[Show abstract][Hide abstract] ABSTRACT: Mesenchymal stem cells (MSCs) can be polarized into two distinct populations, MSC1 and MSC2, by activation of different Toll-like receptors (TLRs). TLR4-primed MSC1 expressedproinflammatory factors, whereas TLR3-primed MSC2 expressed suppressive factors. However, little is known about the function of TLRs on B lymphocyte-related immune modulation. In this study, we investigated the expression of B cell activating factor (BAFF), a member of the tumor necrosis factor ligand superfamily with notable stimulating activity on B cells, in human MSCs (hMSCs) and in murine MSCs (mMSCs) after activation of TLRs. BAFF was increasingly expressed in the presence of TLR4 agonist (Lipopolysaccharide, LPS), while TLR2 agonist (Zymosan) and TLR3-agonist (polyinocinic–polycytidykic acid, poly I:C) had no effect on BAFF expression. In addition, we demonstrated that signaling pathways of NF-κB, p38 MAPK, and JNKwere involved in TLR4-primed BAFF expression. Our results suggested that TLR4 and downstream pathways in MSCs exert an important function in B lymphocyte-related immune regulation. Further defining a homogeneous population of MSCs should provide insight into MSC-based immune-modulating therapy.
Biochemical and Biophysical Research Communications 01/2014; · 2.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Embryonic stem cells (ESCs), characterized by their ability to both self-renew and differentiate into multiple cell lineages, are a powerful model for biomedical research and developmental biology. Human and mouse ESCs share many features, yet have distinctive aspects, including fundamental differences in the signaling pathways and cell cycle controls that support self-renewal. Here, we explore the molecular basis of human ESC self-renewal using Bayesian network machine learning to integrate cell-type-specific, high-throughput data for gene function discovery. We integrated high-throughput ESC data from 83 human studies (~1.8 million data points collected under 1100 conditions) and 62 mouse studies (~2.4 million data points collected under 1085 conditions) into separate human and mouse predictive networks focused on ESC self-renewal to analyze shared and distinct functional relationships among protein-coding gene orthologs. Computational evaluations show that these networks are highly accurate, literature validation confirms their biological relevance, and RT-PCR validation supports our predictions. Our results reflect the importance of key regulatory genes known to be strongly associated with self-renewal and pluripotency in both species (e.g. POU5F1, SOX2, and NANOG), identify metabolic differences between species (e.g. threonine metabolism), clarify differences between human and mouse ESC developmental signaling pathways (e.g. LIF-activated JAK/STAT in mouse; Nodal/Activin-A-activated FGF in human), and reveal many novel genes and pathways predicted to be functionally associated with self-renewal in each species. These interactive networks are available online at www.StemSight.org for stem cell researchers to develop new hypotheses, discover potential mechanisms involving sparsely annotated genes, and prioritize genes of interest for experimental validation. Stem Cells 2013.
[Show abstract][Hide abstract] ABSTRACT: Derived from mesoderm precursors, hemangioblasts are bi-potential common progenitors of hematopoietic cells and endothelial cells. The regulatory events controlling hematopoietic and endothelial lineage specification are largely unknown, especially in humans. In this study, we establish a serum-free and feeder-free system with high efficient embryoid body (EB) generation to investigate the signals that direct differentiation of human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced-pluripotent stem cells (hiPSCs). Consistent with previous studies, the CD34+CD31+VE-cadherin+ cells derived from hPSCs contain hematopoietic and endothelial progenitors. In the presentence of hematopoietic and endothelial growth factors, some of CD34+CD31+VE-cadherin+ cells give rise to blast colony-forming cells (BL-CFCs), which has been used as to characterize bi-potential hemangioblasts. We found that the level of transforming growth factor beta (TGF-ß) 1 protein is increased during hPSC differentiation, and that TGF-ß signaling has the double-edged effect on hematopoietic and endothelial lineage differentiation in hPSCs. An addition of TGF-ß to hPSC differentiation before mesoderm induction promotes the development of mesoderm and the generation of CD34+CD31+VE-cadherin+ cells. An addition of TGF-ß inhibitor, SB431542, before mesoderm induction downregulates the expression of mesodermal markers and reduces the number of CD34+CD31+VE-cadherin+ progenitor cells. However, inhibition of TGF-ß signaling after mesoderm induction increases CD34+CD31+VE-cadherin+ progenitors and BL-CFCs. These data provide evidence that balance of positive and negative effects of TGF-ß signaling at appropriate timing is critical, and potential means to improve hematopoiesis and vasculogenesis from hPSCs.
Stem cells and development 06/2013; · 4.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
PLoS ONE 01/2013; 8(2):e56810. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: China's output in fundamental stem cell research has increased markedly in recent years. Vigorous public investment and infrastructure development have enabled major productivity gains, but challenges in regulation, governance, and the management of clinical expectations must be addressed to ensure scientific quality and sustainable growth.
[Show abstract][Hide abstract] ABSTRACT: Robust expansion and genetic manipulation of human embryonic stem cells (hESCs) and induced-pluripotent stem (iPS) cells are limited by poor cell survival after enzymatic dissociation into single cells. Although inhibition of apoptosis is implicated for the single-cell survival of hESCs, the protective role of attenuation of apoptosis in hESC survival has not been elucidated. Bcl-xL is one of several anti-apoptotic proteins, which are members of the Bcl-2 family of proteins. Using an inducible system, we ectopically expressed Bcl-xL gene in hESCs, and found a significant increase of hESC colonies in the single-cell suspension cultures. Overexpression of Bcl-xL in hESCs decreased apoptotic caspase-3(+) cells, suggesting attenuation of apoptosis in hESCs. Without altering the kinetics of pluripotent gene expression, the efficiency to generate embryoid bodies (EBs) in vitro and the formation of teratoma in vivo were significantly increased in Bcl-xL-overexpressing hESCs after single-cell dissociation. Interestingly, the number and size of hESC colonies from cluster cultures were not affected by Bcl-xL overexpression. Several genes of extracellular matrix and adhesion molecules were upregulated by Bcl-xL in hESCs without single-cell dissociation, suggesting that Bcl-xL regulates adhesion molecular expression independent of cell dissociation. In addition, the gene expressions of FAS and several TNF signaling mediators were downregulated by Bcl-xL. These data support a model in which Bcl-xL promotes cell survival and increases cloning efficiency of dissociated hESCs without altering hESC self-renewal by i) attenuation of apoptosis, and ii) upregulation of adhesion molecules to facilitate cell-cell or cell-matrix interactions.
Stem Cell Research 01/2012; 8(1):26-37. · 4.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Direct reprogramming of human somatic cells into induced pluripotent stem (iPS) cells by defined transcription factors (TFs) provides great potential for regenerative medicine and biomedical research. This procedure has many challenges, including low reprogramming efficiency, many partially reprogrammed colonies, somatic coding mutations in the genome, etc. Here, we describe a simple approach for generating fully reprogrammed human iPS cells by using a single polycistronic retroviral vector expressing four human TFs in a single open reading frame (ORF), combined with a cocktail containing three small molecules (Sodium butyrate, SB431542, and PD0325901). Our results demonstrate that human iPS cells generated by this approach express human ES cells markers and exhibit pluripotency demonstrated by their abilities to differentiate into the three germ layers in vitro and in vivo. Notably, this approach not only provides a much faster reprogramming process but also significantly diminishes partially reprogrammed iPS cell colonies, thus facilitating efficient isolation of desired fully reprogrammed iPS cell colonies.
PLoS ONE 01/2011; 6(10):e26592. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The molecules and environment that direct pluripotent stem cell differentiation into cardiomyocytes are largely unknown. Here, we determined a critical role of receptor tyrosine kinase, EphB4, in regulating cardiomyocyte generation from embryonic stem (ES) cells through endothelial cells. The number of spontaneous contracting cardiomyocytes, and the expression of cardiac-specific genes, including alpha-MHC and MLC-2V, was significantly decreased in EphB4-null ES cells. EphB4 was expressed in endothelial cells underneath contracting cardiomyocytes, but not in cardiomyocytes. Angiogenic inhibitors, including endostatin and angiostatin, inhibited endothelial cell differentiation and diminished cardiomyogenesis in ES cells. Generation of functional cardiomyocytes and the expression of cardiac-specific genes were significantly enhanced by co-culture of ES cells with human endothelial cells. Furthermore, the defects of cardiomyocyte differentiation in EphB4-deficient ES cells were rescued by human endothelial cells. For the first time, our study demonstrated that endothelial cells play an essential role in facilitating cardiomyocyte differentiation from pluripotent stem cells. EphB4 signaling is a critical component of the endothelial niche to regulate regeneration of cardiomyocytes.
Journal of Cellular Biochemistry 09/2010; 111(1):29-39. · 3.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bone marrow injury is a major adverse side effect of radiation and chemotherapy. Attempts to limit such damage are warranted, but their success requires a better understanding of how radiation and anticancer drugs harm the bone marrow. Here, we report one pivotal role of the BH3-only protein Puma in the radiosensitivity of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). Puma deficiency in mice confers resistance to high-dose radiation in a hematopoietic cell-autonomous manner. Unexpectedly, loss of one Puma allele is sufficient to confer mice radioresistance. Interestingly, null mutation in Puma protects both primitive and differentiated hematopoietic cells from damage caused by low-dose radiation but selectively protects HSCs and HPCs against high-dose radiation, thereby accelerating hematopoietic regeneration. Consistent with these findings, Puma is required for radiation-induced apoptosis in HSCs and HPCs, and Puma is selectively induced by irradiation in primitive hematopoietic cells, and this induction is impaired in Puma-heterozygous cells. Together, our data indicate that selective targeting of p53 downstream apoptotic targets may represent a novel strategy to protecting HSCs and HPCs in patients undergoing intensive cancer radiotherapy and chemotherapy.
[Show abstract][Hide abstract] ABSTRACT: Eph receptors and their ephrin ligands are involved in normal hematopoietic development and tumorigenesis. Using methylated CpG island amplification/DNA promoter microarray, we identified several EPH receptor and EPHRIN genes as potential hypermethylation targets in acute lymphoblastic leukemia (ALL). We subsequently studied the DNA methylation status of the Eph/ephrin family by bisulfite pyrosequencing. Hypermethylation of EPHA2, -A4, -A5, -A6, -A7, -A10, EPHB1, -B2, -B3, -B4, EFNA1, -A3, -A5, and EFNB1 and -B2 genes was detected in leukemia cell lines and primary ALL bone marrow samples. Expression analysis of EPHB4, EFNB2, and EFNA5 genes demonstrated that DNA methylation was associated with gene silencing. We cloned the promoter region of EPHB4 and demonstrated that promoter hypermethylation can result in EPHB4 transcriptional silencing. Restoration of EPHB4 expression by lentiviral transduction resulted in reduced proliferation and apoptotic cell death in Raji cells in which EPHB4 is methylated and silenced. Finally, we demonstrated that phosphorylated Akt is down-regulated in Raji cells transduced with EPHB4. These results suggest that epigenetic silencing by hypermethylation of EPH/EPHRIN family genes contributes to ALL pathogenesis and that EPHB4 can function as a tumor suppressor in ALL.
[Show abstract][Hide abstract] ABSTRACT: Both extrinsic and intrinsic mechanisms tightly govern hematopoietic stem cell (HSC) decisions of self-renewal and differentiation. However, transcription factors that can selectively regulate HSC self-renewal division after stress remain to be identified. Slug is an evolutionarily conserved zinc-finger transcription factor that is highly expressed in primitive hematopoietic cells and is critical for the radioprotection of these key cells. We studied the effect of Slug in the regulation of HSCs in Slug-deficient mice under normal and stress conditions using serial functional assays. Here, we show that Slug deficiency does not disturb hematopoiesis or alter HSC homeostasis and differentiation in bone marrow but increases the numbers of primitive hematopoietic cells in the extramedullary spleen site. Deletion of Slug enhances HSC repopulating potential but not its homing and differentiation ability. Furthermore, Slug deficiency increases HSC proliferation and repopulating potential in vivo after myelosuppression and accelerates HSC expansion during in vitro culture. Therefore, we propose that Slug is essential for controlling the transition of HSCs from relative quiescence under steady-state condition to rapid proliferation under stress conditions. Our data suggest that inhibition of Slug in HSCs may present a novel strategy for accelerating hematopoietic recovery, thus providing therapeutic benefits for patients after clinical myelosuppressive treatment.
[Show abstract][Hide abstract] ABSTRACT: Human embryonic stem (hES) cells are able to give rise to a variety of cell lineages under specific culture condition. An effective strategy for stable genetic modification in hES cells may provide a powerful tool for study of human embryogenesis and cell-based therapies. However, gene silences are documented in hES cells. In current study, we investigated whether genes controlled under ubiquitin promoter are expressed during hematopoietic-endothelial differentiation in hES cells. Undifferentiated hES cells (H1) were transduced by lentivirus encoding green fluorescent protein (GFP) gene under ubiquitin promoter. GFP-expressing hES cells (GFP-H1) were established after several rounds of mechanical selection under fluorescence microscope. GFP gene was stably expressed in hES cells throughout prolonged (> 50 passages) cultivation, and in differentiated embryo body (EB) and teratoma. Hematopoietic and endothelial markers, including KDR (VEGFR2), CD34, CD31, Tie-2, GATA-1 and GATA-2, were expressed at similar levels during hES cell differentiation in parent hES cells and GFP-H1 hES cells. CD34(+) cells isolated from GFP-H1 hES cells were capable to generate hematopoietic colony-forming cells and tubular structure-forming cells. Differentiated GFP-EB formed vasculature structures in a semi-solid sprouting EB model. These results indicated that a transgene under ubiquitin promoter in lentiviral transduced hES cells retained its expression in undifferentiated hES cells and in hES-derived hematopoietic and endothelial cells. With the view of embryonic mesodermal developing events in humans, genetic modification of hES cells by lentiviral vectors provides a powerful tool for study of hematopoiesis and vasculogenesis.
[Show abstract][Hide abstract] ABSTRACT: The signals that direct pluripotent stem cell differentiation into lineage-specific cells remain largely unknown. Here, we investigated the roles of BMP on vascular progenitor development from human embryonic stem cells (hESCs). In a serum-free condition, hESCs sequentially differentiated into CD34+CD31-, CD34+CD31+, and then CD34-CD31+ cells during vascular cell development. CD34+CD31+ cells contained vascular progenitor population that gives rise to endothelial cells and smooth muscle cells. BMP4 promoted hESC differentiation into CD34+CD31+ cells at an early stage. In contrast, TGFbeta suppressed BMP4-induced CD34+CD31+ cell development, and promoted CD34+CD31- cells that failed to give rise to either endothelial or smooth muscle cells. The BMP-Smad inhibitor, dorsomorphin, inhibited phosphorylation of Smad1/5/8, and blocked hESC differentiation to CD34+CD31+ progenitor cells, suggesting that BMP Smad-dependent signaling is critical for CD34+CD31+ vascular progenitor development. Our findings provide new insight into how pluripotent hESCs differentiate into vascular cells.
Journal of Cellular Biochemistry 11/2009; 109(2):363-74. · 3.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cardiovascular disease is one of leading causes of death throughout the U.S. and the world. The damage of cardiomyocytes resulting from ischemic injury is irreversible and leads to the development of progressive heart failure, which is characterized by the loss of functional cardiomyocytes. Because cardiomyocytes are unable to regenerate in the adult heart, cell-based therapy of transplantation provides a potential alternative approach to replace damaged myocardial tissue and restore cardiac function. A major roadblock toward this goal is the lack of donor cells; therefore, it is urgent to identify the cardiovascular cells that are necessary for achieving cardiac muscle regeneration. Pluripotent embryonic stem (ES) cells have enormous potential as a source of therapeutic tissues, including cardiovascular cells; however, the regulatory elements mediating ES cell differentiation to cardiomyocytes are largely unknown. In this review, we will focus on extrinsic factors that play a role in regulating different stages of cardiomyocyte differentiation of ES cells.
Journal of Cellular Biochemistry 06/2008; 104(1):119-28. · 3.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Stem cells have two common properties: the capacity for self-renewal and the potential to differentiate into one or more specialized cell types. In general, stem cells can be divided into two broad categories: adult (somatic) stem cells and embryonic stem cells. Recent evidence suggested that tumors may contain "cancer stem cells" with indefinite potential for self-renewal. In this review, we will focus on the molecular mechanisms regulating embryonic stem cell self-renewal and differentiation, and discuss how these mechanisms may be relevant in cancer cells.
Journal of Cellular Biochemistry 03/2008; 103(3):709-18. · 3.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We describe the differentiation of human embryonic stem (hES) cells into endothelial cells using a scalable two-dimensional method that avoids an embryoid-body intermediate. After transplantation into severe combined immunodeficient (SCID) mice, the differentiated cells contributed to arborized blood vessels that integrated into the host circulatory system and served as blood conduits for 150 d.
[Show abstract][Hide abstract] ABSTRACT: The molecular mechanisms that regulate human blood vessel formation during early development are largely unknown. Here we used human ESCs (hESCs) as an in vitro model to explore early human vasculogenesis. We demonstrated that stromal cell-derived factor-1 (SDF-1) and CXCR4 were expressed concurrently with hESC-derived embryonic endothelial differentiation. Human ESC-derived embryonic endothelial cells underwent dose-dependent chemotaxis to SDF-1, which enhanced vascular network formation in Matrigel. Blocking of CXCR4 signaling abolished capillary-like structures induced by SDF-1. Inhibition of the SDF-1/CXCR4 signaling pathway by AMD3100, a CXCR4 antagonist, disrupted the endothelial sprouting outgrowth from human embryoid bodies, suggesting that the SDF-1/CXCR4 axis plays a critical role in regulating initial vessel formation, and may function as a morphogen during human embryonic vascular development.
[Show abstract][Hide abstract] ABSTRACT: Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is widely used as a marker during vasculogenesis and angiogenesis from embryonic stem (ES) cells. However, the expression of PECAM-1 isoforms in ES cells has not been determined. The present study was designed to determine the role of PECAM-1 isoforms during in vitro endothelial differentiation of ES cells. It was found that undifferentiated ES cells expressed high level of PECAM-1, which primarily located at cell-cell junction, but the expression of PECAM-1 was sharply down-regulated during early ES cell differentiation. In addition, undifferentiated ES cells were found the expressed all eight known alternatively spliced PECAM-1 isoforms, among them the expression of PECAM-1 isoforms lacking exon 15 or 14&15 was predominant. Quantitative analysis revealed a significant increase in the expression of PECAM-1 isoform lacking exon 12&14&15 as vascular development of ES cells. These results indicate a constitutive expression of PECAM-1 in undifferentiated murine ES cells and suggest a developmental role of PECAM-1 isoform changes during vasculogenesis and angiogenesis.
Journal of Cellular Biochemistry 06/2005; 95(3):559-70. · 3.06 Impact Factor