bridges microscopy and genome-wide sequencing approaches to study 3D chromatin organization.

PubMed

Ulianov, Sergey V; Tachibana-Konwalski, Kikue; Razin, Sergey V

2017-10-01

Recent years have witnessed an explosion of the single-cell biochemical toolbox including chromosome conformation capture (3C)-based methods that provide novel insights into chromatin spatial organization in individual cells. The observations made with these techniques revealed that topologically associating domains emerge from cell population averages and do not exist as static structures in individual cells. Stochastic nature of the genome folding is likely to be biologically relevant and may reflect the ability of chromatin fibers to adopt a number of alternative configurations, some of which could be transiently stabilized and serve regulatory purposes. Single-cell Hi-C approaches provide an opportunity to analyze chromatin folding in rare cell types such as stem cells, tumor progenitors, oocytes, and totipotent cells, contributing to a deeper understanding of basic mechanisms in development and disease. Here, we review key findings of single-cell Hi-C and discuss possible biological reasons and consequences of the inferred dynamic chromatin spatial organization. © 2017 WILEY Periodicals, Inc.

In search of the skeletal stem cell: isolation and separation strategies at the macro/micro scale for skeletal regeneration.

PubMed

Gothard, David; Tare, Rahul S; Mitchell, Peter D; Dawson, Jonathan I; Oreffo, Richard O C

2011-04-07

Skeletal stem cells (SSCs) show great capacity for bone and cartilage repair however, current in vitro cultures are heterogeneous displaying a hierarchy of differentiation potential. SSCs represent the diminutive true multipotent stem cell fraction of bone marrow mononuclear cell (BMMNC) populations. Endeavours to isolate SSCs have generated a multitude of separation methodologies. SSCs were first identified and isolated by their ability to adhere to culture plastic. Once isolated, further separation is achieved via culture in selective or conditioned media (CM). Indeed, preferential SSC growth has been demonstrated through selective in vitro culture conditions. Other approaches have utilised cell morphology (size and shape) as selection criteria. Studies have also targeted SSCs based on their preferential adhesion to specified compounds, individually or in combination, on both macro and microscale platforms. Nevertheless, most of these methods which represent macroscale function with relatively high throughput, yield insufficient purity. Consequently, research has sought to downsize isolation methodologies to the microscale for single cell analysis. The central approach is identification of the requisite cell populations of SSC-specific surface markers that can be targeted for isolation by either positive or negative selection. SELEX and phage display technology provide apt means to sift through substantial numbers of candidate markers. In contrast, single cell analysis is the paramount advantage of microfluidics, a relatively new field for cell biology. Here cells can be separated under continuous or discontinuous flow according to intrinsic phenotypic and physicochemical properties. The combination of macroscale quantity with microscale specificity to generate robust high-throughput (HT) technology for pure SSC sorting, isolation and enrichment offers significant implications therein for skeletal regenerative strategies as a consequence of lab on chip derived methodology.

Preparation of anti-inflammatory mesenchymal stem/precursor cells (MSCs) through sphere formation using hanging-drop culture technique.

PubMed

Bartosh, Thomas J; Ylostalo, Joni H

2014-02-06

Herein, we describe a protocol for preparation of pre-activated anti-inflammatory human mesenchymal stem/precursor cells (MSCs) in 3-D culture without addition of exogenous chemicals or gene-transfer approaches. MSCs are an easily procurable source of multipotent adult stem cells with therapeutic potential largely attributed to their paracrine regulation of inflammation and immunity. However, the culture conditions to prepare the ideal MSCs for cell therapy remain elusive. Furthermore, the reported lag time for activation in experimental models has prompted investigations on pre-activating the cells prior to their administration. In this protocol, standard 2-D culture-expanded MSCs are activated by aggregation into 3-D spheres using hanging-drop cultures. MSC activation is evaluated by real-time PCR and/or ELISA for anti-inflammatory factors (TSG-6, STC-1, PGE2), and by a functional assay using lipopolysaccharide-stimulated macrophage cultures. Further, we elucidate methods to prepare MSC-sphere conditioned medium, intact spheres, and suspension of single cells from spheres for experimental and clinical applications. Copyright © 2014 John Wiley & Sons, Inc.

Preparation of anti-inflammatory mesenchymal stem/precursor cells (MSCs) through sphere formation using hanging drop culture technique

PubMed Central

Bartosh, Thomas J.

2014-01-01

Herein, we describe a protocol for preparation of pre-activated anti-inflammatory human mesenchymal stem/precursor cells (MSCs) in 3D culture without addition of exogenous chemicals or gene transfer approaches. MSCs are an easily procurable source of multipotent adult stem cells with therapeutic potential largely attributed to their paracrine regulation of inflammation and immunity. However, the culture conditions to prepare the ideal MSCs for cell therapy remain elusive. Furthermore, reported lag time for activation in experimental models have prompted investigations to pre-activate the cells prior to their administration. In this protocol, standard 2D culture expanded MSCs are activated by aggregation into 3D spheres using hanging drop cultures. MSC activation is evaluated by real-time PCR and/or ELISA for anti-inflammatory factors (TSG-6, STC-1, PGE2), and by a functional assay using lipopolysaccharide-stimulated macrophage cultures. Furthermore, we elucidate methods to prepare MSC sphere conditioned medium, intact spheres, and suspension of single cells from spheres for experimental and clinical applications. PMID:24510769

Outcomes of autologous or allogeneic stem cell transplantation for non-Hodgkin lymphoma.

PubMed

Reddy, Nishitha M; Oluwole, Olalekan; Greer, John P; Engelhardt, Brian G; Jagasia, Madan H; Savani, Bipin N

2014-01-01

Transplant outcomes of autologous or allogeneic stem cell transplantation (SCT) have not been elucidated as a single cohort in non-Hodgkin lymphoma (NHL). We analyzed the outcomes of 270 adult recipients receiving autologous (auto) SCT (n = 198) or allogeneic (allo) SCT (n = 72) for NHL between the years 2000 and 2010. Five-year overall survival rates for B and T cell NHL were 58% and 50%, respectively (allo-SCT 51% vs. 54% for B and T-cell NHL, and auto-SCT 60% vs. 47% for B and T cell lymphoma, respectively). In multivariate analysis, the number of chemotherapy regimens and disease status pre-SCT were independently associated with long-term outcome after SCT (for both auto- and allo-SCT). We conclude that the type of transplantation offered to patients, based on patient selection and disease-related factors, can achieve long-term survival, highlighting the importance of further improvement in disease control and reducing procedure-related mortality. The role of transplantation needs to be reevaluated in the era of targeted therapy. Copyright © 2014 ISEH - Society for Hematology and Stem Cells. All rights reserved.

CXCR1 remodels the vascular niche to promote hematopoietic stem and progenitor cell engraftment

PubMed Central

Blaser, Bradley W.; Moore, Jessica L.; Hagedorn, Elliott J.; Li, Brian; Riquelme, Raquel; Yang, Song; Zhou, Yi; Tamplin, Owen J.; Binder, Vera

2017-01-01

The microenvironment is an important regulator of hematopoietic stem and progenitor cell (HSPC) biology. Recent advances marking fluorescent HSPCs have allowed exquisite visualization of HSPCs in the caudal hematopoietic tissue (CHT) of the developing zebrafish. Here, we show that the chemokine cxcl8 and its receptor, cxcr1, are expressed by zebrafish endothelial cells, and we identify cxcl8/cxcr1 signaling as a positive regulator of HSPC colonization. Single-cell tracking experiments demonstrated that this is a result of increases in HSPC–endothelial cell “cuddling,” HSPC residency time within the CHT, and HSPC mitotic rate. Enhanced cxcl8/cxcr1 signaling was associated with an increase in the volume of the CHT and induction of cxcl12a expression. Finally, using parabiotic zebrafish, we show that cxcr1 acts HSPC nonautonomously to improve the efficiency of donor HSPC engraftment. This work identifies a mechanism by which the hematopoietic niche remodels to promote HSPC engraftment and suggests that cxcl8/cxcr1 signaling is a potential therapeutic target in patients undergoing hematopoietic stem cell transplantation. PMID:28351983

Vitamin A-Retinoic Acid Signaling Regulates Hematopoietic Stem Cell Dormancy.

PubMed

Cabezas-Wallscheid, Nina; Buettner, Florian; Sommerkamp, Pia; Klimmeck, Daniel; Ladel, Luisa; Thalheimer, Frederic B; Pastor-Flores, Daniel; Roma, Leticia P; Renders, Simon; Zeisberger, Petra; Przybylla, Adriana; Schönberger, Katharina; Scognamiglio, Roberta; Altamura, Sandro; Florian, Carolina M; Fawaz, Malak; Vonficht, Dominik; Tesio, Melania; Collier, Paul; Pavlinic, Dinko; Geiger, Hartmut; Schroeder, Timm; Benes, Vladimir; Dick, Tobias P; Rieger, Michael A; Stegle, Oliver; Trumpp, Andreas

2017-05-18

Dormant hematopoietic stem cells (dHSCs) are atop the hematopoietic hierarchy. The molecular identity of dHSCs and the mechanisms regulating their maintenance or exit from dormancy remain uncertain. Here, we use single-cell RNA sequencing (RNA-seq) analysis to show that the transition from dormancy toward cell-cycle entry is a continuous developmental path associated with upregulation of biosynthetic processes rather than a stepwise progression. In addition, low Myc levels and high expression of a retinoic acid program are characteristic for dHSCs. To follow the behavior of dHSCs in situ, a Gprc5c-controlled reporter mouse was established. Treatment with all-trans retinoic acid antagonizes stress-induced activation of dHSCs by restricting protein translation and levels of reactive oxygen species (ROS) and Myc. Mice maintained on a vitamin A-free diet lose HSCs and show a disrupted re-entry into dormancy after exposure to inflammatory stress stimuli. Our results highlight the impact of dietary vitamin A on the regulation of cell-cycle-mediated stem cell plasticity. VIDEO ABSTRACT. Copyright © 2017. Published by Elsevier Inc.

Cell therapies: realizing the potential of this new dimension to medical therapeutics.

PubMed

Singh, Pawanbir; Williams, David J

2008-08-01

Stem cells promise to treat conditions poorly served by conventional therapeutics. Cells from both embryonic and somatic tissues are being used to create cell therapies for genetic, traumatic and degenerative conditions. The current human, healthcare and fiscal costs of these conditions are significant. This review summarizes the use of stem cells for neurological and cardiac disorders and diabetes to determine the requirements for generic translational research to assist such therapies to be a reality. While there are multiple strategies in each disease area, with no clear favourite, there are clear opportunities in treatments that use a single cell type. A key requirement is to work with pluripotent progenitor cells to cultivate and differentiate a sufficiently large population of functioning cells. Challenges also arise in determining and achieving timely delivery of the correct dose of cells to where they can most effectively treat the disease and best benefit individual patients. (c) 2008 John Wiley & Sons, Ltd.

The evolution of stem-cell transplantation in multiple myeloma.

PubMed

Mahajan, Sarakshi; Tandon, Nidhi; Kumar, Shaji

2018-05-01

Autologous stem-cell transplantation (ASCT) remains an integral part of treatment for previously untreated, and may have value in the treatment of relapsed patients with, multiple myeloma (MM). The addition of novel agents like immunomodulators and proteasome inhibitors as induction therapy before and as consolidation/maintenance therapy after ASCT has led to an improvement in complete response (CR) rates, progression-free survival (PFS) and overall survival (OS). With advances in supportive care, older patients and patients with renal insufficiency are now able to safely undergo the procedure. The data concerning the timing of ASCT (early in the disease course or at first relapse), single versus tandem (double) ASCT and the role and duration of consolidation and maintenance therapy post ASCT remain conflicting. This review aims to discuss the evolution of stem-cell transplant over the past 3 decades and its current role in the context of newer, safer and more effective therapeutic agents.

Treatment with adipose derived mesenchymal stem cells and their conditioned media reverse carrageenan induced paw oedema in db/db mice.

PubMed

Shree, Nitya; Venkategowda, Sunil; Venkatranganna, M V; Bhonde, Ramesh R

2017-06-01

Mesenchymal stem cells are known for anti inflammatory and immunomodulatory activities. The aim of our study was to evaluate the effect of human adipose derived mesenchymal stem cells (hADMSCs) and its conditioned media (CM) on carrageenan induced acute inflammation in db/db mice. We injected 5×10 5 ADMSCs or the CM in the inflamed paw. We assessed the paw volume, serum IL6 levels and histopathology of the paw to reveal the anti inflammatory effect. We observed a single injection of hADMSCs or CM could reverse the inflammation within 24h as evidenced by reduction in paw volume, IL6 levels and histological examination. Our result equivocally demonstrates the role of CM in normalising the inflammation better than hADMSCs. This study will pave way for an alternative to anti inflammatory drugs. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Validation of a single-platform method for hematopoietic CD34+ stem cells enumeration according to accreditation procedure.

PubMed

Massin, Frédéric; Huili, Cai; Decot, Véronique; Stoltz, Jean-François; Bensoussan, Danièle; Latger-Cannard, Véronique

2015-01-01

Stem cells for autologous and allogenic transplantation are obtained from several sources including bone marrow, peripheral blood or cord blood. Accurate enumeration of viable CD34+ hematopoietic stem cells (HSC) is routinely used in clinical settings, especially to monitor progenitor cell mobilization and apheresis. The number of viable CD34+ HSC has also been shown to be the most critical factor in haematopoietic engraftment. The International Society for Cellular Therapy actually recommends the use of single-platform flow cytometry system using 7-AAD as a viability dye. In a way to move routine analysis from a BD FACSCaliburTM instrument to a BD FACSCantoTM II, according to ISO 15189 standard guidelines, we define laboratory performance data of the BDTM Stem Cell Enumeration (SCE) kit on a CE-IVD system including a BD FACSCanto II flow cytometer and the BD FACSCantoTM Clinical Software. InterQCTM software, a real time internet laboratory QC management system developed by VitroTM and distributed by Becton DickinsonTM, was also tested to monitor daily QC data, to define the internal laboratory statistics and to compare them to external laboratories. Precision was evaluated with BDTM Stem Cell Control (high and low) results and the InterQC software, an internet laboratory QC management system by Vitro. This last one drew Levey-Jennings curves and generated numeral statistical parameters allowing detection of potential changes in the system performances as well as interlaboratory comparisons. Repeatability, linearity and lower limits of detection were obtained with routine samples from different origins. Agreement evaluation between BD FACSCanto II system versus BD FACSCalibur system was tested on fresh peripheral blood, freeze-thawed apheresis, fresh bone marrow and fresh cord blood samples. Instrument's measure and staining repeatability clearly evidenced acceptable variability on the different samples tested. Intra- and inter-laboratory CV in CD34+ cell absolute count are consistent and reproducible. Linearity analysis, established between 2 and 329 cells/μl showed a linear relation between expected counts and measured counts (R2=0.97). Linear regression and Bland-Altman representations showed an excellent correlation on samples from different sources between the two systems and allowed the transfer of routine analysis from BD FACSCalibur to BD FACSCanto II. The BD SCE kit provides an accurate measure of the CD34 HSC, and can be used in daily routine to optimize the enumeration of hematopoietic CD34+ stem cells by flow cytometry. Moreover, the InterQC system seems to be a very useful tool for laboratory daily quality monitoring and thus for accreditation.

Development of Defective and Persistent Sendai Virus Vector

PubMed Central

Nishimura, Ken; Sano, Masayuki; Ohtaka, Manami; Furuta, Birei; Umemura, Yoko; Nakajima, Yoshiro; Ikehara, Yuzuru; Kobayashi, Toshihiro; Segawa, Hiroaki; Takayasu, Satoko; Sato, Hideyuki; Motomura, Kaori; Uchida, Eriko; Kanayasu-Toyoda, Toshie; Asashima, Makoto; Nakauchi, Hiromitsu; Yamaguchi, Teruhide; Nakanishi, Mahito

2011-01-01

The ectopic expression of transcription factors can reprogram differentiated tissue cells into induced pluripotent stem cells. However, this is a slow and inefficient process, depending on the simultaneous delivery of multiple genes encoding essential reprogramming factors and on their sustained expression in target cells. Moreover, once cell reprogramming is accomplished, these exogenous reprogramming factors should be replaced with their endogenous counterparts for establishing autoregulated pluripotency. Complete and designed removal of the exogenous genes from the reprogrammed cells would be an ideal option for satisfying this latter requisite as well as for minimizing the risk of malignant cell transformation. However, no single gene delivery/expression system has ever been equipped with these contradictory characteristics. Here we report the development of a novel replication-defective and persistent Sendai virus (SeVdp) vector based on a noncytopathic variant virus, which fulfills all of these requirements for cell reprogramming. The SeVdp vector could accommodate up to four exogenous genes, deliver them efficiently into various mammalian cells (including primary tissue cells and human hematopoietic stem cells) and express them stably in the cytoplasm at a prefixed balance. Furthermore, interfering with viral transcription/replication using siRNA could erase the genomic RNA of SeVdp vector from the target cells quickly and thoroughly. A SeVdp vector installed with Oct4/Sox2/Klf4/c-Myc could reprogram mouse primary fibroblasts quite efficiently; ∼1% of the cells were reprogrammed to Nanog-positive induced pluripotent stem cells without chromosomal gene integration. Thus, this SeVdp vector has potential as a tool for advanced cell reprogramming and for stem cell research. PMID:21138846

Distinctive features of single nucleotide alterations in induced pluripotent stem cells with different types of DNA repair deficiency disorders

PubMed Central

Okamura, Kohji; Sakaguchi, Hironari; Sakamoto-Abutani, Rie; Nakanishi, Mahito; Nishimura, Ken; Yamazaki-Inoue, Mayu; Ohtaka, Manami; Periasamy, Vaiyapuri Subbarayan; Alshatwi, Ali Abdullah; Higuchi, Akon; Hanaoka, Kazunori; Nakabayashi, Kazuhiko; Takada, Shuji; Hata, Kenichiro; Toyoda, Masashi; Umezawa, Akihiro

2016-01-01

Disease-specific induced pluripotent stem cells (iPSCs) have been used as a model to analyze pathogenesis of disease. In this study, we generated iPSCs derived from a fibroblastic cell line of xeroderma pigmentosum (XP) group A (XPA-iPSCs), a rare autosomal recessive hereditary disease in which patients develop skin cancer in the areas of skin exposed to sunlight. XPA-iPSCs exhibited hypersensitivity to ultraviolet exposure and accumulation of single-nucleotide substitutions when compared with ataxia telangiectasia-derived iPSCs that were established in a previous study. However, XPA-iPSCs did not show any chromosomal instability in vitro, i.e. intact chromosomes were maintained. The results were mutually compensating for examining two major sources of mutations, nucleotide excision repair deficiency and double-strand break repair deficiency. Like XP patients, XPA-iPSCs accumulated single-nucleotide substitutions that are associated with malignant melanoma, a manifestation of XP. These results indicate that XPA-iPSCs may serve a monitoring tool (analogous to the Ames test but using mammalian cells) to measure single-nucleotide alterations, and may be a good model to clarify pathogenesis of XP. In addition, XPA-iPSCs may allow us to facilitate development of drugs that delay genetic alteration and decrease hypersensitivity to ultraviolet for therapeutic applications. PMID:27197874

GABA and Gap Junctions in the Development of Synchronized Activity in Human Pluripotent Stem Cell-Derived Neural Networks

PubMed Central

Mäkinen, Meeri Eeva-Liisa; Ylä-Outinen, Laura; Narkilahti, Susanna

2018-01-01

The electrical activity of the brain arises from single neurons communicating with each other. However, how single neurons interact during early development to give rise to neural network activity remains poorly understood. We studied the emergence of synchronous neural activity in human pluripotent stem cell (hPSC)-derived neural networks simultaneously on a single-neuron level and network level. The contribution of gamma-aminobutyric acid (GABA) and gap junctions to the development of synchronous activity in hPSC-derived neural networks was studied with GABA agonist and antagonist and by blocking gap junctional communication, respectively. We characterized the dynamics of the network-wide synchrony in hPSC-derived neural networks with high spatial resolution (calcium imaging) and temporal resolution microelectrode array (MEA). We found that the emergence of synchrony correlates with a decrease in very strong GABA excitation. However, the synchronous network was found to consist of a heterogeneous mixture of synchronously active cells with variable responses to GABA, GABA agonists and gap junction blockers. Furthermore, we show how single-cell distributions give rise to the network effect of GABA, GABA agonists and gap junction blockers. Finally, based on our observations, we suggest that the earliest form of synchronous neuronal activity depends on gap junctions and a decrease in GABA induced depolarization but not on GABAA mediated signaling. PMID:29559893

Targeting insulin resistance in type 2 diabetes via immune modulation of cord blood-derived multipotent stem cells (CB-SCs) in stem cell educator therapy: phase I/II clinical trial.

PubMed

Zhao, Yong; Jiang, Zhaoshun; Zhao, Tingbao; Ye, Mingliang; Hu, Chengjin; Zhou, Huimin; Yin, Zhaohui; Chen, Yana; Zhang, Ye; Wang, Shanfeng; Shen, Jie; Thaker, Hatim; Jain, Summit; Li, Yunxiang; Diao, Yalin; Chen, Yingjian; Sun, Xiaoming; Fisk, Mary Beth; Li, Heng

2013-07-09

The prevalence of type 2 diabetes (T2D) is increasing worldwide and creating a significant burden on health systems, highlighting the need for the development of innovative therapeutic approaches to overcome immune dysfunction, which is likely a key factor in the development of insulin resistance in T2D. It suggests that immune modulation may be a useful tool in treating the disease. In an open-label, phase 1/phase 2 study, patients (N=36) with long-standing T2D were divided into three groups (Group A, oral medications, n=18; Group B, oral medications+insulin injections, n=11; Group C having impaired β-cell function with oral medications+insulin injections, n=7). All patients received one treatment with the Stem Cell Educator therapy in which a patient's blood is circulated through a closed-loop system that separates mononuclear cells from the whole blood, briefly co-cultures them with adherent cord blood-derived multipotent stem cells (CB-SCs), and returns the educated autologous cells to the patient's circulation. Clinical findings indicate that T2D patients achieve improved metabolic control and reduced inflammation markers after receiving Stem Cell Educator therapy. Median glycated hemoglobin (HbA1C) in Group A and B was significantly reduced from 8.61%±1.12 at baseline to 7.25%±0.58 at 12 weeks (P=2.62E-06), and 7.33%±1.02 at one year post-treatment (P=0.0002). Homeostasis model assessment (HOMA) of insulin resistance (HOMA-IR) demonstrated that insulin sensitivity was improved post-treatment. Notably, the islet beta-cell function in Group C subjects was markedly recovered, as demonstrated by the restoration of C-peptide levels. Mechanistic studies revealed that Stem Cell Educator therapy reverses immune dysfunctions through immune modulation on monocytes and balancing Th1/Th2/Th3 cytokine production. Clinical data from the current phase 1/phase 2 study demonstrate that Stem Cell Educator therapy is a safe approach that produces lasting improvement in metabolic control for individuals with moderate or severe T2D who receive a single treatment. In addition, this approach does not appear to have the safety and ethical concerns associated with conventional stem cell-based approaches. ClinicalTrials.gov number, NCT01415726.

Germ stem cells are active in postnatal mouse ovary under physiological conditions

PubMed Central

Guo, Kun; Li, Chao-hui; Wang, Xin-yi; He, Da-jian; Zheng, Ping

2016-01-01

STUDY HYPOTHESIS Are active ovarian germ stem cells present in postnatal mouse ovaries under physiological conditions? STUDY FINDING Active ovarian germ stem cells exist and function in adult mouse ovaries under physiological conditions. WHAT IS KNOWN ALREADY In vitro studies suggested the existence of germ stem cells in postnatal ovaries of mouse, pig and human. However, in vivo studies provided evidence against the existence of active germ stem cells in postnatal mouse ovaries. Thus, it remains controversial whether such germ stem cells really exist and function in vivo in postnatal mammalian ovaries. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Octamer-binding transcription factor 4 (Oct4)-MerCreMer transgenic mice were crossed with R26R-enhanced yellow fluorescent protein (EYFP) mice to establish a tamoxifen-inducible tracing system so that Oct4-expressing potential ovarian germ stem cells in young adult mice (5–6 weeks old) can be labeled with EYFP. The germ cell activities of DNA replication, mitotic division, entry into meiosis and progression to primordial follicle stage were investigated by means of immunofluorescent staining of ovarian tissues collected at different time points post-tamoxifen injection (1 day, 3 days, 2 months and 4 months). Meiosis entry and primordial follicle formation were also measured by EYFP-labeled single-cell RT–PCR. Germ cell proliferation and mitotic division were examined through 5-bromodeoxyuridine triphosphate incorporation assay. At each time point, ovaries from two to three animals were used for each set of experiment. MAIN RESULTS AND THE ROLE OF CHANCE By labeling the Oct4-expressing small germ cells and tracing their fates for up to 4 months, we observed persistent meiosis entry and primordial follicle replenishment. Furthermore, we captured the transient processes of mitotic DNA replication as well as mitotic division of the marked germ cells at various time periods after tracing. These lines of evidence unambiguously support the presence of active germ stem cells in postnatal ovaries and their function in replenishing primordial follicle pool under physiological conditions. Moreover, we pointed out that Oct4+ deleted in azoospermia-like (Dazl)− but not Oct4+Dazl+ or Oct4+ DEAD (Asp–Glu–Ala–Asp) Box Polypeptide 4 (Ddx4)+ cells contain a population of germ stem cells in mouse ovary. LIMITATIONS, REASONS FOR CAUTION This study was conducted in mice. Whether or not the results are applicable to human remain unclear. The future work should aim at identifying the specific ovarian germ stem cell marker and evaluating the significance of these stem cells to normal ovarian function. WIDER IMPLICATIONS OF THE FINDINGS Clarifying the existence of active germ stem cells and their functional significance in postnatal mammalian ovaries could provide new insights in understanding the mechanism of ovarian aging and failure. LARGE SCALE DATA Not applicable. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the National Key Basic Research Program of China (grant number 2012CBA01300) and the National Natural Science Foundation of China to P.Z. (31571484). No competing interests are reported. PMID:26916381

Naïve Induced Pluripotent Stem Cells Generated From β-Thalassemia Fibroblasts Allow Efficient Gene Correction With CRISPR/Cas9.

PubMed

Yang, Yuanyuan; Zhang, Xiaobai; Yi, Li; Hou, Zhenzhen; Chen, Jiayu; Kou, Xiaochen; Zhao, Yanhong; Wang, Hong; Sun, Xiao-Fang; Jiang, Cizhong; Wang, Yixuan; Gao, Shaorong

2016-01-01

Conventional primed human embryonic stem cells and induced pluripotent stem cells (iPSCs) exhibit molecular and biological characteristics distinct from pluripotent stem cells in the naïve state. Although naïve pluripotent stem cells show much higher levels of self-renewal ability and multidifferentiation capacity, it is unknown whether naïve iPSCs can be generated directly from patient somatic cells and will be superior to primed iPSCs. In the present study, we used an established 5i/L/FA system to directly reprogram fibroblasts of a patient with β-thalassemia into transgene-free naïve iPSCs with molecular signatures of ground-state pluripotency. Furthermore, these naïve iPSCs can efficiently produce cross-species chimeras. Importantly, using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease genome editing system, these naïve iPSCs exhibit significantly improved gene-correction efficiencies compared with the corresponding primed iPSCs. Furthermore, human naïve iPSCs could be directly generated from noninvasively collected urinary cells, which are easily acquired and thus represent an excellent cell resource for further clinical trials. Therefore, our findings demonstrate the feasibility and superiority of using patient-specific iPSCs in the naïve state for disease modeling, gene editing, and future clinical therapy. In the present study, transgene-free naïve induced pluripotent stem cells (iPSCs) directly converted from the fibroblasts of a patient with β-thalassemia in a defined culture system were generated. These naïve iPSCs, which show ground-state pluripotency, exhibited significantly improved single-cell cloning ability, recovery capacity, and gene-targeting efficiency compared with conventional primed iPSCs. These results provide an improved strategy for personalized treatment of genetic diseases such as β-thalassemia. ©AlphaMed Press.

Naïve Induced Pluripotent Stem Cells Generated From β-Thalassemia Fibroblasts Allow Efficient Gene Correction With CRISPR/Cas9

PubMed Central

Yang, Yuanyuan; Zhang, Xiaobai; Yi, Li; Hou, Zhenzhen; Chen, Jiayu; Kou, Xiaochen; Zhao, Yanhong; Wang, Hong; Sun, Xiao-Fang; Jiang, Cizhong

2016-01-01

Conventional primed human embryonic stem cells and induced pluripotent stem cells (iPSCs) exhibit molecular and biological characteristics distinct from pluripotent stem cells in the naïve state. Although naïve pluripotent stem cells show much higher levels of self-renewal ability and multidifferentiation capacity, it is unknown whether naïve iPSCs can be generated directly from patient somatic cells and will be superior to primed iPSCs. In the present study, we used an established 5i/L/FA system to directly reprogram fibroblasts of a patient with β-thalassemia into transgene-free naïve iPSCs with molecular signatures of ground-state pluripotency. Furthermore, these naïve iPSCs can efficiently produce cross-species chimeras. Importantly, using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease genome editing system, these naïve iPSCs exhibit significantly improved gene-correction efficiencies compared with the corresponding primed iPSCs. Furthermore, human naïve iPSCs could be directly generated from noninvasively collected urinary cells, which are easily acquired and thus represent an excellent cell resource for further clinical trials. Therefore, our findings demonstrate the feasibility and superiority of using patient-specific iPSCs in the naïve state for disease modeling, gene editing, and future clinical therapy. Significance In the present study, transgene-free naïve induced pluripotent stem cells (iPSCs) directly converted from the fibroblasts of a patient with β-thalassemia in a defined culture system were generated. These naïve iPSCs, which show ground-state pluripotency, exhibited significantly improved single-cell cloning ability, recovery capacity, and gene-targeting efficiency compared with conventional primed iPSCs. These results provide an improved strategy for personalized treatment of genetic diseases such as β-thalassemia. PMID:26676643

Skeletal muscle-derived interstitial progenitor cells (PICs) display stem cell properties, being clonogenic, self-renewing, and multi-potent in vitro and in vivo.

PubMed

Cottle, Beverley J; Lewis, Fiona C; Shone, Victoria; Ellison-Hughes, Georgina M

2017-07-04

The development of cellular therapies to treat muscle wastage with disease or age is paramount. Resident muscle satellite cells are not currently regarded as a viable cell source due to their limited migration and growth capability ex vivo. This study investigated the potential of muscle-derived PW1 + /Pax7 - interstitial progenitor cells (PICs) as a source of tissue-specific stem/progenitor cells with stem cell properties and multipotency. Sca-1 + /PW1 + PICs were identified on tissue sections from hind limb muscle of 21-day-old mice, isolated by magnetic-activated cell sorting (MACS) technology and their phenotype and characteristics assessed over time in culture. Green fluorescent protein (GFP)-labelled PICs were used to determine multipotency in vivo in a tumour formation assay. Isolated PICs expressed markers of pluripotency (Oct3/4, Sox2, and Nanog), were clonogenic, and self-renewing with >60 population doublings, and a population doubling time of 15.8 ± 2.9 h. PICs demonstrated an ability to generate both striated and smooth muscle, whilst also displaying the potential to differentiate into cell types of the three germ layers both in vitro and in vivo. Moreover, PICs did not form tumours in vivo. These findings open new avenues for a variety of solid tissue engineering and regeneration approaches, utilising a single multipotent stem cell type isolated from an easily accessible source such as skeletal muscle.

The union of somatic gonad precursors and primordial germ cells during C. elegans embryogenesis

PubMed Central

Rohrschneider, Monica R.; Nance, Jeremy

2013-01-01

Somatic gonadal niche cells control the survival, differentiation, and proliferation of germline stem cells. The establishment of this niche-stem cell relationship is critical, and yet the precursors to these two cell types are often born at a distance from one another. The simple C. elegans gonadal primordium, which contains two somatic gonad precursors (SGPs) and two primordial germ cells (PGCs), provides an accessible model for determining how stem cell and niche cell precursors first assemble during development. To visualize the morphogenetic events that lead to formation of the gonadal primordium, we generated transgenic strains to label the cell membranes of the SGPs and PGCs and captured time-lapse movies as the gonadal primordium formed. We identify three distinct phases of SGP behavior: posterior migration along the endoderm towards the PGCs, extension of a single long projection around the adjacent PGC, and a dramatic wrapping over the PGC surfaces. We show that the endoderm and PGCs are dispensable for SGP posterior migration and initiation of projections. However, both tissues are required for the final positioning of the SGPs and the morphology of their projections, and PGCs are absolutely required for SGP wrapping behaviors. Finally, we demonstrate that the basement membrane component laminin, which localizes adjacent to the developing gonadal primordium, is required to prevent the SGPs from over-extending past the PGCs. Our findings provide a foundation for understanding the cellular and molecular regulation of the establishment of a niche-stem cell relationship. PMID:23562590

Unfinished business: ongoing ethical exceptionalism in the oversight of human pluripotent stem cell research in Canada.

PubMed

Baylis, Françoise; Downie, Jocelyn

2012-01-01

In this article, we critically examine the arguments for and against the exceptional status given human pluripotent stem cell research in Canada (through the latest [December 2010] revision of the Tri-Council Policy Statement: Ethical Conduct for Research Involving Humans), and conclude that this exceptionalism is unwarranted and ethically unsound. In our view, the three federal research granting agencies should honor their longstanding commitment that researchers, research sponsors, and Research Ethics Boards in Canada have access to "a single reference document for all research involving humans conducted under the auspices of institutions eligible for Agency funding." As well, responsibility for the development, interpretation, and implementation of Canada's research ethics guidelines should be under the authority of a single oversight body that is independent of the federal research granting Agencies.

Generation of murine induced pluripotent stem cells by using high-density distributed electrodes network.

PubMed

Lu, Ming-Yu; Li, Zhihong; Hwang, Shiaw-Min; Linju Yen, B; Lee, Gwo-Bin

2015-09-01

This study reports a robust method of gene transfection in a murine primary cell model by using a high-density electrodes network (HDEN). By demonstrating high cell viability after gene transfection and successful expression of transgenes including fluorescent proteins, the HDEN device shows great promise as a solution in which reprogramming efficiency using non-viral induction for generation of murine induced pluripotent stem cells (iPSCs) is optimized. High and steady transgene expression levels in host cells of iPSCs can be demonstrated using this method. Moreover, the HDEN device achieved successful gene transfection with a low voltage of less than 180 V while requiring relatively low cell numbers (less than 1.5 × 10(4) cells). The results are comparable to current conventional methods, demonstrating a reasonable fluorescent-plasmid transfection rate (42.4% in single transfection and 24.5% in triple transfection) and high cell viability of over 95%. The gene expression levels of each iPSC factor was measured to be over 10-fold higher than that reported in previous studies using a single mouse embryonic fibroblast cell. Our results demonstrate that the generation of iPSCs using HDEN transfection of plasmid DNA may be a feasible and safe alternative to using viral transfection methods in the near future.

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

Epigenetics of cell fate reprogramming and its implications for neurological disorders modelling.

PubMed

Grzybek, Maciej; Golonko, Aleksandra; Walczak, Marta; Lisowski, Pawel

2017-03-01

The reprogramming of human induced pluripotent stem cells (hiPSCs) proceeds in a stepwise manner with reprogramming factors binding and epigenetic composition changes during transition to maintain the epigenetic landscape, important for pluripotency. There arises a question as to whether the aberrant epigenetic state after reprogramming leads to epigenetic defects in induced stem cells causing unpredictable long term effects in differentiated cells. In this review, we present a comprehensive view of epigenetic alterations accompanying reprogramming, cell maintenance and differentiation as factors that influence applications of hiPSCs in stem cell based technologies. We conclude that sample heterogeneity masks DNA methylation signatures in subpopulations of cells and thus believe that beside a genetic evaluation, extensive epigenomic screening should become a standard procedure to ensure hiPSCs state before they are used for genome editing and differentiation into neurons of interest. In particular, we suggest that exploitation of the single-cell composition of the epigenome will provide important insights into heterogeneity within hiPSCs subpopulations to fast forward development of reliable hiPSC-based analytical platforms in neurological disorders modelling and before completed hiPSC technology will be implemented in clinical approaches. Copyright © 2016 Elsevier Inc. All rights reserved.

Treatment of AVN Using Autologous BM Stem Cells and Activated Platelet-Derived Growth Factor Concentrates.

PubMed

Nandeesh, Nagaraj H; Janardhan, Kiranmayee; Subramanian, Vignesh; Ashtekar, Abhishek Bhushan; Srikruthi, Nandagiri; Koka, Prasad S; Deb, Kaushik

Avascular Necrosis (AVN) of hip is a devastating condition seen in younger individuals. It is the ischemic death of the constituents of the bone cartilage of the hip. The femoral head (FH) is the most common site for AVN. It results from interruption of the normal blood flow to the FH that fits into the hip socket. Earlier studies using autologous bone marrow stem cell concentrate injections have shown encouraging results with average success rates. The current study was designed to improve significantly the cartilage regeneration and clinical outcome. Total of 48 patients underwent autologous bone marrow stem cell and activated platelet-rich plasma derived growth factor concentrate (PRP-GFC) therapy for early and advanced stages AVN of femoral head in a single multi-specialty center. The total treatment was divided into three phases. In the phase I, all the clinical diagnostic measurements such as magnetic resonance imaging (MRI), computed tomography (CT) etc. with respect to the AVN patients and bone marrow aspiration from posterior iliac spine from the patients were carried out. In the phase II, isolation of stem cells and preparation from the patients were performed. Subsequently, in phase III, the stem cells and PRP- GFCs were transplanted in the enrolled patients. Ninety three percent of the enrolled AVN patients showed marked enhancement in the hip bone joint space (more than 3mm) after combined stem cells and PRP-GFC treatment as evidenced by comparison of the pre- and post-treatment MRI data thus indicative of regeneration of cartilage. The treated patients showed significant improvement in their motor function, cartilage regrowth (3 to 10mm), and high satisfaction in the two-year follow-up. Combination of stem cell and PRP-GFC therapy has shown promising cartilage regeneration in 45 out of 48 patients of AVN. This study clearly demonstrates the safety and efficacy of this treatment. Larger numbers of patients need to be evaluated to better understand the efficacy of the combined stem cell and PRP-GFC therapy on AVN patients.

Next-generation sequencing traces human induced pluripotent stem cell lines clonally generated from heterogeneous cancer tissue.

PubMed

Ishikawa, Tetsuya

2017-05-26

To investigate genotype variation among induced pluripotent stem cell (iPSC) lines that were clonally generated from heterogeneous colon cancer tissues using next-generation sequencing. Human iPSC lines were clonally established by selecting independent single colonies expanded from heterogeneous primary cells of S-shaped colon cancer tissues by retroviral gene transfer ( OCT3/4 , SOX2 , and KLF4 ). The ten iPSC lines, their starting cancer tissues, and the matched adjacent non-cancerous tissues were analyzed using next-generation sequencing and bioinformatics analysis using the human reference genome hg19. Non-synonymous single-nucleotide variants (SNVs) (missense, nonsense, and read-through) were identified within the target region of 612 genes related to cancer and the human kinome. All SNVs were annotated using dbSNP135, CCDS, RefSeq, GENCODE, and 1000 Genomes. The SNVs of the iPSC lines were compared with the genotypes of the cancerous and non-cancerous tissues. The putative genotypes were validated using allelic depth and genotype quality. For final confirmation, mutated genotypes were manually curated using the Integrative Genomics Viewer. In eight of the ten iPSC lines, one or two non-synonymous SNVs in EIF2AK2 , TTN , ULK4 , TSSK1B , FLT4 , STK19 , STK31 , TRRAP , WNK1 , PLK1 or PIK3R5 were identified as novel SNVs and were not identical to the genotypes found in the cancer and non-cancerous tissues. This result suggests that the SNVs were de novo or pre-existing mutations that originated from minor populations, such as multifocal pre-cancer (stem) cells or pre-metastatic cancer cells from multiple, different clonal evolutions, present within the heterogeneous cancer tissue. The genotypes of all ten iPSC lines were different from the mutated ERBB2 and MKNK2 genotypes of the cancer tissues and were identical to those of the non-cancerous tissues and that found in the human reference genome hg19. Furthermore, two of the ten iPSC lines did not have any confirmed mutated genotypes, despite being derived from cancerous tissue. These results suggest that the traceability and preference of the starting single cells being derived from pre-cancer (stem) cells, stroma cells such as cancer-associated fibroblasts, and immune cells that co-existed in the tissues along with the mature cancer cells. The genotypes of iPSC lines derived from heterogeneous cancer tissues can provide information on the type of starting cell that the iPSC line was generated from.

Enhancement of the repair of dog alveolar cleft by an autologous iliac bone, bone marrow-derived mesenchymal stem cell, and platelet-rich fibrin mixture.

PubMed

Yuanzheng, Chen; Yan, Gao; Ting, Li; Yanjie, Fu; Peng, Wu; Nan, Bai

2015-05-01

Autologous bone graft has been regarded as the criterion standard for the repair of alveolar cleft. However, the most prominent issue in alveolar cleft treatment is the high absorption rate of the bone graft. The authors' objective was to investigate the effects of an autologous iliac bone, bone marrow-derived mesenchymal stem cell, and platelet-rich fibrin mixture on the repair of dog alveolar cleft. Twenty beagle dogs with unilateral alveolar clefts created by surgery were divided randomly into four groups: group A underwent repair with an autologous iliac bone, bone marrow-derived mesenchymal stem cell, and platelet-rich fibrin mixture; group B underwent repair with autologous iliac bone and bone marrow-derived mesenchymal stem cells; group C underwent repair with autologous iliac bone and platelet-rich fibrin; and group D underwent repair with autologous iliac bone as the control. One day and 6 months after transplantation, the transplant volumes and bone mineral density were assessed by quantitative computed tomography. All of the transplants were harvested for hematoxylin and eosin staining 6 months later. Bone marrow-derived mesenchymal stem cells and platelet-rich fibrin transplants formed the greatest amounts of new bone among the four groups. The new bone formed an extensive union with the underlying maxilla in groups A, B, and C. Transplants with the bone marrow-derived mesenchymal stem cells, platelet-rich fibrin, and their mixture retained the majority of their initial volume, whereas the transplants in the control group showed the highest absorption rate. Bone mineral density of transplants with the bone marrow-derived mesenchymal stem cells, platelet-rich fibrin, and their mixture 6 months later was significantly higher than in the control group (p < 0.05), and was the highest in bone marrow-derived mesenchymal stem cells and platelet-rich fibrin mixed transplants. Hematoxylin and eosin staining showed that the structure of new bones formed the best in group A. Both bone marrow-derived mesenchymal stem cells and platelet-rich fibrin are capable of improving the repair of dog alveolar cleft, and the mixture of them is more potent than each one of them used singly for enhancing new bone regeneration.

Laminin enhances the growth of human neural stem cells in defined culture media

PubMed Central

Hall, Peter E; Lathia, Justin D; Caldwell, Maeve A; ffrench-Constant, Charles

2008-01-01

Background Human neural stem cells (hNSC) have the potential to provide novel cell-based therapies for neurodegenerative conditions such as multiple sclerosis and Parkinson's disease. In order to realise this goal, protocols need to be developed that allow for large quantities of hNSC to be cultured efficiently. As such, it is important to identify factors which enhance the growth of hNSC. In vivo, stem cells reside in distinct microenvironments or niches that are responsible for the maintenance of stem cell populations. A common feature of niches is the presence of the extracellular matrix molecule, laminin. Therefore, this study investigated the effect of exogenous laminin on hNSC growth. Results To measure hNSC growth, we established culture conditions using B27-supplemented medium that enable neurospheres to grow from human neural cells plated at clonal densities. Limiting dilution assays confirmed that neurospheres were derived from single cells at these densities. Laminin was found to increase hNSC numbers as measured by this neurosphere formation. The effect of laminin was to augment the proliferation/survival of the hNSC, rather than promoting the undifferentiated state. In agreement, apoptosis was reduced in dissociated neurospheres by laminin in an integrin β1-dependent manner. Conclusion The addition of laminin to the culture medium enhances the growth of hNSC, and may therefore aid their large-scale production. PMID:18651950

Controlling major cellular processes of human mesenchymal stem cells using microwell structures.

PubMed

Xu, Xun; Wang, Weiwei; Kratz, Karl; Fang, Liang; Li, Zhengdong; Kurtz, Andreas; Ma, Nan; Lendlein, Andreas

2014-12-01

Directing stem cells towards a desired location and function by utilizing the structural cues of biomaterials is a promising approach for inducing effective tissue regeneration. Here, the cellular response of human adipose-derived mesenchymal stem cells (hADSCs) to structural signals from microstructured substrates comprising arrays of square-shaped or round-shaped microwells is explored as a transitional model between 2D and 3D systems. Microwells with a side length/diameter of 50 μm show advantages over 10 μm and 25 μm microwells for accommodating hADSCs within single microwells rather than in the inter-microwell area. The cell morphologies are three-dimensionally modulated by the microwell structure due to differences in focal adhesion and consequent alterations of the cytoskeleton. In contrast to the substrate with 50 μm round-shaped microwells, the substrate with 50 μm square-shaped microwells promotes the proliferation and osteogenic differentiation potential of hADSCs but reduces the cell migration velocity and distance. Such microwell shape-dependent modulatory effects are highly associated with Rho/ROCK signaling. Following ROCK inhibition, the differences in migration, proliferation, and osteogenesis between cells on different substrates are diminished. These results highlight the possibility to control stem cell functions through the use of structured microwells combined with the manipulation of Rho/ROCK signaling. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

An RNA tool kit to study the status of mouse ES cells: sex determination and stemness.

PubMed

Jay, F; Ciaudo, C

2013-09-01

Mouse embryonic stem cells (mESCs) are pluripotent stem cells derived from the inner cell mass of the blastocyst. They can be maintained under controlled culture conditions in a pluripotent state, or be induced to differentiate into all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm. Several studies have characterised the coding and non-coding (nc) RNA repertoires of mESCs, uncovering highly dynamic variations during the process of differentiation, but also qualitative differences pertaining to sex. For example, up-regulation of the long non-coding RNA Xist on the X chromosome induces gene silencing and X inactivation exclusively during female mESC differentiation. In contrast, specific small RNAs have been shown to be up-regulated during male mESC differentiation. Here, we illustrate how a small set of key coding and ncRNAs can be exploited as dynamic and sensitive markers of the stemness and/or the differentiation status of male or female mESC lines. We describe adapted techniques for the extended characterization and analysis of mESCs from as little material as that cultured in a single 75cm(2) flask. Copyright © 2013 Elsevier Inc. All rights reserved.

Stem Cells: A Renaissance in Human Biology Research.

PubMed

Wu, Jun; Izpisua Belmonte, Juan Carlos

2016-06-16

The understanding of human biology and how it relates to that of other species represents an ancient quest. Limited access to human material, particularly during early development, has restricted researchers to only scratching the surface of this inherently challenging subject. Recent technological innovations, such as single cell "omics" and human stem cell derivation, have now greatly accelerated our ability to gain insights into uniquely human biology. The opportunities afforded to delve molecularly into scarce material and to model human embryogenesis and pathophysiological processes are leading to new insights of human development and are changing our understanding of disease and choice of therapy options. Copyright © 2016 Elsevier Inc. All rights reserved.

Laser bioprinting of human induced pluripotent stem cells-the effect of printing and biomaterials on cell survival, pluripotency, and differentiation.

PubMed

Koch, Lothar; Deiwick, Andrea; Franke, Annika; Schwanke, Kristin; Haverich, Axel; Zweigerdt, Robert; Chichkov, Boris

2018-04-25

Research on human induced pluripotent stem cells (hiPSCs) is one of the fastest growing fields in biomedicine. Generated from patient's own somatic cells, hiPSCs can be differentiated towards all functional cell types and returned to the patient without immunological concerns. 3D printing of hiPSCs could enable the generation of functional organs for replacement therapies or realization of organ-on-chip systems for individualized medicine. Printing of living cells was demonstrated with immortalized cell lines, primary cells, and adult stem cells with different printing technologies and biomaterials. However, hiPSCs are more sensitive to handling procedures, in particular, when dissociated into single cells. Both pluripotency and directed differentiation are influenced by numerous environmental factors including culture media, biomaterials, and cell density. Notably, existing literature on the effect of applied biomaterials on pluripotency is rather ambiguous. In this study, laser bioprinting of undifferentiated hiPSCs in combination with different biomaterials was performed and the impact on cells' behavior, pluripotency, and differentiation was investigated. Our findings suggest that hiPSCs are indeed more sensitive to the applied biomaterials, but not to laser printing itself. With appropriate biomaterials, such as the hyaluronic acid based solutions applied in this study, hiPSCs can be successfully laser printed without losing their pluripotency.

Evaluating Interaction of Cord Blood Hematopoietic Stem/Progenitor Cells with Functionally Integrated Three-Dimensional Microenvironments.

PubMed

Mokhtari, Saloomeh; Baptista, Pedro M; Vyas, Dipen A; Freeman, Charles Jordan; Moran, Emma; Brovold, Matthew; Llamazares, Guillermo A; Lamar, Zanneta; Porada, Christopher D; Soker, Shay; Almeida-Porada, Graça

2018-03-01

Despite advances in ex vivo expansion of cord blood-derived hematopoietic stem/progenitor cells (CB-HSPC), challenges still remain regarding the ability to obtain, from a single unit, sufficient numbers of cells to treat an adolescent or adult patient. We and others have shown that CB-HSPC can be expanded ex vivo in two-dimensional (2D) cultures, but the absolute percentage of the more primitive stem cells decreases with time. During development, the fetal liver is the main site of HSPC expansion. Therefore, here we investigated, in vitro, the outcome of interactions of primitive HSPC with surrogate fetal liver environments. We compared bioengineered liver constructs made from a natural three-dimensional-liver-extracellular-matrix (3D-ECM) seeded with hepatoblasts, fetal liver-derived (LvSt), or bone marrow-derived stromal cells, to their respective 2D culture counterparts. We showed that the inclusion of cellular components within the 3D-ECM scaffolds was necessary for maintenance of HSPC viability in culture, and that irrespective of the microenvironment used, the 3D-ECM structures led to the maintenance of a more primitive subpopulation of HSPC, as determined by flow cytometry and colony forming assays. In addition, we showed that the timing and extent of expansion depends upon the biological component used, with LvSt providing the optimal balance between preservation of primitive CB HSPC and cellular differentiation. Stem Cells Translational Medicine 2018;7:271-282. © 2018 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

The influence of prostatic anatomy and neurotrophins on basal prostate epithelial progenitor cells.

PubMed

Höfner, Thomas; Klein, Corinna; Eisen, Christian; Rigo-Watermeier, Teresa; Haferkamp, Axel; Trumpp, Andreas; Sprick, Martin R

2016-01-01

Based on findings of surface marker, protein screens as well as the postulated near-urethral location of the prostate stem cell niche, we were interested whether androgen ablation, distinct anatomic regions within the prostate or neurotrophins have an influence on basal prostate epithelial progenitor cells (PESCs). Microdissection of the prostate, enzymatic digestion, and preparation of single cells was performed from murine and human prostates. Adult PESC marker expressions were compared between a group of C57BL/6 mice and a separate group of castrated C57BL/6 mice. Surface markers CD13/CD271 on human prostate epithelial progenitor cells were evaluated by FACS analyses in cells cultured under novel stem cell conditions. The effect of neurotrophins NGF, NT3, and BDNF were evaluated with respect to their influence on proliferation and activation of human basal PESCs in vitro. We demonstrate the highest percentage of CD49f+ and Trop2+ expressing cells in the urethra near prostatic regions of WT mice (Trop2+ proximal: 10% vs. distal to the urethra: 3%, P < 0.001). While a marked increase of Trop2 expressing cells can be measured both in the proximal and distal prostatic regions after castration, the most prominent increase in Trop2+ cells can be measured in the prostatic tissue distant to the urethra. Furthermore, we demonstrate that the proportion of syndecan-1 expressing cells greatly increases in the regions proximal to the urethra after castration (WT: 5% vs. castrated: 40%). We identified heterogeneous CD13 and nerve growth factor receptor (p75(NGFR), CD271) expression on CD49f(+)/TROP2(high) human basal PESCs. Addition of the neurotrophins NT3, BDNF, and NGF to the stem cell media led to a marked temporary increase in the proliferation of human basal PESCs. Our results in mice support the model, in which the proximal urethral region contains the prostate stem cell niche while a stronger androgen-dependent regulation of adult prostate stem cells can be found in the peripheral prostatic tissue. Neutrophin signaling via nerve growth factor receptor is possibly involved in human prostate stem cell homeostasis. © 2015 Wiley Periodicals, Inc.

i-bodies, Human Single Domain Antibodies That Antagonize Chemokine Receptor CXCR4*

PubMed Central

Dolezal, Olan; Cao, Benjamin; See, Heng B.; Pfleger, Kevin D. G.; Gorry, Paul R.; Pow, Andrew; Viduka, Katerina; Lim, Kevin; Lu, Bernadine G. C.; Chang, Denison H. C.; Murray-Rust, Thomas; Dogovski, Con; Doerflinger, Marcel; Zhang, Yuan; Parisi, Kathy; Casey, Joanne L.; Nuttall, Stewart D.; Foley, Michael

2016-01-01

CXCR4 is a G protein-coupled receptor with excellent potential as a therapeutic target for a range of clinical conditions, including stem cell mobilization, cancer prognosis and treatment, fibrosis therapy, and HIV infection. We report here the development of a fully human single-domain antibody-like scaffold termed an “i-body,” the engineering of which produces an i-body library possessing a long complementarity determining region binding loop, and the isolation and characterization of a panel of i-bodies with activity against human CXCR4. The CXCR4-specific i-bodies show antagonistic activity in a range of in vitro and in vivo assays, including inhibition of HIV infection, cell migration, and leukocyte recruitment but, importantly, not the mobilization of hematopoietic stem cells. Epitope mapping of the three CXCR4 i-bodies AM3-114, AM4-272, and AM3-523 revealed binding deep in the binding pocket of the receptor. PMID:27036939

Delayed effects of rhG-CSF mobilization treatment and apheresis on circulating CD34+ and CD34+ Thy-1dim CD38- progenitor cells, and lymphoid subsets in normal stem cell donors for allogeneic transplantation.

PubMed

Körbling, M; Anderlini, P; Durett, A; Maadani, F; Bojko, P; Seong, D; Giralt, S; Khouri, I; Andersson, B; Mehra, R; vanBesien, K; Mirza, N; Przepiorka, D; Champlin, R

1996-12-01

Allogeneic transplantation of peripheral blood progenitor cells (PBPC) is emerging as a new stem cell transplant modality. Rather than undergoing general anesthesia for bone marrow harvest, normal blood stem cell donors are subjected to rhG-CSF mobilization treatment followed by single or multiple apheresis. Whereas the effects of cytokine treatment and apheresis on stem cell peripheralization and collection have been described, little is known about delayed effects of rhG-CSF treatment and apheresis on a normal hematopoietic system, and there are no long-term data that address safety issues. Ten normal, patient-related donors underwent a 3 or 4 day rhG-CSF (filgrastim) treatment (12 micrograms/kg/day) followed by single or tandem apheresis. We monitored peripheral blood (PB) cellularity including CD34+ and lymphoid subsets at baseline, during cytokine treatment, prior to apheresis, and at days 2, 4, 7, 30 and 100 post-apheresis. The PB progenitor cell concentration peak prior to apheresis was followed by a nadir by day 7 and normalized by day 30, with the exception of the most primitive CD34+ Thy-1dim CD38- progenitor subset that reached a nadir by day 30. Lymphoid subsets such as CD3, 4, 8, suppressor cells (CD3+ 4- 8- TCR+ alpha beta), and B cells (CD19+) showed a similar pattern with a nadir concentration by day 7, followed, except for B cells, by a rebound by day 30 and subnormal counts at day 100. The PB concentrations of hemoglobin and platelets dropped mainly due to the apheresis procedure itself, and normalized by day 30. With cytokine treatment, the PB alkaline phosphatase and lactate dehydrogenase concentrations increased 2.2- and 2.8-fold, respectively, over baseline, and returned to normal range by day 30. Based on the preliminary nature of this study, the clinical relevance of these findings is still unclear.

Single-cell RNA-sequencing: The future of genome biology is now

PubMed Central

Picelli, Simone

2017-01-01

ABSTRACT Genome-wide single-cell analysis represents the ultimate frontier of genomics research. In particular, single-cell RNA-sequencing (scRNA-seq) studies have been boosted in the last few years by an explosion of new technologies enabling the study of the transcriptomic landscape of thousands of single cells in complex multicellular organisms. More sensitive and automated methods are being continuously developed and promise to deliver better data quality and higher throughput with less hands-on time. The outstanding amount of knowledge that is going to be gained from present and future studies will have a profound impact in many aspects of our society, from the introduction of truly tailored cancer treatments, to a better understanding of antibiotic resistance and host-pathogen interactions; from the discovery of the mechanisms regulating stem cell differentiation to the characterization of the early event of human embryogenesis. PMID:27442339

Emerging Imaging and Genomic Tools for Developmental Systems Biology.

PubMed

Liu, Zhe; Keller, Philipp J

2016-03-21

Animal development is a complex and dynamic process orchestrated by exquisitely timed cell lineage commitment, divisions, migration, and morphological changes at the single-cell level. In the past decade, extensive genetic, stem cell, and genomic studies provided crucial insights into molecular underpinnings and the functional importance of genetic pathways governing various cellular differentiation processes. However, it is still largely unknown how the precise coordination of these pathways is achieved at the whole-organism level and how the highly regulated spatiotemporal choreography of development is established in turn. Here, we discuss the latest technological advances in imaging and single-cell genomics that hold great promise for advancing our understanding of this intricate process. We propose an integrated approach that combines such methods to quantitatively decipher in vivo cellular dynamic behaviors and their underlying molecular mechanisms at the systems level with single-cell, single-molecule resolution. Copyright © 2016 Elsevier Inc. All rights reserved.

A New Cell Separation Method Based on Antibody-Immobilized Nanoneedle Arrays for the Detection of Intracellular Markers.

PubMed

Kawamura, Ryuzo; Miyazaki, Minami; Shimizu, Keita; Matsumoto, Yuta; Silberberg, Yaron R; Sathuluri, Ramachandra Rao; Iijima, Masumi; Kuroda, Shun'ichi; Iwata, Futoshi; Kobayashi, Takeshi; Nakamura, Chikashi

2017-11-08

Focusing on intracellular targets, we propose a new cell separation technique based on a nanoneedle array (NNA) device, which allows simultaneous insertion of multiple needles into multiple cells. The device is designed to target and lift ("fish") individual cells from a mixed population of cells on a substrate using an antibody-functionalized NNA. The mechanics underlying this approach were validated by force analysis using an atomic force microscope. Accurate high-throughput separation was achieved using one-to-one contacts between the nanoneedles and the cells by preparing a single-cell array in which the positions of the cells were aligned with 10,000 nanoneedles in the NNA. Cell-type-specific separation was realized by controlling the adhesion force so that the cells could be detached in cell-type-independent manner. Separation of nestin-expressing neural stem cells (NSCs) derived from human induced pluripotent stem cells (hiPSCs) was demonstrated using the proposed technology, and successful differentiation to neuronal cells was confirmed.

In vivo evaluation of adipose- and muscle-derived stem cells as a treatment for nonhealing diabetic wounds using multimodal microscopy

NASA Astrophysics Data System (ADS)

Li, Joanne; Pincu, Yair; Marjanovic, Marina; Bower, Andrew J.; Chaney, Eric J.; Jensen, Tor; Boppart, Marni D.; Boppart, Stephen A.

2016-08-01

Impaired skin wound healing is a significant comorbid condition of diabetes, which often results in nonhealing diabetic ulcers due to poor peripheral microcirculation, among other factors. The effectiveness of the regeneration of adipose-derived stem cells (ADSCs) and muscle-derived stem cells (MDSCs) was assessed using an integrated multimodal microscopy system equipped with two-photon fluorescence and second-harmonic generation imaging. These imaging modalities, integrated in a single platform for spatial and temporal coregistration, allowed us to monitor in vivo changes in the collagen network and cell dynamics in a skin wound. Fluorescently labeled ADSCs and MDSCs were applied topically to the wound bed of wild-type and diabetic (db/db) mice following punch biopsy. Longitudinal imaging demonstrated that ADSCs and MDSCs provided remarkable capacity for improved diabetic wound healing, and integrated microscopy revealed a more organized collagen remodeling in the wound bed of treated mice. The results from this study verify the regenerative capacity of stem cells toward healing and, with multimodal microscopy, provide insight regarding their impact on the skin microenvironment. The optical method outlined in this study, which has the potential for in vivo human use, may optimize the care and treatment of diabetic nonhealing wounds.

Reciprocal activation between STAT3 and miR-181b regulates the proliferation of esophageal cancer stem-like cells via the CYLD pathway.

PubMed

Xu, Dan-Dan; Zhou, Peng-Jun; Wang, Ying; Zhang, Li; Fu, Wu-Yu; Ruan, Bi-Bo; Xu, Hai-Peng; Hu, Chao-Zhi; Tian, Lu; Qin, Jin-Hong; Wang, Sheng; Wang, Xiao; Li, Yi-Cheng; Liu, Qiu-Ying; Ren, Zhe; Zhang, Rong; Wang, Yi-Fei

2016-05-17

Recent studies have suggested that cancer cells contain subpopulations that can initiate tumor growth, self-renew, and maintain tumor cell growth. However, for esophageal cancer cells, the relationship between STAT3, microRNAs and cancer stem cells remains unclear. Serum-free culture was used to enrich esophageal cancer stem-like cells (ECSLC). Flow cytometry determined the proportion of ECSLC. qPCR were performed to examine expression level of stemness factors, mesenchymal markers, ATP-binding cassette (ABC) transporters, STAT3, miR-181b, CYLD. Western blot were performed to analyze the expression of STAT3, p-STAT3 and CYLD (cylindromatosis). BALB/c mice xenograft studies were conducted to evaluate the tumorigenicity of enriched ECSLC. Sphere formation assay and colony formation assays were employed to analyze the relationship between STAT3 and miR-181b. Luciferase assays were used to evaluate activity which CYLD is a target of miR-181b. Sphere formation cells (SFCs) with properties of ECSLC were enriched. Enriched SFCs in serum-free suspension culture exhibited cancer stem-like cell properties and increased single-positive CD44 + CD24-, stemness factor, mesenchymal marker expression ABC transporters and tumorigenicity in vivo compared with the parental cells. Additionally, we found that reciprocal activation between STAT3 and miR-181b regulated SFCs proliferation. Moreover, STAT3 directly activated miR-181b transcription in SFCs and miR-181b then potentiated p-STAT3 activity. Luciferase assays indicated that CYLD was a direct and functional target of miR-181b. The mutual regulation between STAT3 and miR-181b in SFCs was required for proliferation and apoptosis resistance. STAT3 and miR-181b control each other's expression in a positive feedback loop that regulates SFCs via CYLD pathway. These findings maybe is helpful for targeting ECSLC and providing approach for esophageal cancer treatments.

A paper-based scaffold for enhanced osteogenic differentiation of equine adipose-derived stem cells.

PubMed

Petersen, Gayle F; Hilbert, Bryan J; Trope, Gareth D; Kalle, Wouter H J; Strappe, Padraig M

2015-11-01

We investigated the applicability of single layer paper-based scaffolds for the three-dimensional (3D) growth and osteogenic differentiation of equine adipose-derived stem cells (EADSC), with comparison against conventional two-dimensional (2D) culture on polystyrene tissue culture vessels. Viable culture of EADSC was achieved using paper-based scaffolds, with EADSC grown and differentiated in 3D culture retaining high cell viability (>94 %), similarly to EADSC in 2D culture. Osteogenic differentiation of EADSC was significantly enhanced in 3D culture, with Alizarin Red S staining and quantification demonstrating increased mineralisation (p < 0.0001), and an associated increase in expression of the osteogenic-specific markers alkaline phosphatase (p < 0.0001), osteopontin (p < 0.0001), and runx2 (p < 0.01). Furthermore, scanning electron microscopy revealed a spherical morphology of EADSC in 3D culture, compared to a flat morphology of EADSC in 2D culture. Single layer paper-based scaffolds provide an enhanced environment for the in vitro 3D growth and osteogenic differentiation of EADSC, with high cell viability, and a spherical morphology.

Embryoid bodies formation and differentiation from mouse embryonic stem cells in collagen/Matrigel scaffolds.

PubMed

Zhou, Jin; Zhang, Ye; Lin, Qiuxia; Liu, Zhiqiang; Wang, Haibin; Duan, Cuimi; Wang, Yanmeng; Hao, Tong; Wu, Kuiwu; Wang, Changyong

2010-07-01

Embryonic stem (ES) cells have the potential to develop into any type of tissue and are considered as a promising source of seeding cells for tissue engineering and transplantation therapy. The main catalyst for ES cells differentiation is the growth into embryoid bodies (EBs), which are utilized widely as the trigger of in vitro differentiation. In this study, a novel method for generating EBs from mouse ES cells through culture in collagen/Matrigel scaffolds was successfully established. When single ES cells were seeded in three dimensional collagen/Matrigel scaffolds, they grew into aggregates gradually and formed simple EBs with circular structures. After 7 days' culture, they formed into cystic EBs that would eventually differentiate into the three embryonic germ layers. Evaluation of the EBs in terms of morphology and potential to differentiate indicated that they were typical in structure and could generate various cell types; they were also able to form into tissue-like structures. Moreover, with introduction of ascorbic acid, ES cells differentiated into cardiomyocytes efficiently and started contracting synchronously at day 19. The results demonstrated that collagen/Matrigel scaffolds supported EBs formation and their subsequent differentiation in a single three dimensional environment. Copyright 2010 Institute of Genetics and Developmental Biology and the Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

Optimization of a therapeutic protocol for intravenous injection of human mesenchymal stem cells after cerebral ischemia in adult rats.

PubMed

Omori, Yoshinori; Honmou, Osamu; Harada, Kuniaki; Suzuki, Junpei; Houkin, Kiyohiro; Kocsis, Jeffery D

2008-10-21

The systemic injection of human mesenchymal stem cells (hMSCs) prepared from adult bone marrow has therapeutic benefits after cerebral artery occlusion in rats, and may have multiple therapeutic effects at various sites and times within the lesion as the cells respond to a particular pathological microenvironment. However, the comparative therapeutic benefits of multiple injections of hMSCs at different time points after cerebral artery occlusion in rats remain unclear. In this study, we induced middle cerebral artery occlusion (MCAO) in rats using intra-luminal vascular occlusion, and infused hMSCs intravenously at a single 6 h time point (low and high cell doses) and various multiple time points after MCAO. From MRI analyses lesion volume was reduced in all hMSC cell injection groups as compared to serum alone injections. However, the greatest therapeutic benefit was achieved following a single high cell dose injection at 6 h post-MCAO, rather than multiple lower cell infusions over multiple time points. Three-dimensional analysis of capillary vessels in the lesion indicated that the capillary volume was equally increased in all of the cell-injected groups. Thus, differences in functional outcome in the hMSC transplantation subgroups are not likely the result of differences in angiogenesis, but rather from differences in neuroprotective effects.

Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis

PubMed Central

Huch, Meritxell; Bonfanti, Paola; Boj, Sylvia F; Sato, Toshiro; Loomans, Cindy J M; van de Wetering, Marc; Sojoodi, Mozhdeh; Li, Vivian S W; Schuijers, Jurian; Gracanin, Ana; Ringnalda, Femke; Begthel, Harry; Hamer, Karien; Mulder, Joyce; van Es, Johan H; de Koning, Eelco; Vries, Robert G J; Heimberg, Harry; Clevers, Hans

2013-01-01

Lgr5 marks adult stem cells in multiple adult organs and is a receptor for the Wnt-agonistic R-spondins (RSPOs). Intestinal, stomach and liver Lgr5+ stem cells grow in 3D cultures to form ever-expanding organoids, which resemble the tissues of origin. Wnt signalling is inactive and Lgr5 is not expressed under physiological conditions in the adult pancreas. However, we now report that the Wnt pathway is robustly activated upon injury by partial duct ligation (PDL), concomitant with the appearance of Lgr5 expression in regenerating pancreatic ducts. In vitro, duct fragments from mouse pancreas initiate Lgr5 expression in RSPO1-based cultures, and develop into budding cyst-like structures (organoids) that expand five-fold weekly for >40 weeks. Single isolated duct cells can also be cultured into pancreatic organoids, containing Lgr5 stem/progenitor cells that can be clonally expanded. Clonal pancreas organoids can be induced to differentiate into duct as well as endocrine cells upon transplantation, thus proving their bi-potentiality. PMID:24045232

Single marker identification of head and neck squamous cell carcinoma cancer stem cells with aldehyde dehyrdrogenase

PubMed Central

Clay, MR.; Tabor, M.; Owen, J.; Carey, TE.; Bradford, CR.; Wolf, GT.; Wicha, MS.; Prince, ME.

2010-01-01

Background According to the cancer stem cell (CSC) theory only a small subset of cancer cells are capable of forming tumors. We previously reported that CD44 isolates tumorigenic cells from HNSCC. Recent studies indicate that aldehyde dehydrogenase (ALDH) activity may represent a more specific marker of CSCs. Methods Six primary HNSCC were collected. Cells with high and low ALDH activity (ALDHhigh/ALDHlow) were isolated. ALDHhigh and ALDHlow populations were implanted into NOD/SCID mice and monitored for tumor development. Results ALDHhigh cells represented a small percentage of the tumor cells (1-7.8%). ALDHhigh cells formed tumors from as few as 500 cells in 24/45 implantations while only 3/37 implantations of ALDHlow cells formed tumors. Conclusions ALDHhigh cells comprise a subpopulation cells in HNSCC that are tumorigenic and capable of producing tumors at very low numbers. This finding indicates that ALDH activity on its own is a highly selective marker for CSCs in HNSCC. PMID:20073073

Slow and sustained nitric oxide releasing compounds inhibit multipotent vascular stem cell proliferation and differentiation without causing cell death.

PubMed

Curtis, Brandon M; Leix, Kyle Alexander; Ji, Yajing; Glaves, Richard Samuel Elliot; Ash, David E; Mohanty, Dillip K

2014-07-18

Atherosclerosis is the leading cause of cerebral and myocardial infarction. It is believed that neointimal growth common in the later stages of atherosclerosis is a result of vascular smooth muscle cell (SMC) de-differentiation in response to endothelial injury. However, the claims of the SMC de-differentiation theory have not been substantiated by monitoring the fate of mature SMCs in response to such injuries. A recent study suggests that atherosclerosis is a consequence of multipotent vascular stem cell (MVSC) differentiation. Nitric oxide (NO) is a well-known mediator against atherosclerosis, in part because of its inhibitory effect on SMC proliferation. Using three different NO-donors, we have investigated the effects of NO on MVSC proliferation. Results indicate that NO inhibits MVSC proliferation in a concentration dependent manner. A slow and sustained delivery of NO proved to inhibit proliferation without causing cell death. On the other hand, larger, single-burst NO concentrations, inhibits proliferation, with concurrent significant cell death. Furthermore, our results indicate that endogenously produced NO inhibits MVSC differentiation to mesenchymal-like stem cells (MSCs) and subsequently to SMC as well. Published by Elsevier Inc.

[Adipose-derived stromal cells (ASC) - basics and therapeutic approaches in otorhinolaryngology].

PubMed

Frölich, K; Hagen, R; Kleinsasser, N

2014-06-01

Adipose-derived Stromal Cells (ASC) - Basics and Therapeutic Approaches in Otorhinolaryngology Mesenchymal stem cells from adipose tissue can be easily harvested with less discomfort, low donor-site morbidity and high amount compared to bone marrow-derived stem cells. Due to their multilineage differentiation potential in various cell types, immunmodulatory properties and their capability to enhance wound healing, ASC are a promising cell source for tissue engineering approaches and regenerative medicine. They are characterized by the expression of specific surface marker proteins and their differentiation potential into the mesenchymal lineages. Whereas only preclinical studies are published for otorhinolaryngology-related therapeutic options using ASC, various diseases, for instance graft-versus-host disease, have already been treated with ASC in single cases or clinical trials. Safety and genomic stability of ASC as well as the risk of spontaneous malignant transformation are still disputed. This review summarizes the current literature on characterization and anatomic localization of ASC. In addition, beside the presentation of preclinical studies concerning therapeutic approaches in otorhinolaryngology as well as of current clinical applications, the issue of safety of ASC in human stem cell therapy is discussed. © Georg Thieme Verlag KG Stuttgart · New York.

Clonal heterogeneity as a driver of disease variability in the evolution of myeloproliferative neoplasms.

PubMed

Prick, Janine; de Haan, Gerald; Green, Anthony R; Kent, David G

2014-10-01

Myeloproliferative neoplasms (MPNs) are clonal hematological diseases in which cells of the myelo-erythroid lineage are overproduced and patients are predisposed to leukemic transformation. Hematopoietic stem cells are the suspected disease-initiating cells, and these cells must acquire a clonal advantage relative to nonmutant hematopoietic stem cells to perpetuate disease. In 2005, several groups identified a single gain-of-function point mutation in JAK2 that associated with the majority of MPNs, and subsequent studies have led to a comprehensive understanding of the mutational landscape in MPNs. However, confusion still exists as to how a single genetic aberration can be associated with multiple distinct disease entities. Many explanations have been proposed, including JAK2V617F homozygosity, individual patient heterogeneity, and the differential regulation of downstream JAK2 signaling pathways. Several groups have made knock-in mouse models expressing JAK2V617F and have observed divergent phenotypes, each recapitulating some aspects of disease. Intriguingly, most of these models do not observe a strong hematopoietic stem cell self-renewal advantage compared with wild-type littermate controls, raising the question of how a clonal advantage is established in patients with MPNs. This review summarizes the current molecular understanding of MPNs and the diversity of disease phenotypes and proposes that the increased proliferation induced by JAK2V617F applies a selection pressure on the mutant clone that results in highly diverse clonal evolution in individuals. Copyright © 2014 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

Gene expression profiling of single cells on large-scale oligonucleotide arrays

PubMed Central

Hartmann, Claudia H.; Klein, Christoph A.

2006-01-01

Over the last decade, important insights into the regulation of cellular responses to various stimuli were gained by global gene expression analyses of cell populations. More recently, specific cell functions and underlying regulatory networks of rare cells isolated from their natural environment moved to the center of attention. However, low cell numbers still hinder gene expression profiling of rare ex vivo material in biomedical research. Therefore, we developed a robust method for gene expression profiling of single cells on high-density oligonucleotide arrays with excellent coverage of low abundance transcripts. The protocol was extensively tested with freshly isolated single cells of very low mRNA content including single epithelial, mature and immature dendritic cells and hematopoietic stem cells. Quantitative PCR confirmed that the PCR-based global amplification method did not change the relative ratios of transcript abundance and unsupervised hierarchical cluster analysis revealed that the histogenetic origin of an individual cell is correctly reflected by the gene expression profile. Moreover, the gene expression data from dendritic cells demonstrate that cellular differentiation and pathway activation can be monitored in individual cells. PMID:17071717

On-chip Magnetic Separation and Cell Encapsulation in Droplets

NASA Astrophysics Data System (ADS)

Chen, A.; Byvank, T.; Bharde, A.; Miller, B. L.; Chalmers, J. J.; Sooryakumar, R.; Chang, W.-J.; Bashir, R.

2012-02-01

The demand for high-throughput single cell assays is gaining importance because of the heterogeneity of many cell suspensions, even after significant initial sorting. These suspensions may display cell-to-cell variability at the gene expression level that could impact single cell functional genomics, cancer, stem-cell research and drug screening. The on-chip monitoring of individual cells in an isolated environment could prevent cross-contamination, provide high recovery yield and ability to study biological traits at a single cell level These advantages of on-chip biological experiments contrast to conventional methods, which require bulk samples that provide only averaged information on cell metabolism. We report on a device that integrates microfluidic technology with a magnetic tweezers array to combine the functionality of separation and encapsulation of objects such as immunomagnetically labeled cells or magnetic beads into pico-liter droplets on the same chip. The ability to control the separation throughput that is independent of the hydrodynamic droplet generation rate allows the encapsulation efficiency to be optimized. The device can potentially be integrated with on-chip labeling and/or bio-detection to become a powerful single-cell analysis device.

Histone Deacetylase Inhibitors Stimulate Dedifferentiation of Human Breast Cancer Cells through WNT/β-catenin Signaling

PubMed Central

Debeb, Bisrat G; Lacerda, Lara; Xu, Wei; Larson, Richard; Solley, Travis; Atkinson, Rachel; Sulman, Erik P.; Ueno, Naoto T; Krishnamurthy, Savitri; Reuben, James M; Buchholz, Thomas A; Woodward, Wendy A

2015-01-01

Recent studies have shown that differentiated cancer cells can de-differentiate into cancer stem cells (CSCs) although to date no studies have reported whether this transition is influenced by systemic anti-cancer agents. Valproic acid (VA) is a histone deacetylase (HDAC) inhibitor that promotes self renewal and expansion of hematopietic stem cells and facilitates the generation of induced pluripotent stem cells from somatic cells and is currently being investigated in breast cancer clinical trials. We hypothesized that HDAC inhibitors reprogram differentiated cancer cells towards the more resistant stem cell-like state. Two highly aggressive breast cancer cell lines, SUM159 and MDA-231, were FACS-sorted based on ALDH activity and subsequently ALDH-negative and ALDH-positive cells were treated with one of two known HDAC inhibitors, VA or SAHA (suberoylanilide hydroxamic acid). In addition, primary tumor cells from patients with metastatic breast cancer were evaluated for ALDH activity following treatment with HDAC inhibitors. We demonstrate that single cell sorted ALDH- negative cells spontaneously generated ALDH-positive cells in vitro. Treatment of ALDH-negative cells with HDAC inhibitors promoted the expansion of ALDH-positive cells and increased mammosphere forming efficiency. Most importantly, it significantly increased the tumor-initiating capacity of ALDH- negative cells in limiting dilution outgrowth assays. Moreover, while HDAC inhibitors upregulated β-catenin expression and significantly increased WNT reporter activity, a TCF4 dominant negative construct abolished HDAC-inhibitor induced expansion of CSCs. These results demonstrate that HDAC inhibitors promote the expansion of breast CSCs through dedifferentiation and have important clinical implications for the use of HDAC inhibitors in the treatment of cancer. PMID:22961641

Stem cells from human fat as cellular delivery vehicles in an athymic rat posterolateral spine fusion model.

PubMed

Hsu, Wellington K; Wang, Jeffrey C; Liu, Nancy Q; Krenek, Lucie; Zuk, Patricia A; Hedrick, Marc H; Benhaim, Prosper; Lieberman, Jay R

2008-05-01

Mesenchymal stem cells derived from human liposuction aspirates, termed processed lipoaspirate cells, have been utilized as cellular delivery vehicles for the induction of bone formation in tissue engineering and gene therapy strategies. In this study, we sought to evaluate the efficacy of bone morphogenetic protein (BMP)-2-producing adipose-derived stem cells in inducing a posterolateral spine fusion in an athymic rat model. Single-level (L4-L5) intertransverse spinal arthrodesis was attempted with use of a type-I collagen matrix in five groups of athymic rats, with eight animals in each group. Group I was treated with 5 x 10(6) adipose-derived stem cells transduced with an adenoviral vector containing the BMP-2 gene; group II, with 5 x 10(6) adipose-derived stem cells treated with osteogenic media and 1 microg/mL of recombinant BMP-2 (rhBMP-2); group III, with 10 microg of rhBMP-2; group IV, with 1 microg of rhBMP-2; and group V, with 5 x 10(6) adipose-derived stem cells alone. The animals that showed radiographic evidence of healing were killed four weeks after cell implantation and were examined with plain radiographs, manual palpation, microcomputed tomography scanning, and histological analysis. All eight animals in group I demonstrated successful spinal fusion, with a large fusion mass, four weeks postoperatively. Furthermore, group-I specimens consistently revealed spinal fusion at the cephalad level (L3 and L4), where no fusion bed had been prepared surgically. In contrast, despite substantial BMP-2 production measured in vitro, group-II animals demonstrated minimal bone formation even eight weeks after implantation. Of the groups treated with the application of rhBMP-2 alone, the one that received a relatively high dose (group III) had a higher rate of fusion (seen in all eight specimens) than the one that received the low dose (group IV, in which fusion was seen in four of the eight specimens). None of the group-V animals (treated with adipose-derived stem cells alone) demonstrated successful spine fusion eight weeks after the surgery. Adipose-derived stem cells show promise as gene transduction targets for inducing bone formation to enhance spinal fusion in biologically stringent environments.

The stem cell organisation, and the proliferative and gene expression profile of Barrett's epithelium, replicates pyloric-type gastric glands

PubMed Central

Lavery, Danielle L; Nicholson, Anna M; Poulsom, Richard; Jeffery, Rosemary; Hussain, Alia; Gay, Laura J; Jankowski, Janusz A; Zeki, Sebastian S; Barr, Hugh; Harrison, Rebecca; Going, James; Kadirkamanathan, Sritharan; Davis, Peter; Underwood, Timothy; Novelli, Marco R; Rodriguez–Justo, Manuel; Shepherd, Neil; Jansen, Marnix; Wright, Nicholas A; McDonald, Stuart A C

2014-01-01

Objective Barrett's oesophagus shows appearances described as ‘intestinal metaplasia’, in structures called ‘crypts’ but do not typically display crypt architecture. Here, we investigate their relationship to gastric glands. Methods Cell proliferation and migration within Barrett's glands was assessed by Ki67 and iododeoxyuridine (IdU) labelling. Expression of mucin core proteins (MUC), trefoil family factor (TFF) peptides and LGR5 mRNA was determined by immunohistochemistry or by in situ hybridisation, and clonality was elucidated using mitochondrial DNA (mtDNA) mutations combined with mucin histochemistry. Results Proliferation predominantly occurs in the middle of Barrett's glands, diminishing towards the surface and the base: IdU dynamics demonstrate bidirectional migration, similar to gastric glands. Distribution of MUC5AC, TFF1, MUC6 and TFF2 in Barrett's mirrors pyloric glands and is preserved in Barrett's dysplasia. MUC2-positive goblet cells are localised above the neck in Barrett's glands, and TFF3 is concentrated in the same region. LGR5 mRNA is detected in the middle of Barrett's glands suggesting a stem cell niche in this locale, similar to that in the gastric pylorus, and distinct from gastric intestinal metaplasia. Gastric and intestinal cell lineages within Barrett's glands are clonal, indicating derivation from a single stem cell. Conclusions Barrett's shows the proliferative and stem cell architecture, and pattern of gene expression of pyloric gastric glands, maintained by stem cells showing gastric and intestinal differentiation: neutral drift may suggest that intestinal differentiation advances with time, a concept critical for the understanding of the origin and development of Barrett's oesophagus. PMID:24550372

Immunophenotypical characterization of canine mesenchymal stem cells from perivisceral and subcutaneous adipose tissue by a species-specific panel of antibodies.

PubMed

Ivanovska, Ana; Grolli, Stefano; Borghetti, Paolo; Ravanetti, Francesca; Conti, Virna; De Angelis, Elena; Macchi, Francesca; Ramoni, Roberto; Martelli, Paolo; Gazza, Ferdinando; Cacchioli, Antonio

2017-10-01

Immunophenotypical characterization of mesenchymal stem cells is fundamental for the design and execution of sound experimental and clinical studies. The scarce availability of species-specific antibodies for canine antigens has hampered the immunophenotypical characterization of canine mesenchymal stem cells (MSC). The aim of this study was to select a panel of species-specific direct antibodies readily useful for canine mesenchymal stem cells characterization. They were isolated from perivisceral and subcutaneous adipose tissue samples collected during regular surgeries from 8 dogs. Single color flow cytometric analysis of mesenchymal stem cells (P3) deriving from subcutaneous and perivisceral adipose tissue with a panel of 7 direct anti-canine antibodies revealed two largely homogenous cell populations with a similar pattern: CD29 + , CD44 + , CD73 + , CD90 + , CD34 - , CD45 - and MHC-II - with no statistically significant differences among them. Antibody reactivity was demonstrated on canine peripheral blood mononuclear cells. The similarities are reinforced by their in vitro cell morphology, trilineage differentiation ability and RT-PCR analysis (CD90 + , CD73 + , CD105 + , CD44 + , CD13 + , CD29 + , Oct-4 + gene and CD31 - and CD45 - expression). Our results report for the first time a comparison between the immunophenotypic profile of canine MSC deriving from perivisceral and subcutaneous adipose tissue. The substantial equivalence between the two populations has practical implication on clinical applications, giving the opportunity to choose the source depending on the patient needs. The results contribute to routine characterization of MSC populations grown in vitro, a mandatory process for the definition of solid and reproducible laboratory and therapeutic procedures. Copyright © 2017 Elsevier Ltd. All rights reserved.

The stem cell organisation, and the proliferative and gene expression profile of Barrett's epithelium, replicates pyloric-type gastric glands.

PubMed

Lavery, Danielle L; Nicholson, Anna M; Poulsom, Richard; Jeffery, Rosemary; Hussain, Alia; Gay, Laura J; Jankowski, Janusz A; Zeki, Sebastian S; Barr, Hugh; Harrison, Rebecca; Going, James; Kadirkamanathan, Sritharan; Davis, Peter; Underwood, Timothy; Novelli, Marco R; Rodriguez-Justo, Manuel; Shepherd, Neil; Jansen, Marnix; Wright, Nicholas A; McDonald, Stuart A C

2014-12-01

Barrett's oesophagus shows appearances described as 'intestinal metaplasia', in structures called 'crypts' but do not typically display crypt architecture. Here, we investigate their relationship to gastric glands. Cell proliferation and migration within Barrett's glands was assessed by Ki67 and iododeoxyuridine (IdU) labelling. Expression of mucin core proteins (MUC), trefoil family factor (TFF) peptides and LGR5 mRNA was determined by immunohistochemistry or by in situ hybridisation, and clonality was elucidated using mitochondrial DNA (mtDNA) mutations combined with mucin histochemistry. Proliferation predominantly occurs in the middle of Barrett's glands, diminishing towards the surface and the base: IdU dynamics demonstrate bidirectional migration, similar to gastric glands. Distribution of MUC5AC, TFF1, MUC6 and TFF2 in Barrett's mirrors pyloric glands and is preserved in Barrett's dysplasia. MUC2-positive goblet cells are localised above the neck in Barrett's glands, and TFF3 is concentrated in the same region. LGR5 mRNA is detected in the middle of Barrett's glands suggesting a stem cell niche in this locale, similar to that in the gastric pylorus, and distinct from gastric intestinal metaplasia. Gastric and intestinal cell lineages within Barrett's glands are clonal, indicating derivation from a single stem cell. Barrett's shows the proliferative and stem cell architecture, and pattern of gene expression of pyloric gastric glands, maintained by stem cells showing gastric and intestinal differentiation: neutral drift may suggest that intestinal differentiation advances with time, a concept critical for the understanding of the origin and development of Barrett's oesophagus. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Haematopoietic stem cell transplantation as first-line treatment in myeloma: a global perspective of current concepts and future possibilities

PubMed Central

Mactier, Catriona Elizabeth; Islam, Md Serajul

2012-01-01

Stem cell transplantation forms an integral part of the treatment for multiple myeloma. This paper reviews the current role of transplantation and the progress that has been made in order to optimize the success of this therapy. Effective induction chemotherapy is important and a combination regimen incorporating the novel agent bortezomib is now favorable. Adequate induction is a crucial adjunct to stem cell transplantation and in some cases may potentially postpone the need for transplant. Different conditioning agents prior to transplantation have been explored: high-dose melphalan is most commonly used and bortezomib is a promising additional agent. There is no well-defined superior transplantation protocol but single or tandem autologous stem cell transplantations are those most commonly used, with allogeneic transplantation only used in clinical trials. The appropriate timing of transplantation in the treatment plan is a matter of debate. Consolidation and maintenance chemotherapies, particularly thalidomide and bortezomib, aim to improve and prolong disease response to transplantation and delay recurrence. Prognostic factors for the outcome of stem cell transplant in myeloma have been highlighted. Despite good responses to chemotherapy and transplantation, the problem of disease recurrence persists. Thus, there is still much room for improvement. Treatments which harness the graft-versus-myeloma effect may offer a potential cure for this disease. Trials of novel agents are underway, including targeted therapies for specific antigens such as vaccines and monoclonal antibodies. PMID:25992212

Ocular surface involvements in ectrodactyly-ectodermal dysplasia-cleft syndrome.

PubMed

Kennedy, David P; Chandler, John W; McCulley, James P

2015-06-01

To present the ocular manifestation of 2 cases of ectrodactyly-ectodermal dysplasia-cleft syndrome, a multiple congenital anomaly syndrome caused by a single point mutation of the p63 gene that controls epidermal development and homeostasis and to present treatment options. Patient 1 presented with mild signs and symptoms of dry eye and limbal stem cell deficiency with retention of 20/30 vision. Patient 2 presented with severe signs and symptoms of limbal stem cell deficiency with diffuse corneal scarring and counting fingers vision. This second patient's course was complicated by allergic conjunctivitis and advanced steroid-induced glaucoma. The cause of visual loss in ectrodactyly-ectodermal dysplasia-cleft syndrome appears to be multifactorial and likely includes inflammation of the ocular surface, tear film abnormalities, eyelid abnormalities, and limbal stem cell deficiency. Treatment modalities including lubrication, contact lenses, and limbal stem cell transplantation are reviewed. The ophthalmic conditions seen in ectrodactyly-ectodermal dysplasia-cleft syndrome frequently lead to vision loss. Early correct diagnosis and appropriate therapy are paramount because p63 gene mutations have a critical role in maintaining the integrity of the ocular surface in the setting of limbal stem cell deficiency, especially if there are other ocular surface insults such as lid disease, meibomian gland dysfunction and toxicity from topical medications. Patients should be monitored at regular, frequent intervals; and particular attention should be taken to avoid adverse secondary effects of these conditions and medications. Copyright © 2015 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.

Paradoxical drop in circulating neutrophil count following granulocyte-colony stimulating factor and stem cell factor administration in rhesus macaques.

PubMed

Gordon, Brent C; Revenis, Amy M; Bonifacino, Aylin C; Sander, William E; Metzger, Mark E; Krouse, Allen E; Usherson, Tatiana N; Donahue, Robert E

2007-06-01

Granulocyte colony-stimulating factor (G-CSF) is frequently used therapeutically to treat chronic or transient neutropenia and to mobilize hematopoietic stem cells. Shortly following G-CSF administration, we observed a dramatic transient drop in circulating neutrophil number. This article characterizes this effect in a rhesus macaque animal model. Hematologic changes were monitored following subcutaneous (SQ) administration of G-CSF. G-CSF was administered as a single SQ dose at 10 microg/kg or 50 microg/kg. It was also administered (10 microg/kg) in combination with stem cell factor (SCF; 200 microg/kg) over 5 days. Flow cytometry was performed on serial blood samples to detect changes in cell surface adhesion protein expression. Neutrophil count dramatically declined 30 minutes after G-CSF administration. This decline was observed whether 10 microg/kg G-CSF was administered in combination with SCF over 5 days, or given as a single 10 microg/kg dose. At a single 50 microg/kg dose, the decline accelerated to 15 minutes. Neutrophil count returned to baseline after 120 minutes and rapidly increased thereafter. An increase in CD11a and CD49d expression coincided with the drop in neutrophil count. A transient paradoxical decline in neutrophil count was observed following administration of G-CSF either alone or in combination with SCF. This decline accelerated with the administration of a higher dose of G-CSF and was associated with an increase in CD11a and CD49d expression. It remains to be determined whether this decline in circulating neutrophils is associated with an increase in endothelial margination and/or entrance into extravascular compartments.

Gli3 is a negative regulator of Tas1r3-expressing taste cells

PubMed Central

Jyotaki, Masafumi; Redding, Kevin; Jiang, Peihua

2018-01-01

Mouse taste receptor cells survive from 3–24 days, necessitating their regeneration throughout adulthood. In anterior tongue, sonic hedgehog (SHH), released by a subpopulation of basal taste cells, regulates transcription factors Gli2 and Gli3 in stem cells to control taste cell regeneration. Using single-cell RNA-Seq we found that Gli3 is highly expressed in Tas1r3-expressing taste receptor cells and Lgr5+ taste stem cells in posterior tongue. By PCR and immunohistochemistry we found that Gli3 was expressed in taste buds in all taste fields. Conditional knockout mice lacking Gli3 in the posterior tongue (Gli3CKO) had larger taste buds containing more taste cells than did control wild-type (Gli3WT) mice. In comparison to wild-type mice, Gli3CKO mice had more Lgr5+ and Tas1r3+ cells, but fewer type III cells. Similar changes were observed ex vivo in Gli3CKO taste organoids cultured from Lgr5+ taste stem cells. Further, the expression of several taste marker and Gli3 target genes was altered in Gli3CKO mice and/or organoids. Mirroring these changes, Gli3CKO mice had increased lick responses to sweet and umami stimuli, decreased lick responses to bitter and sour taste stimuli, and increased glossopharyngeal taste nerve responses to sweet and bitter compounds. Our results indicate that Gli3 is a suppressor of stem cell proliferation that affects the number and function of mature taste cells, especially Tas1r3+ cells, in adult posterior tongue. Our findings shed light on the role of the Shh pathway in adult taste cell regeneration and may help devise strategies for treating taste distortions from chemotherapy and aging. PMID:29415007

Effect of Secreted Molecules of Human Embryonic Stem Cell-Derived Mesenchymal Stem Cells on Acute Hepatic Failure Model.

PubMed

Lotfinia, Majid; Kadivar, Mehdi; Piryaei, Abbas; Pournasr, Behshad; Sardari, Soroush; Sodeifi, Niloofar; Sayahpour, Forugh-Azam; Baharvand, Hossein

2016-12-15

Adult tissue-derived mesenchymal stem cells (MSCs) show tremendous promise for a wide array of therapeutic applications predominantly through paracrine activity. Recent reports showed that human embryonic stem cell (ESC)-derived MSCs are an alternative for regenerative cellular therapy due to manufacturing large quantities of MSCs from a single donor. However, no study has been reported to uncover the secretome of human ESC-MSCs as treatment of an acute liver failure (ALF) mouse model. We demonstrated that human ESC-MSCs showed similar morphology and cell surface markers compared with bone marrow-derived MSCs. ESC-MSCs exhibited a higher growth rate during early in vitro expansion, along with adipogenic and osteogenic differentiation potential. Treatment with ESC-MSC-conditioned medium (CM) led to statistically significant enhancement of primary hepatocyte viability and increased immunomodulatory interleukin-10 secretion from lipopolysaccharide-induced human blood mononuclear cells. Analysis of the MSCs secretome by a protein array screen showed an association between higher frequencies of secretory proteins such as vascular endothelial growth factor (VEGF) and regulation of cell proliferation, cell migration, the development process, immune system process, and apoptosis. In this thioacetamide-induced mouse model of acute liver injury, we observed that systemic infusion of VEGF led to significant survival. These data have provided the first experimental evidence of the therapeutic potential of human ESC-MSC-derived molecules. These molecules show trophic support to hepatocytes, which potentially creates new avenues for the treatment of ALF, as an inflammatory condition.

Types of Stem Cells

MedlinePlus

... Cell Glossary Search Toggle Nav Types of Stem Cells Stem cells are the foundation from which all ... About Stem Cells > Types of Stem Cells Stem cells Stem cells are the foundation for every organ ...

Macrophage involvement affects matrix stiffness-related influences on cell osteogenesis under three-dimensional culture conditions.

PubMed

He, Xiao-Tao; Wu, Rui-Xin; Xu, Xin-Yue; Wang, Jia; Yin, Yuan; Chen, Fa-Ming

2018-04-15

Accumulating evidence indicates that the physicochemical properties of biomaterials exert profound influences on stem cell fate decisions. However, matrix-based regulation selected through in vitro analyses based on a given cell population do not genuinely reflect the in vivo conditions, in which multiple cell types are involved and interact dynamically. This study constitutes the first investigation of how macrophages (Mφs) in stiffness-tunable transglutaminase cross-linked gelatin (TG-gel) affect the osteogenesis of bone marrow-derived mesenchymal stem cells (BMMSCs). When a single cell type was cultured, low-stiffness TG-gels promoted BMMSC proliferation, whereas high-stiffness TG-gels supported cell osteogenic differentiation. However, Mφs in high-stiffness TG-gels were more likely to polarize toward the pro-inflammatory M1 phenotype. Using either conditioned medium (CM)-based incubation or Transwell-based co-culture, we found that Mφs encapsulated in the low-stiffness matrix exerted a positive effect on the osteogenesis of co-cultured BMMSCs. Conversely, Mφs in high-stiffness TG-gels negatively affected cell osteogenic differentiation. When both cell types were cultured in the same TG-gel type and placed into the Transwell system, the stiffness-related influences of Mφs on BMMSCs were significantly altered; both the low- and high-stiffness matrix induced similar levels of BMMSC osteogenesis. Although the best material parameter for synergistically affecting Mφs and BMMSCs remains unknown, our data suggest that Mφ involvement in the co-culture system alters previously identified material-related influences on BMMSCs, such as matrix stiffness-related effects, which were identified based on a culture system involving a single cell type. Such Mφ-stem cell interactions should be considered when establishing proper matrix parameter-associated cell regulation in the development of biomimetic biomaterials for regenerative applications. The substrate stiffness of a scaffold plays critical roles in modulating both reparative cells, such as mesenchymal stem cells (MSCs), and immune cells, such as macrophages (Mφs). Although the influences of material stiffness on either Mφs or MSCs, have been extensively described, how the two cell types respond to matrix cues to dynamically affect each other in a three-dimensional (3D) biosystem remains largely unknown. Here, we report our findings that, in a platform wherein Mφs and bone marrow-derived MSCs coexist, matrix stiffness can influence stem cell fate through both direct matrix-associated regulation and indirect Mφ-based modulation. Our data support future studies of the MSC-Mφ-matrix interplay in the 3D context to optimize matrix parameters for the development of the next biomaterial. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Systematic computation with functional gene-sets among leukemic and hematopoietic stem cells reveals a favorable prognostic signature for acute myeloid leukemia.

PubMed

Yang, Xinan Holly; Li, Meiyi; Wang, Bin; Zhu, Wanqi; Desgardin, Aurelie; Onel, Kenan; de Jong, Jill; Chen, Jianjun; Chen, Luonan; Cunningham, John M

2015-03-24

Genes that regulate stem cell function are suspected to exert adverse effects on prognosis in malignancy. However, diverse cancer stem cell signatures are difficult for physicians to interpret and apply clinically. To connect the transcriptome and stem cell biology, with potential clinical applications, we propose a novel computational "gene-to-function, snapshot-to-dynamics, and biology-to-clinic" framework to uncover core functional gene-sets signatures. This framework incorporates three function-centric gene-set analysis strategies: a meta-analysis of both microarray and RNA-seq data, novel dynamic network mechanism (DNM) identification, and a personalized prognostic indicator analysis. This work uses complex disease acute myeloid leukemia (AML) as a research platform. We introduced an adjustable "soft threshold" to a functional gene-set algorithm and found that two different analysis methods identified distinct gene-set signatures from the same samples. We identified a 30-gene cluster that characterizes leukemic stem cell (LSC)-depleted cells and a 25-gene cluster that characterizes LSC-enriched cells in parallel; both mark favorable-prognosis in AML. Genes within each signature significantly share common biological processes and/or molecular functions (empirical p = 6e-5 and 0.03 respectively). The 25-gene signature reflects the abnormal development of stem cells in AML, such as AURKA over-expression. We subsequently determined that the clinical relevance of both signatures is independent of known clinical risk classifications in 214 patients with cytogenetically normal AML. We successfully validated the prognosis of both signatures in two independent cohorts of 91 and 242 patients respectively (log-rank p < 0.0015 and 0.05; empirical p < 0.015 and 0.08). The proposed algorithms and computational framework will harness systems biology research because they efficiently translate gene-sets (rather than single genes) into biological discoveries about AML and other complex diseases.

TRIM28 and β-Actin Identified via Nanobody-Based Reverse Proteomics Approach as Possible Human Glioblastoma Biomarkers

PubMed Central

Jovčevska, Ivana; Zupanec, Neja; Kočevar, Nina; Cesselli, Daniela; Podergajs, Neža; Stokin, Clara Limbaeck; Myers, Michael P.; Muyldermans, Serge; Ghassabeh, Gholamreza Hassanzadeh; Motaln, Helena; Ruaro, Maria Elisabetta; Bourkoula, Evgenia; Turnšek, Tamara Lah; Komel, Radovan

2014-01-01

Malignant gliomas are among the rarest brain tumours, and they have the worst prognosis. Grade IV astrocytoma, known as glioblastoma multiforme (GBM), is a highly lethal disease where the standard therapies of surgery, followed by radiation and chemotherapy, cannot significantly prolong the life expectancy of the patients. Tumour recurrence shows more aggressive form compared to the primary tumour, and results in patient survival from 12 to 15 months only. Although still controversial, the cancer stem cell hypothesis postulates that cancer stem cells are responsible for early relapse of the disease after surgical intervention due to their high resistance to therapy. Alternative strategies for GBM therapy are thus urgently needed. Nanobodies are single-domain antigen-binding fragments of heavy-chain antibodies, and together with classical antibodies, they are part of the camelid immune system. Nanobodies are small and stable, and they share a high degree of sequence identity to the human heavy chain variable domain, and these characteristics offer them advantages over classical antibodies or antibody fragments. We first immunised an alpaca with a human GBM stem-like cell line prepared from primary GBM cultures. Next, a nanobody library was constructed in a phage-display vector. Using nanobody phage-display technology, we selected specific GBM stem-like cell binders through a number of affinity selections, using whole cell protein extracts and membrane protein-enriched extracts from eight different GBM patients, and membrane protein-enriched extracts from two established GBM stem-like cell lines (NCH644 and NCH421K cells). After the enrichment, periplasmic extract ELISA was used to screen for specific clones. These nanobody clones were recloned into the pHEN6 vector, expressed in Escherichia coli WK6, and purified using immobilised metal affinity chromatography and size-exclusion chromatography. Specific nanobody:antigen pairs were obtained and mass spectrometry analysis revealed two proteins, TRIM28 and β-actin, that were up-regulated in the GBM stem-like cells compared to the controls. PMID:25419715

Donor lymphocyte apheresis for adoptive immunotherapy compared with blood stem cell apheresis.

PubMed

Körbling, M; Giralt, S; Khouri, I; Mirza, N; Donato, M; Anderlini, P; Fischer, H; Andreeff, M; McMannis, J; Champlin, R

2001-01-01

Donor lymphocyte transfusion has gained considerable interest as adoptive cellular immunotherapy for prevention or treatment of relapse after allogeneic stem cell transplantation. This study was designed to compare the yield of CD3(+), CD3(+)4(+), CD3(+)8(+), CD19(+), CD3(-)56(+)16(+), and CD34(+) cells contained in apheresis products from 61 consecutive non-cytokine treated, human leukocyte antigen (HLA)-matched donors for lymphocyte collection with the corresponding apheresis-derived cell yield from 112 consecutive, HLA-matched donors for blood stem cell collection who received recombinant human granulocyte colony stimulating factor (rhG-CSF, filgrastim) 6 microg/kg every 12 hours until cell collection was completed. Apheresis was started on day 4 or 5 of rhG-CSF treatment. The yield of lymphoid subsets was significantly different in the two sample groups, rhG-CSF treated product yields exceeding untreated product yields by a median of 2.1-fold (range: 1.3-2.6). However, the CD34(+) cell yield in rhG-CSF-treated apheresis products exceeded untreated products by 26-fold. A single untreated apheresis procedure was usually sufficient to collect a target dose of 1 x 10(8)/kg CD3(+) cells. Untreated apheresis products contained a median of 0.2 x 10(6)/kg CD34(+) cells. A potential engraftment dose of > or =0.5 x 10(6) CD34(+) cells per kg of recipient body weight was contained in 16% of 57 untreated apheresis products. One single apheresis performed in a normal, untreated donor provides a sufficient amount of CD3(+) cells for adoptive immunotherapy. Compared with that of an rhG-CSF stimulated apheresis product, the CD34(+) cell count is usually, but not always, below the engraftment dose range. RhG-CSF treatment has little effect on the yield of lymphoid subsets collected by apheresis but is highly selective of the release of CD34(+) cells. This report provides baseline data for studies that will show whether other cytokines such as granulocyte macrophage colony stimulating factor (GM-CSF) and/or Flt-3 Ligand can immunomodulate allotransfusates in vivo to improve the graft-vs.-leukemia (GVL) effect after allogeneic stem cell transplantation, while lowering the incidence and severity of graft-vs.-host disease (GVHD). Copyright 2001 Wiley-Liss, Inc.

Abundant cytomegalovirus (CMV) reactive clonotypes in the CD8(+) T cell receptor alpha repertoire following allogeneic transplantation.

PubMed

Link, C S; Eugster, A; Heidenreich, F; Rücker-Braun, E; Schmiedgen, M; Oelschlägel, U; Kühn, D; Dietz, S; Fuchs, Y; Dahl, A; Domingues, A M J; Klesse, C; Schmitz, M; Ehninger, G; Bornhäuser, M; Schetelig, J; Bonifacio, E

2016-06-01

Allogeneic stem cell transplantation is potentially curative, but associated with post-transplantation complications, including cytomegalovirus (CMV) infections. An effective immune response requires T cells recognizing CMV epitopes via their T cell receptors (TCRs). Little is known about the TCR repertoire, in particular the TCR-α repertoire and its clinical relevance in patients following stem cell transplantation. Using next-generation sequencing we examined the TCR-α repertoire of CD8(+) T cells and CMV-specific CD8(+) T cells in four patients. Additionally, we performed single-cell TCR-αβ sequencing of CMV-specific CD8(+) T cells. The TCR-α composition of human leucocyte antigen (HLA)-A*0201 CMVpp65- and CMVIE -specific T cells was oligoclonal and defined by few dominant clonotypes. Frequencies of single clonotypes reached up to 11% of all CD8(+) T cells and half of the total CD8(+) T cell repertoire was dominated by few CMV-reactive clonotypes. Some TCR-α clonotypes were shared between patients. Gene expression of the circulating CMV-specific CD8(+) T cells was consistent with chronically activated effector memory T cells. The CD8(+) T cell response to CMV reactivation resulted in an expansion of a few TCR-α clonotypes to dominate the CD8(+) repertoires. These results warrant further larger studies to define the ability of oligoclonally expanded T cell clones to achieve an effective anti-viral T cell response in this setting. © 2016 British Society for Immunology.

miRNA-regulated cancer stem cells: understanding the property and the role of miRNA in carcinogenesis.

PubMed

Chakraborty, Chiranjib; Chin, Kok-Yong; Das, Srijit

2016-10-01

Over the last few years, microRNAs (miRNA)-controlled cancer stem cells have drawn enormous attention. Cancer stem cells are a small population of tumor cells that possess the stem cell property of self-renewal. Recent data shows that miRNA regulates this small population of stem cells. In the present review, we explained different characteristics of cancer stem cells as well as miRNA regulation of self-renewal and differentiation in cancer stem cells. We also described the migration and tumor formation. Finally, we described the different miRNAs that regulate various types of cancer stem cells, such as prostate cancer stem cells, head and neck cancer stem cells, breast cancer stem cells, colorectal cancer stem cells, lung cancer stem cells, gastric cancer stem cells, pancreatic cancer stem cells, etc. Extensive research is needed in order to employ miRNA-based therapeutics to control cancer stem cell population in various cancers in the future.

Light-sheet Bayesian microscopy enables deep-cell super-resolution imaging of heterochromatin in live human embryonic stem cells.

PubMed

Hu, Ying S; Zhu, Quan; Elkins, Keri; Tse, Kevin; Li, Yu; Fitzpatrick, James A J; Verma, Inder M; Cang, Hu

2013-01-01

Heterochromatin in the nucleus of human embryonic cells plays an important role in the epigenetic regulation of gene expression. The architecture of heterochromatin and its dynamic organization remain elusive because of the lack of fast and high-resolution deep-cell imaging tools. We enable this task by advancing instrumental and algorithmic implementation of the localization-based super-resolution technique. We present light-sheet Bayesian super-resolution microscopy (LSBM). We adapt light-sheet illumination for super-resolution imaging by using a novel prism-coupled condenser design to illuminate a thin slice of the nucleus with high signal-to-noise ratio. Coupled with a Bayesian algorithm that resolves overlapping fluorophores from high-density areas, we show, for the first time, nanoscopic features of the heterochromatin structure in both fixed and live human embryonic stem cells. The enhanced temporal resolution allows capturing the dynamic change of heterochromatin with a lateral resolution of 50-60 nm on a time scale of 2.3 s. Light-sheet Bayesian microscopy opens up broad new possibilities of probing nanometer-scale nuclear structures and real-time sub-cellular processes and other previously difficult-to-access intracellular regions of living cells at the single-molecule, and single cell level.

Light-sheet Bayesian microscopy enables deep-cell super-resolution imaging of heterochromatin in live human embryonic stem cells

PubMed Central

Hu, Ying S; Zhu, Quan; Elkins, Keri; Tse, Kevin; Li, Yu; Fitzpatrick, James A J; Verma, Inder M; Cang, Hu

2016-01-01

Background Heterochromatin in the nucleus of human embryonic cells plays an important role in the epigenetic regulation of gene expression. The architecture of heterochromatin and its dynamic organization remain elusive because of the lack of fast and high-resolution deep-cell imaging tools. We enable this task by advancing instrumental and algorithmic implementation of the localization-based super-resolution technique. Results We present light-sheet Bayesian super-resolution microscopy (LSBM). We adapt light-sheet illumination for super-resolution imaging by using a novel prism-coupled condenser design to illuminate a thin slice of the nucleus with high signal-to-noise ratio. Coupled with a Bayesian algorithm that resolves overlapping fluorophores from high-density areas, we show, for the first time, nanoscopic features of the heterochromatin structure in both fixed and live human embryonic stem cells. The enhanced temporal resolution allows capturing the dynamic change of heterochromatin with a lateral resolution of 50–60 nm on a time scale of 2.3 s. Conclusion Light-sheet Bayesian microscopy opens up broad new possibilities of probing nanometer-scale nuclear structures and real-time sub-cellular processes and other previously difficult-to-access intracellular regions of living cells at the single-molecule, and single cell level. PMID:27795878

Matrigel Mattress: A Method for the Generation of Single Contracting Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

PubMed

Feaster, Tromondae K; Cadar, Adrian G; Wang, Lili; Williams, Charles H; Chun, Young Wook; Hempel, Jonathan E; Bloodworth, Nathaniel; Merryman, W David; Lim, Chee Chew; Wu, Joseph C; Knollmann, Björn C; Hong, Charles C

2015-12-04

The lack of measurable single-cell contractility of human-induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) currently limits the utility of hiPSC-CMs for evaluating contractile performance for both basic research and drug discovery. To develop a culture method that rapidly generates contracting single hiPSC-CMs and allows quantification of cell shortening with standard equipment used for studying adult CMs. Single hiPSC-CMs were cultured for 5 to 7 days on a 0.4- to 0.8-mm thick mattress of undiluted Matrigel (mattress hiPSC-CMs) and compared with hiPSC-CMs maintained on a control substrate (<0.1-mm thick 1:60 diluted Matrigel, control hiPSC-CMs). Compared with control hiPSC-CMs, mattress hiPSC-CMs had more rod-shape morphology and significantly increased sarcomere length. Contractile parameters of mattress hiPSC-CMs measured with video-based edge detection were comparable with those of freshly isolated adult rabbit ventricular CMs. Morphological and contractile properties of mattress hiPSC-CMs were consistent across cryopreserved hiPSC-CMs generated independently at another institution. Unlike control hiPSC-CMs, mattress hiPSC-CMs display robust contractile responses to positive inotropic agents, such as myofilament calcium sensitizers. Mattress hiPSC-CMs exhibit molecular changes that include increased expression of the maturation marker cardiac troponin I and significantly increased action potential upstroke velocity because of a 2-fold increase in sodium current (INa). The Matrigel mattress method enables the rapid generation of robustly contracting hiPSC-CMs and enhances maturation. This new method allows quantification of contractile performance at the single-cell level, which should be valuable to disease modeling, drug discovery, and preclinical cardiotoxicity testing. © 2015 American Heart Association, Inc.

What is a stem cell?

PubMed

Slack, Jonathan M W

2018-05-15

The historical roots of the stem cell concept are traced with respect to its usage in embryology and in hematology. The modern consensus definition of stem cells, comprising both pluripotent stem cells in culture and tissue-specific stem cells in vivo, is explained and explored. Methods for identifying stem cells are discussed with respect to cell surface markers, telomerase, label retention and transplantability, and properties of the stem cell niche are explored. The CreER method for identifying stem cells in vivo is explained, as is evidence in favor of a stochastic rather than an obligate asymmetric form of cell division. In conclusion, it is found that stem cells do not possess any unique and specific molecular markers; and stem cell behavior depends on the environment of the cell as well as the stem cell's intrinsic qualities. Furthermore, the stochastic mode of division implies that stem cell behavior is a property of a cell population not of an individual cell. In this sense, stem cells do not exist in isolation but only as a part of multicellular system. This article is categorized under: Adult Stem Cells, Tissue Renewal, and Regeneration > Tissue Stem Cells and Niches Adult Stem Cells, Tissue Renewal, and Regeneration > Methods and Principles Adult Stem Cells, Tissue Renewal, and Regeneration > Environmental Control of Stem Cells. © 2018 Wiley Periodicals, Inc.

Nanotechnology & human stem cells: Applications in cardiogenesis and neurogenesis

NASA Astrophysics Data System (ADS)

Tomov, Martin L.

Human stem cell research holds an unprecedented promise to revolutionize the way we approach medicine and healthcare in general, moving us from a position of mostly addressing the symptoms to a state where treatments can focus on removing the underlying causes of a condition. Stem cell research can shed light into normal developmental pathways, as we are beginning to replicate them in a petri dish and can also be used to model diseases and abnormal conditions. Direct applications can range from finding cures for single or multigene diseases to demonstrating that we can replace these genes with a normal copy. We can even begin to model lifelong conditions such as aging by iPSC technology by relying on fetal, young, adult, and centenarian populations to provide insights into the process. We have also begun to understand the microenvironment in which specific cell populations reside. Being able to replicate the chemical, physical mechanical, and spatial needs of those cells, research groups are successfully generating full organs using cadaver scaffolds of heart and kidney, and there is promising research to reach the same success with other organs, such as the liver, and pancreas. Advances in those areas open an enormous potential to study organs, organoids, organ valves, tubes or other functional elements such as beating cardiomyocytes in vitro. There is also the need to evaluate the whole genome of induced and differentiated cells, with its myriad of interacting pathways. Bioinformatics can help our understanding of embryogenesis, organ differentiation and function. It can also help optimize our stem cell and bio-scaffold tools to advance closer to functional organs and tissues. Such a combination approach will also include pluripotency evaluation and multi-lineage differentiation, as well as platforms that may assist in cell therapies: 3D structures, micro-ribbons, directed patterning to name a few. There is now a clearer path forward with stem cell research than was ever before possible. My research has made fundamental contributions to the stem cell field by detailed analysis of uniformly generated 3D stem cell intermediates that are embryoid bodies. I have also contributed to the derivation of the first fully characterized ethnically diverse induced pluripotent stem cells from minority populations (ED-iPSCs), and advances in generating functional beating cardiomyocytes in vitro to aid cardiomyoplasty therapies. My work has also explored scaffolds for directing neural cell assembly or encouraging self-assembly for applications in CNS neurodegeneration, addiction, and spinal cord injury. These contributions to the field are outlined in my Specific Aims below and detailed in the chapters of my thesis.

Single-cell gene expression profiling reveals functional heterogeneity of undifferentiated human epidermal cells

PubMed Central

Tan, David W. M.; Jensen, Kim B.; Trotter, Matthew W. B.; Connelly, John T.; Broad, Simon; Watt, Fiona M.

2013-01-01

Human epidermal stem cells express high levels of β1 integrins, delta-like 1 (DLL1) and the EGFR antagonist LRIG1. However, there is cell-to-cell variation in the relative abundance of DLL1 and LRIG1 mRNA transcripts. Single-cell global gene expression profiling showed that undifferentiated cells fell into two clusters delineated by expression of DLL1 and its binding partner syntenin. The DLL1+ cluster had elevated expression of genes associated with endocytosis, integrin-mediated adhesion and receptor tyrosine kinase signalling. Differentially expressed genes were not independently regulated, as overexpression of DLL1 alone or together with LRIG1 led to the upregulation of other genes in the DLL1+ cluster. Overexpression of DLL1 and LRIG1 resulted in enhanced extracellular matrix adhesion and increased caveolin-dependent EGFR endocytosis. Further characterisation of CD46, one of the genes upregulated in the DLL1+ cluster, revealed it to be a novel cell surface marker of human epidermal stem cells. Cells with high endogenous levels of CD46 expressed high levels of β1 integrin and DLL1 and were highly adhesive and clonogenic. Knockdown of CD46 decreased proliferative potential and β1 integrin-mediated adhesion. Thus, the previously unknown heterogeneity revealed by our studies results in differences in the interaction of undifferentiated basal keratinocytes with their environment. PMID:23482486

Zika Virus Selectively Kills Aggressive Human Embryonal CNS Tumor Cells In Vitro and In Vivo.

PubMed

Kaid, Carolini; Goulart, Ernesto; Caires-Júnior, Luiz C; Araujo, Bruno H S; Soares-Schanoski, Alessandra; Bueno, Heloisa M S; Telles-Silva, Kayque A; Astray, Renato M; Assoni, Amanda F; Júnior, Antônio F R; Ventini, Daniella C; Puglia, Ana L P; Gomes, Roselane P; Zatz, Mayana; Okamoto, Oswaldo K

2018-06-15

Zika virus (ZIKV) is largely known for causing brain abnormalities due to its ability to infect neural progenitor stem cells during early development. Here, we show that ZIKV is also capable of infecting and destroying stem-like cancer cells from aggressive human embryonal tumors of the central nervous system (CNS). When evaluating the oncolytic properties of Brazilian Zika virus strain (ZIKV BR ) against human breast, prostate, colorectal, and embryonal CNS tumor cell lines, we verified a selective infection of CNS tumor cells followed by massive tumor cell death. ZIKV BR was more efficient in destroying embryonal CNS tumorspheres than normal stem cell neurospheres. A single intracerebroventricular injection of ZIKV BR in BALB/c nude mice bearing orthotopic human embryonal CNS tumor xenografts resulted in a significantly longer survival, decreased tumor burden, fewer metastasis, and complete remission in some animals. Tumor cells closely resembling neural stem cells at the molecular level with activated Wnt signaling were more susceptible to the oncolytic effects of ZIKV BR Furthermore, modulation of Wnt signaling pathway significantly affected ZIKV BR -induced tumor cell death and viral shedding. Altogether, these preclinical findings indicate that ZIKV BR could be an efficient agent to treat aggressive forms of embryonal CNS tumors and could provide mechanistic insights regarding its oncolytic effects. Significance: Brazilian Zika virus strain kills aggressive metastatic forms of human CNS tumors and could be a potential oncolytic agent for cancer therapy. Cancer Res; 78(12); 3363-74. ©2018 AACR . ©2018 American Association for Cancer Research.

Imaging cellular pharmacokinetics of 18F-FDG and 6-NBDG uptake by inflammatory and stem cells.

PubMed

Zaman, Raiyan T; Tuerkcan, Silvan; Mahmoudi, Morteza; Saito, Toshinobu; Yang, Phillip C; Chin, Frederick T; McConnell, Michael V; Xing, Lei

2018-01-01

Myocardial infarction (MI) causes significant loss of cardiomyocytes, myocardial tissue damage, and impairment of myocardial function. The inability of cardiomyocytes to proliferate prevents the heart from self-regeneration. The treatment for advanced heart failure following an MI is heart transplantation despite the limited availability of the organs. Thus, stem-cell-based cardiac therapies could ultimately prevent heart failure by repairing injured myocardium that reverses cardiomyocyte loss. However, stem-cell-based therapies lack understanding of the mechanisms behind a successful therapy, including difficulty tracking stem cells to provide information on cell migration, proliferation and differentiation. In this study, we have investigated the interaction between different types of stem and inflammatory cells and cell-targeted imaging molecules, 18F-FDG and 6-NBDG, to identify uptake patterns and pharmacokinetics in vitro. Macrophages (both M1 and M2), human induced pluripotent stem cells (hiPSCs), and human amniotic mesenchymal stem cells (hAMSCs) were incubated with either 18F-FDG or 6-NBDG. Excess radiotracer and fluorescence were removed and a 100 μm-thin CdWO4 scintillator plate was placed on top of the cells for radioluminescence microscopy imaging of 18F-FDG uptake, while no scintillator was needed for fluorescence imaging of 6-NBDG uptake. Light produced following beta decay was imaged with a highly sensitive inverted microscope (LV200, Olympus) and an Electron Multiplying Charge-Couple Device (EM-CCD) camera. Custom-written software was developed in MATLAB for image processing. The average cellular activity of 18F-FDG in a single cell of hAMSCs (0.670±0.028 fCi/μm2, P = 0.001) was 20% and 36% higher compared to uptake in hiPSCs (0.540±0.026 fCi/μm2, P = 0.003) and macrophages (0.430±0.023 fCi/μm2, P = 0.002), respectively. hAMSCs exhibited the slowest influx (0.210 min-1) but the fastest efflux (0.327 min-1) rate compared to the other tested cell lines for 18F-FDG. This cell line also has the highest phosphorylation but exhibited the lowest rate of de-phosphorylation. The uptake pattern for 6-NBDG was very different in these three cell lines. The average cellular activity of 6-NBDG in a single cell of macrophages (0.570±0.230 fM/μm2, P = 0.004) was 38% and 14% higher compared to hiPSCs (0.350±0.160 fM/μm2, P = 0.001) and hAMSCs (0.490±0.028 fM/μm2, P = 0.006), respectively. The influx (0.276 min-1), efflux (0.612 min-1), phosphorylation (0.269 min-1), and de-phosphorylation (0.049 min-1) rates were also highest for macrophages compared to the other two tested cell lines. hAMSCs were found to be 2-3× more sensitive to 18F-FDG molecule compared to hiPSCs/macrophages. However, macrophages exhibited the most sensitivity towards 6-NBDG. Based on this result, hAMSCs targeted with 18F-FDG could be more suitable for understanding the mechanisms behind successful therapy for treating MI patients by gathering information on cell migration, proliferation and differentiation.