Stem Cell Lineage Specification Research Articles

Rare Cell Proteomic Reactor Applied to Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-based Quantitative Proteomics Study of Human Embryonic Stem Cell Differentiation

The molecular basis governing the differentiation of human embryonic stem cells (hESCs) remains largely unknown. Systems-level analysis by proteomics provides a unique approach to tackle this question. However, the requirement of a large number of cells for proteomics analysis (i.e. 10(6)-10(7) cells) makes this assay challenging, especially for the study of rare events during hESCs lineage specification. Here, a fully integrated proteomics sample processing and analysis platform, termed rare cell proteomic reactor (RCPR), was developed for large scale quantitative proteomics analysis of hESCs with ∼50,000 cells. hESCs were completely extracted by a defined lysis buffer, and all of the proteomics sample processing procedures, including protein preconcentration, reduction, alkylation, and digestion, were integrated into one single capillary column with a strong cation exchange monolith matrix. Furthermore, on-line two-dimensional LC-MS/MS analysis was performed directly using RCPR as the first dimension strong cation exchange column. 2,281 unique proteins were identified on this system using only 50,000 hESCs. For stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative study, a ready-to-use and chemically defined medium and an in situ differentiation procedure were developed for complete SILAC labeling of hESCs with well characterized self-renewal and differentiation properties. Mesoderm-enriched differentiation was studied by RCPR using 50,000 hESCs, and 1,086 proteins were quantified with a minimum of two peptides per protein. Of these, 56 proteins exhibited significant changes during mesoderm-enriched differentiation, and eight proteins were demonstrated for the first time to be overexpressed during early mesoderm development. This work provides a new platform for the study of rare cells and in particular for further elucidating proteins that govern the mesoderm lineage specification of human pluripotent stem cells.

Tet1 and Tet2 Regulate 5-Hydroxymethylcytosine Production and Cell Lineage Specification in Mouse Embryonic Stem Cells

TET family enzymes convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. Here, we show that Tet1 and Tet2 are Oct4-regulated enzymes that together sustain 5hmC in mouse embryonic stem cells (ESCs) and are induced concomitantly with 5hmC during reprogramming of fibroblasts to induced pluripotent stem cells. ESCs depleted of Tet1 by RNAi show diminished expression of the Nodal antagonist Lefty1 and display hyperactive Nodal signaling and skewed differentiation into the endoderm-mesoderm lineage in embryoid bodies invitro. In Fgf4- and heparin-supplemented culture conditions, Tet1-depleted ESCs activate the trophoblast stem cell lineage determinant Elf5 and can colonize the placenta in midgestation embryo chimeras. Consistent with these findings, Tet1-depleted ESCsform aggressive hemorrhagic teratomas with increased endoderm, reduced neuroectoderm, and ectopic appearance of trophoblastic giant cells. Thus, 5hmC is an epigenetic modification associated with the pluripotent state, and Tet1 functions to regulate the lineage differentiation potential of ESCs.

Regulation of lineage specification of hematopoietic stem cells by polycomb group gene Bmi1

Regulation of lineage specification of hematopoietic stem cells by polycomb group gene Bmi1

Calcineurin-NFAT Signaling Critically Regulates Early Lineage Specification in Mouse Embryonic Stem Cells and Embryos

Self-renewal and pluripotency are hallmarks of embryonic stem cells (ESCs). However, the signaling pathways that trigger their transition from self-renewal to differentiation remain elusive. Here, we report that calcineurin-NFAT signaling is both necessary and sufficient to switch ESCs from an undifferentiated state to lineage-specific cells and that the inhibition of this pathway can maintain long-term ESC self-renewal independent of leukemia inhibitory factor. Mechanistically, this pathway converges with the Erk1/2 pathway to regulate Src expression and promote the epithelial-mesenchymal transition (EMT), a process required for lineage specification in response to differentiation stimuli. Furthermore, calcineurin-NFAT signaling is activated when the earliest differentiation event occurs in mouse embryos, and its inhibition disrupts extraembryonic lineage development. Collectively, our results demonstrate that the NFAT and Erk1/2 cascades form a signaling switch for early lineage segregation in mouse ESCs and provide significant insights into the regulation of the balance between ESC self-renewal and early lineage specification.

Germ Cells Are Forever: Programming of the Germ Line Genome.

In most organisms, primordial germ cells (PGCs) are set-aside early during embryogenesis. Subsequently, PGCs migrate through the embryo, associate with somatic gonadal cells and form the gonad. While PGCs seem highly specialized, their products are the ultimate stem cells that generate a complete organism generation after generation. We are interested in how germ line stem cell fate is established in the embryo and maintained during adult stages. Transcriptional regulation as well as translational control play an important role in controlling germ cell proliferation and germ line stem cell maintenance. We have used RNA expression screens to identify genes whose RNAs are specifically present in germ cells. We found that the 3′UTRs of these RNAs instruct the time and place when the RNAs are translated during germ cell development. This has let to the identification new targets for the translational regulators Nanos and Pumilio. These targets affect germ cell transcriptional silencing required for primordial germ cell specification and germ line stem cell differentiation in the adult. (platform)

SDF-1-Enhanced Cardiogenesis Requires CXCR4 Induction in Pluripotent Stem Cells

Transformation of pluripotent stem cells into cardiac tissue is the hallmark of cardiogenesis, yet pro-cardiogenic signals remain partially understood. Preceding cardiogenic induction, a surge in CXCR4 chemokine receptor expression in the early stages of stem cell lineage specification coincides with the acquisition of pre-cardiac profiles. Accordingly, CXCR4 selection, in conjunction with mesoderm-specific VEGF type II receptor FLK-1 co-expression, segregates cardiogenic populations. To assess the functionality of the CXCR4 biomarker, targeted activation and disruption were here exploited in the context of embryonic stem cell-derived cardiogenesis. Implicated as a cardiogenic hub through unbiased bioinformatics analysis, induction of the CXCR4/SDF-1 receptor-ligand axis triggered enhanced beating activity in stem cell progeny. Gene expression knockdown of CXCR4 disrupted spontaneous embryoid body differentiation and blunted the expression of cardiogenic markers MEF2C, Nkx2.5, MLC2a, MLC2v, and cardiac-MHC. Exogenous SDF-1 treatment failed to rescue cardiogenic-deficient phenotype, demonstrating a requirement for CXCR4 expression in mediating SDF-1 effects. Thus, a pro-cardiogenic signaling role for the CXCR4/SDF1 axis is herein revealed within pluripotent stem cell progenitors, exposing a functional target to promote lineage-specific differentiation.

Response of Human Embryonic Stem Cell–Derived Mesenchymal Stem Cells to Osteogenic Factors and Architectures of Materials During In Vitro Osteogenesis

One of the major challenges to the application of human embryonic stem cells (hESCs) to the repair of defective tissues is the directed differentiation of cells into specific lineages to avoid the formation of inferior heterogeneous tissues. To accomplish this goal, the lineage-specific stem cell population needs to be isolated and optimal differentiation conditions need to be defined. In this study, homogenous hESC-derived mesenchymal stem cells (hESC-MSCs) were generated and used to construct bone tissue. The effect of osteogenic factors, including dexamethasone (Dex) and bone morphogenetic protein-7 (BMP-7), on the osteogenesis of hESC-MSCs was investigated. It was found that BMP-7 itself had little effect on the in vitro osteogenic differentiation of hESC-MSCs; however, there was a synergic effect between BMP-7 and Dex in promoting osteogenesis. The effect of osteoconductive nanofibrous polylactic acid material on osteogenesis of hESC-MSCs was also investigated. It was found that the nanofibrous matrix architecture promoted alkaline phosphatase activity and calcium deposition of cells cultured under osteogenic conditions. Based on these findings, the hESC-MSCs were cultured on three-dimensional nanofibrous scaffolds in combination with Dex and BMP-7 stimulation in vitro to generate bone-like tissues. After 6 weeks of culture, highly mineralized tissues developed with specific bone marker genes expressed. These data illustrate the promise of hESC-MSCs for bone regeneration under optimal conditions.

Unique Differentiation Profile of Mouse Embryonic Stem Cells in Rotary and Stirred Tank Bioreactors

Embryonic stem (ES)-cell-derived lineage-specific stem cells, for example, hematopoietic stem cells, could provide a potentially unlimited source for transplantable cells, especially for cell-based therapies. However, reproducible methods must be developed to maximize and scale-up ES cell differentiation to produce clinically relevant numbers of therapeutic cells. Bioreactor-based dynamic culture conditions are amenable to large-scale cell production, but few studies have evaluated how various bioreactor types and culture parameters influence ES cell differentiation, especially hematopoiesis. Our results indicate that cell seeding density and bioreactor speed significantly affect embryoid body formation and subsequent generation of hematopoietic stem and progenitor cells in both stirred tank (spinner flask) and rotary microgravity (Synthecon™) type bioreactors. In general, high percentages of hematopoietic stem and progenitor cells were generated in both bioreactors, especially at high cell densities. In addition, Synthecon bioreactors produced more sca-1(+) progenitors and spinner flasks generated more c-Kit(+) progenitors, demonstrating their unique differentiation profiles. cDNA microarray analysis of genes involved in pluripotency, germ layer formation, and hematopoietic differentiation showed that on day 7 of differentiation, embryoid bodies from both bioreactors consisted of all three germ layers of embryonic development. However, unique gene expression profiles were observed in the two bioreactors; for example, expression of specific hematopoietic genes were significantly more upregulated in the Synthecon cultures than in spinner flasks. We conclude that bioreactor type and culture parameters can be used to control ES cell differentiation, enhance unique progenitor cell populations, and provide means for large-scale production of transplantable therapeutic cells.

Poised Lineage Specification in Multipotential Hematopoietic Stem and Progenitor Cells by the Polycomb Protein Bmi1

Polycomb group (PcG) proteins are essential regulators of stem cells. PcG and trithorax group proteins mark developmental regulator gene promoters with bivalent domains consisting of overlapping repressive and activating histone modifications to keep them poised for activation in embryonic stem cells. Bmi1, a component of PcG complexes, maintains the self-renewal capacity of adult stem cells, but its role in multipotency remains obscure. Here we show that Bmi1 is critical for multipotency of hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs). B cell lineage developmental regulator genes, Ebf1 and Pax5, appeared to be transcriptionally repressed by bivalent domains before lineage commitment. Loss of Bmi1 resulted in a resolution of bivalent domains at the Ebf1 and Pax5 loci, leading to their premature expression in HSC/MPPs accompanied by accelerated lymphoid specification and a marked reduction in HSC/MPPs. Thus, Bmi1 is required to reinforce bivalent domains at key developmental regulator gene loci to maintain lineage specification poised for activation in adult stem cells.

Direct Inhibition of Pumilo Activity by Bam and Bgcn in Drosophila Germ Line Stem Cell Differentiation

The fate of stem cells is intricately regulated by numerous extrinsic and intrinsic factors that promote maintenance or differentiation. The RNA-binding translational repressor Pumilio (Pum) in conjunction with Nanos (Nos) is required for self-renewal, whereas Bam (bag-of-marbles) and Bgcn (benign gonial cell neoplasm) promote differentiation of germ line stem cells in the Drosophila ovary. Genetic analysis suggests that Bam and Bgcn antagonize Pum/Nos function to promote differentiation; however, the molecular basis of this epistatic relationship is currently unknown. Here, we show that Bam and Bgcn inhibit Pum function through direct binding. We identified a ternary complex involving Bam, Bgcn, and Pum in which Bam, but not Bgcn, directly interacts with Pum, and this interaction is greatly increased by the presence of Bgcn. In a heterologous reporter assay to monitor Pum activity, Bam, but not Bgcn, inhibits Pum activity. Notably, the N-terminal region of Pum, which lacks the C-terminal RNA-binding Puf domain, mediates both the ternary protein interaction and the Bam inhibition of Pum function. These studies suggest that, in cystoblasts, Bam and Bgcn may directly inhibit Pum/Nos activity to promote differentiation of germ line stem cells.

The regulation of stem cell differentiation by cell-cell contact on micropatterned material surfaces

Using the material technique recently developed by us, we prepared a micropattern on poly(ethylene glycol) (PEG) hydrogel to keep background resistant to cell adhesion for a long time, which made examination of differentiation of localized stem cells available. Our micropattern designed in this paper prevented or ensured contact between cells adhering in arginine-glycine-aspartic acid (RGD) microdomains, and thus afforded a unique way to study the effects of cell-cell contact on the lineage differentiation of stem cells while ruling out the interference of soluble factors or cell seeding concentration etc. As demonstration, mesenchymal stem cells derived from rats were examined in this study, and both osteogenic and adipogenic differentiations were found to be regulated by cell-cell contact. Isolated cells exhibited less significant differentiation than paired or aggregated cells. For those stem cells in contact, the extent of differentiation was fairly linearly related to the extent of contact characterized by coordination number. Additionally, we revealed the existence of some unknown cues besides gap junction responsible for such effects of cell-cell contact.

Alternative Splicing in the Differentiation of Human Embryonic Stem Cells into Cardiac Precursors

The role of alternative splicing in self-renewal, pluripotency and tissue lineage specification of human embryonic stem cells (hESCs) is largely unknown. To better define these regulatory cues, we modified the H9 hESC line to allow selection of pluripotent hESCs by neomycin resistance and cardiac progenitors by puromycin resistance. Exon-level microarray expression data from undifferentiated hESCs and cardiac and neural precursors were used to identify splice isoforms with cardiac-restricted or common cardiac/neural differentiation expression patterns. Splice events for these groups corresponded to the pathways of cytoskeletal remodeling, RNA splicing, muscle specification, and cell cycle checkpoint control as well as genes with serine/threonine kinase and helicase activity. Using a new program named AltAnalyze (http://www.AltAnalyze.org), we identified novel changes in protein domain and microRNA binding site architecture that were predicted to affect protein function and expression. These included an enrichment of splice isoforms that oppose cell-cycle arrest in hESCs and that promote calcium signaling and cardiac development in cardiac precursors. By combining genome-wide predictions of alternative splicing with new functional annotations, our data suggest potential mechanisms that may influence lineage commitment and hESC maintenance at the level of specific splice isoforms and microRNA regulation.

MicroRNAs Regulation Modulated Self-Renewal and Lineage Differentiation of Stem Cells

Stem cells are unique cells in the ability that can self-renew and differentiate into a wide variety of cell types, suggesting that a specific molecular control network underlies these features. To date, stem cells have been applied to many clinical therapeutic approaches. For example, hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) are the cells responding to ischemia or injury and engage in effective revascularization to repair within impairment regions. Transplantation of MSCs after stroke and hindlimb ischemia results in remarkable recovery through enhancing angiogenesis. MicroRNAs are a novel class of endogenous, small, noncoding RNAs that work via translational inhibition or degradation of their target mRNAs to downregulate gene expression. MicroRNAs have been strongly linked to stem cells, which have a remarkable role in development. In this study, we focused on the microRNA regulation in multiple stem cells. For example, miR-520h was upregulated and miR-129 was downregulated in HSC. MiR-103, 107, 140, 143, 638, and 663 were associated with MSCs while miR-302s and miR-136 were associated with ESCs. In NSCs, miR-92b, let-7, and miR-125 were the critical regulators. This overview of the recent advances in the aspects of molecular control of stem cell biology reveals the importance of microRNAs, which may be helpful for future work.

CD15, CD24, and CD29 Define a Surface Biomarker Code for Neural Lineage Differentiation of Stem Cells

Identification and use of cell surface cluster of differentiation (CD) biomarkers have enabled much scientific and clinical progress. We identify a CD surface antigen code for the neural lineage based on combinatorial flow cytometric analysis of three distinct populations derived from human embryonic stem cells: (1) CD15(+)/CD29(HI)/CD24(LO) surface antigen expression defined neural stem cells; (2) CD15(-)/CD29(HI)/CD24(LO) revealed neural crest-like and mesenchymal phenotypes; and (3) CD15(-)/CD29(LO)/CD24(HI) selected neuroblasts and neurons. Fluorescence-activated cell sorting (FACS) for the CD15(-)/CD29(LO)/CD24(HI) profile reduced proliferative cell types in human embryonic stem cell differentiation. This eliminated tumor formation in vivo, resulting in pure neuronal grafts. In conclusion, combinatorial CD15/CD24/CD29 marker profiles define neural lineage development of neural stem cell, neural crest, and neuronal populations from human stem cells. We believe this set of biomarkers enables analysis and selection of neural cell types for developmental studies and pharmacological and therapeutic applications.

Alternative Splicing Modulates Stem Cell Differentiation

Stem cells have the surprising potential to develop into many different cell types. Therefore, major research efforts have focused on transplantation of stem cells and/or derived progenitors for restoring depleted diseased cells in degenerative disorders. Understanding the molecular controls, including alternative splicing, that arise during lineage differentiation of stem cells is crucial for developing stem cell therapeutic approaches in regeneration medicine. Alternative splicing to allow a single gene to encode multiple transcripts with different protein coding sequences and RNA regulatory elements increases genomic complexities. Utilizing differences in alternative splicing as a molecular marker may be more sensitive than simply gene expression in various degrees of stem cell differentiation. Moreover, alternative splicing maybe provide a new concept to acquire induced pluripotent stem cells or promote cell-cell transdifferentiation for restorative therapies and basic medicine researches. In this review, we highlight the recent advances of alternative splicing regulation in stem cells and their progenitors. It will hopefully provide much needed knowledge into realizing stem cell biology and related applications.

Drosophila Maelstrom Ensures Proper Germline Stem Cell Lineage Differentiation by Repressing microRNA-7

Nuage is a germline-unique perinuclear structure conserved throughout the animal kingdom. Maelstrom (Mael) is an unusual nuage component, as it is also found in the nucleus. Mael contains a High Mobility Group box, known to mediate DNA binding. We show that Mael nuclear function is required for proper differentiation in the Drosophila germline stem cell (GSC) lineage. In mael mutant testes, transit-amplifying cysts fail to differentiate into primary spermatocytes, instead breaking down into ectopic GSCs and smaller cysts, due to a depletion of Bag-of-marbles (Bam) protein. Mael regulates Bam via repression of miR-7. Mael binds the miR-7 promoter and is required for the local accumulation of HP1 and H3K9me3. miR-7 targets bam directly at its 3'UTR, and a reduction in miR-7 expression can rescue germline differentiation defects found in mael mutants by alleviating Bam repression. We propose that Mael ensures proper differentiation in the GSC lineage by repressing miR-7.

Altered retinoic acid (RA)‐induced lineage specification of mouse embryonic stem cells (mESCs) following a transient pulse of a histone deacetylase inhibitor.

RA treatment during mESC differentiation results in predominantly neural specification. A transient pulse of the histone deacetylase inhibitor, Trichostatin A (TSA), during early mESC differentiation via embryoid body (EB) formation results in maintenance of undifferentiated characteristics. Our objective was to determine whether a pulse of TSA followed by RA alters neural lineage specification. Undifferentiated mESCs are exposed to 100 nM of TSA during the first 24h of 3 days of EB formation. EBs are plated and RA added for an additional 4 days. Selected RT‐PCR shows repressed neural marker expression and only a small increase in a few endoderm markers. Gene expression array studies analyzed using a lineage annotated gene database demonstrates a marked increase in expression of numerous placental genes and confirms diminished neural gene expression. Immunofluorescence for the trophoblast marker Plac1, shows increased levels in TSA‐pulsed, RA‐treated cells with minimal expression in RA‐only differentiated cells. Conversely, the neural specific product, Doublecortin, is highly expressed in the RA‐only treated cells and shows diminished protein levels in TSA‐pulsed, RA‐treated cells. We conclude that transient TSA treatment prior to RA induces a dramatic lineage switch characterized by repressed neural differentiation and marked trophoblast induction. Supported by NIH grant RO1DK064243 to MKF.

Stochasticity and the Molecular Mechanisms of Induced Pluripotency

The generation of induced pluripotent stem cells from adult somatic cells by ectopic expression of key transcription factors holds significant medical promise. However, current techniques for inducing pluripotency rely on viral infection and are therefore not, at present, viable within a clinical setting. Thus, there is now a need to better understand the molecular basis of stem cell pluripotency and lineage specification in order to investigate alternative methods to induce pluripotency for clinical application. However, the complexity of the underlying molecular circuitry makes this a conceptually difficult task. In order to address these issues, we considered a computational model of transcriptional control of cell fate specification. The model comprises two mutually interacting sub-circuits: a central pluripotency circuit consisting of interactions between stem-cell specific transcription factors OCT4, SOX2 and NANOG coupled to a differentiation circuit consisting of interactions between lineage-specifying master genes.The molecular switches which arise from feedback loops within these circuits give rise to a well-defined sequence of successive gene restrictions corresponding to a controlled differentiation cascade in response to environmental stimuli. Furthermore, we found that this differentiation cascade is strongly unidirectional: once silenced, core transcription factors cannot easily be reactivated. In the context of induced pluripotency, this indicates that differentiated cells are robustly resistant to reprogramming to a more primitive state. However, our model suggests that under certain circumstances, amplification of low-level fluctuations in transcriptional status (transcriptional “noise”) may be sufficient to trigger reactivation of the core pluripotency switch and reprogramming to a pluripotent state. This interpretation offers an explanation of a number of experimental observations concerning the molecular mechanisms of cellular reprogramming by defined factors and suggests a role for stochasticity in reprogramming of somatic cells to pluripotency.

Directing osteogenic and myogenic differentiation of MSCs: interplay of stiffness and adhesive ligand presentation

The mechanical properties of the extracellular matrix (ECM) can exert significant influence in determining cell fate. Human mesenchymal stem cells (MSCs) grown on substrates with varying stiffness have been shown to express various cell lineage markers, without the use of toxic DNA demethylation agents or complex cocktails of expensive growth factors. Here we investigated the myogenic and osteogenic potential of various polyacrylamide gel substrates that were coated with covalently bound tissue-specific ECM proteins (collagen I, collagen IV, laminin, or fibronectin). The gel-protein substrates were shown to support the growth and proliferation of MSCs in a stiffness-dependent manner. Higher stiffness substrates encouraged up to a 10-fold increase in cell number over lower stiffness gels. There appears to be definitive interplay between substrate stiffness and ECM protein with regard to the expression of both osteogenic and myogenic transcription factors by MSCs. Of the 16 gel-protein combinations investigated, osteogenic differentiation was found to occur significantly only on collagen I-coated gels with the highest modulus gel tested (80 kPa). Myogenic differentiation occurred on all gel-protein combinations that had stiffnesses >9 kPa but to varying extents as ascertained by MyoD1 expression. Peak MyoD1 expression was seen on gels with a modulus of 25 kPa coated in fibronectin, with similar levels of expression observed on 80-kPa collagen I-coated gels. The modulation of myogenic and osteogenic transcription factors by various ECM proteins demonstrates that substrate stiffness alone does not direct stem cell lineage specification. This has important implications in the development of tailored biomaterial systems that more closely mimic the microenvironment found in native tissues.

Dorsomorphin, a Selective Small Molecule Inhibitor of BMP Signaling, Promotes Cardiomyogenesis in Embryonic Stem Cells

BackgroundPluripotent embryonic stem (ES) cells, which have the capacity to give rise to all tissue types in the body, show great promise as a versatile source of cells for regenerative therapy. However, the basic mechanisms of lineage specification of pluripotent stem cells are largely unknown, and generating sufficient quantities of desired cell types remains a formidable challenge. Small molecules, particularly those that modulate key developmental pathways like the bone morphogenetic protein (BMP) signaling cascade, hold promise as tools to study in vitro lineage specification and to direct differentiation of stem cells toward particular cell types.Methodology/ Principal FindingsWe describe the use of dorsomorphin, a selective small molecule inhibitor of BMP signaling, to induce myocardial differentiation in mouse ES cells. Cardiac induction is very robust, increasing the yield of spontaneously beating cardiomyocytes by at least 20 fold. Dorsomorphin, unlike the endogenous BMP antagonist Noggin, robustly induces cardiomyogenesis when treatment is limited to the initial 24-hours of ES cell differentiation. Quantitative-PCR analyses of differentiating ES cells indicate that pharmacological inhibition of BMP signaling during the early critical stage promotes the development of the cardiomyocyte lineage, but reduces the differentiation of endothelial, smooth muscle, and hematopoietic cells.Conclusions/ SignificanceAdministration of a selective small molecule BMP inhibitor during the initial stages of ES cell differentiation substantially promotes the differentiation of primitive pluripotent cells toward the cardiomyocytic lineage, apparently at the expense of other mesodermal lineages. Small molecule modulators of developmental pathways like dorsomorphin could become versatile pharmacological tools for stem cell research and regenerative medicine.