Differentiation Of Human ES Cells Research Articles

Maintenance and differentiation of human ES cells on polyvinylidene fluoride scaffolds immobilized with a vitronectin-derived peptide.

Polyvinylidene fluoride (PVDF) is biocompatible, easy to fabricate, and has piezoelectric properties; it has been used for many biomedical applications including stem cell engineering. However, long-term cultivation of human embryonic stem cells (hESCs) and their differentiation toward cardiac lineages on PVDF have not been investigated. Herein, PVDF nanoscaled membrane scaffolds were fabricated by electrospinning; a vitronectin-derived peptide-mussel adhesive protein fusion (VNm) was immobilized on the scaffolds. hESCs cultured on the VNm-coated PVDF scaffold (VNm-PVDF scaffold) were stably expanded for more than 10 passages while maintaining the expression of pluripotency markers and genomic integrity. Under cardiac differentiation conditions, hESCs on the VNm-PVDF scaffold generated more spontaneously beating colonies and showed the upregulation of cardiac-related genes, compared with those cultured on Matrigel and VNm alone. Thus, VNm-PVDF scaffolds may be suitable for the long-term culture of hESCs and their differentiation into cardiac cells, thus expanding their application in regenerative medicine.

Efficient differentiation of human ES and iPS cells into cardiomyocytes on biomaterials under xeno-free conditions.

Current xeno-free and chemically defined methods for the differentiation of hPSCs (human pluripotent stem cells) into cardiomyocytes are not efficient and are sometimes not reproducible. Therefore, it is necessary to develop reliable and efficient methods for the differentiation of hPSCs into cardiomyocytes for future use in cardiovascular research related to drug discovery, cardiotoxicity screening, and disease modeling. We evaluated two representative differentiation methods that were reported previously, and we further developed original, more efficient methods for the differentiation of hPSCs into cardiomyocytes under xeno-free, chemically defined conditions. The developed protocol successively differentiated hPSCs into cardiomyocytes, approximately 90-97% of which expressed the cardiac marker cTnT, with beating speeds and sarcomere lengths that were similar to those of a healthy adult human heart. The optimal cell culture biomaterials for the cardiac differentiation of hPSCs were also evaluated using extracellular matrix-mimetic material-coated dishes. Synthemax II-coated and Laminin-521-coated dishes were found to be the most effective and efficient biomaterials for the cardiac differentiation of hPSCs according to the observation of hPSC-derived cardiomyocytes with high survival ratios, high beating colony numbers, a similar beating frequency to that of a healthy adult human heart, high purity levels (high cTnT expression) and longer sarcomere lengths similar to those of a healthy adult human heart.

Abstract 441: Role of Nitric Oxide and S-nitrosylation in Cardiomyogenesis by iPSCs

Introduction: The mechanism by which signaling pathways, such as Wnt and BMP interact and modulate each other’s function is crucial to our understanding of cardiomyogenesis and cardiomyocyte proliferation. Nitric oxide (NO) is a signaling molecule that can trigger cardiac differentiation of stem cells, suggesting a cardiogenic function of NO synthase(s) (NOS). Hypothesis: NO modulates transcription factor function during pluripotency and differentiation toward a cardiac phenotype. Methods: Induced pluripotent stem cells (iPSCs) were derived from fibroblasts from wildtype mice and mice lacking S-nitrosoglutathione reductase (GSNOR -/- ), a denitrosylase that regulates protein S-nitrosylation. iPSCs were differentiated into functional cardiomyocytes from embryoid bodies (EBs) via the hanging-drop method. Results: During differentiation into cardiomyocytes, GSNOR -/- iPSC-derived cardiomyocytes exhibited reduced expression of mesoderm induction-related ( Brachyury ), cardiac mesoderm (Kdr , Isl-1 ) and cardiac progenitor genes ( Nkx2.5 , GATA4 ). Axin-1, an inducer of apoptosis and negative regulator of the Wnt signaling pathway and MAPK pathways, specifically p38, were increased on EB-Day (D)4. In contrast, SMAD1/5/8, members of the BMP canonical signaling pathway, were reduced beginning on EB-D8. Increased p38 is associated with reduced GATA4 expression and differentiation of human ES cells into cardiomyocytes. Decreased SMAD1/5/8 is likely at least in part responsible for the reduced expression of Nkx2.5. Conclusions: Our findings support that the absence of GSNOR modulates Wnt/β-catenin and BMP signaling pathways during cardiogenesis, resulting in reduced expression of mesoderm, cardiac mesoderm and cardiac progenitor genes. These findings are expected to have important implications for regenerative medicine and can provide new targets for iPS cell-based therapy.

Hemangioblast, hemogenic endothelium, and primitive versus definitive hematopoiesis.

The types of progenitors generated during the successive stages of embryonic blood development are now fairly well characterized. The terminology used to describe these waves, however,can still be confusing. What is truly primitive? What is uniquely definitive? These questions become even more challenging to answer when blood progenitors are derived invitro upon the differentiation of embryonic stem cells or induced pluripotent stem cells. Similarly, the cellular origin of these blood progenitors can be controversial. Are all blood cells, including the primitive wave, derived from hemogenic endothelium? Is the hemangioblast an invitro artifact or is this mesoderm entity also present in the developing embryo? Here, we discuss the latest findings and propose some consensus relating to these controversial issues.

Inhibition of Lysine-Specific Demethylase-1 (LSD1/KDM1A) Promotes the Adipogenic Differentiation of hESCs Through H3K4 Methylation.

Given their totipotency, human embryonic stem cells (hESCs) can differentiate into all types of cells, including adipocytes, and provide an excellent research model for studying diseases associated with the metabolism of adipocytes, such as obesity and diabetes mellitus. Epigenetic regulation, including DNA methylation and histone modification, plays an essential role in the development and differentiation of hESCs. Lysine-specific demethylase 1 (LSD1), a well-characterized histone-modifying enzyme, demethylates dimethylated histone H3 lysine 4 (H3K4) through a flavin adenine dinucleotide (FAD)-dependent oxidative reaction. LSD1 affects the growth and differentiation of human and mouse ES cells, and the deletion of this gene in mice leads to embryonic lethality. Here, we investigated the functional role of LSD1 during the adipogenic differentiation of hESCs involving the demethylation of H3K4. We also found that treating hESCs with the LSD1 inhibitor CBB1007 promotes the adipogenic differentiation of hESCs.

OCT4 Coordinates with WNT Signaling to Pre-pattern Chromatin at the SOX17 Locus during Human ES Cell Differentiation into Definitive Endoderm

SummaryWe demonstrate that the pluripotency gene OCT4 has a role in regulating differentiation via Wnt signaling. OCT4 expression levels in human embryonic stem cells increases transiently during the first 24 hr of in vitro differentiation, with OCT4 occupancy increasing at endoderm regulators such as SOX17 and FOXA2. This increased occupancy correlates with loss of the PRC2 complex and the inhibitory histone mark H3K27me3. Knockdown of OCT4 during differentiation inhibits mesendoderm formation and removal of the H3K27me3 mark from the SOX17 promoter, suggesting that OCT4 acts to induce removal of the PRC2 complex. Furthermore, OCT4 and β-catenin can be co-immunoprecipitated upon differentiation, and Wnt stimulation is required for the enhanced OCT4 occupancy and loss of the PRC2 complex from the SOX17 promoter. In conclusion, our study reveals that OCT4, a master regulator of pluripotency, may also collaborate with Wnt signaling to drive endoderm induction by pre-patterning epigenetic markers on endodermal promoters.

Limited hair cell induction from human induced pluripotent stem cells using a simple stepwise method

Disease-specific induced pluripotent stem cells (iPS) cells are expected to contribute to exploring useful tools for studying the pathophysiology of inner ear diseases and to drug discovery for treating inner ear diseases. For this purpose, stable induction methods for the differentiation of human iPS cells into inner ear hair cells are required. In the present study, we examined the efficacy of a simple induction method for inducing the differentiation of human iPS cells into hair cells. The induction of inner ear hair cell-like cells was performed using a stepwise method mimicking inner ear development. Human iPS cells were sequentially transformed into the preplacodal ectoderm, otic placode, and hair cell-like cells. As a first step, preplacodal ectoderm induction, human iPS cells were seeded on a Matrigel-coated plate and cultured in a serum free N2/B27 medium for 8 days according to a previous study that demonstrated spontaneous differentiation of human ES cells into the preplacodal ectoderm. As the second step, the cells after preplacodal ectoderm induction were treated with basic fibroblast growth factor (bFGF) for induction of differentiation into otic-placode-like cells for 15 days. As the final step, cultured cells were incubated in a serum free medium containing Matrigel for 48 days. After preplacodal ectoderm induction, over 90% of cultured cells expressed the genes that express in preplacodal ectoderm. By culture with bFGF, otic placode marker-positive cells were obtained, although their number was limited. Further 48-day culture in serum free media resulted in the induction of hair cell-like cells, which expressed a hair cell marker and had stereocilia bundle-like constructions on their apical surface. Our results indicate that hair cell-like cells are induced from human iPS cells using a simple stepwise method with only bFGF, without the use of xenogeneic cells.

The lncRNA DEANR1 facilitates human endoderm differentiation by activating FOXA2 expression.

Long non-coding RNAs (lncRNAs) regulate diverse biological processes, including cell lineage specification. Here, we report transcriptome profiling of human endoderm and pancreatic cell lineages using purified cell populations. Analysis of the data sets allows us to identify hundreds of lncRNAs that exhibit differentiation-stage-specific expression patterns. As a first step in characterizing these lncRNAs, we focus on an endoderm-specific lncRNA, definitiveendoderm-associated lncRNA1 (DEANR1), and demonstrate that it plays an important role in human endoderm differentiation. DEANR1 contributes to endoderm differentiation by positively regulating expression of the endoderm factor FOXA2. Importantly, overexpression of FOXA2 is able to rescue endoderm differentiation defects caused byDEANR1 depletion. Mechanistically, DEANR1 facilitates FOXA2 activation by facilitating SMAD2/3 recruitment to the FOXA2 promoter. Thus, our studynot only reveals a large set of differentiation-stage-specific lncRNAs but also characterizes a functional lncRNA that is important for endoderm differentiation.

Transcription factor binding dynamics during human ES cell differentiation.

SummaryPluripotent stem cells provide a powerful system to dissect the underlying molecular dynamics that regulate cell fate changes during mammalian development. Here we report the integrative analysis of genome wide binding data for 38 transcription factors with extensive epigenome and transcriptional data across the differentiation of human embryonic stem cells to the three germ layers. We describe core regulatory dynamics and show the lineage specific behavior of selected factors. In addition to the orchestrated remodeling of the chromatin landscape, we find that the binding of several transcription factors is strongly associated with specific loss of DNA methylation in one germ layer and in many cases a reciprocal gain in the other layers. Taken together, our work shows context-dependent rewiring of transcription factor binding, downstream signaling effectors, and the epigenome during human embryonic stem cell differentiation.

The generation of definitive endoderm from human embryonic stem cells is initially independent from activin A but requires canonical Wnt-signaling.

The activation of the TGF-beta pathway by activin A directs ES cells into the definitive endoderm germ layer. However, there is evidence that activin A/TGF-beta is not solely responsible for differentiation into definitive endoderm. GSK3beta inhibition has recently been shown to generate definitive endoderm-like cells from human ES cells via activation of the canonical Wnt-pathway. The GSK3beta inhibitor CHIR-99021 has been reported to generate mesoderm from human iPS cells. Thus, the specific role of the GSK3beta inhibitor CHIR-99021 was analyzed during the differentiation of human ES cells and compared against a classic endoderm differentiation protocol. At high concentrations of CHIR-99021, the cells were directed towards mesodermal cell fates, while low concentrations permitted mesodermal and endodermal differentiation. Finally, the analyses revealed that GSK3beta inhibition rapidly directed human ES cells into a primitive streak-like cell type independently from the TGF-beta pathway with mesoderm and endoderm differentiation potential. Addition of low activin A concentrations effectively differentiated these primitive streak-like cells into definitive endoderm. Thus, the in vitro differentiation of human ES cells into definitive endoderm is initially independent from the activin A/TGF-beta pathway but requires high canonical Wnt-signaling activity.

Intestinal Differentiation of Human ESCs

ES cells (ESCs) are pluripotent and offer a good tool to study early embryonic development. Intestinal cells are derived from the definitive endoderm. In contrast to adult tissue stem cells, embryonic development and differentiation from ES cells have not been as well investigated in the intestine. There are four differentiated cell types of non-proliferative epithelial cells: enterocytes, goblet cells, enteroendocrine cells, and Paneth cells. Intestinal stem cells (ISCs) and progenitor cells reside in crypts, proliferate vigorously, and function as the source of differentiated epithelial cells. Here, we describe a protocol, in which differentiated cell types of the intestine are derived from human ESCs. In this protocol, we describe a protocol to differentiate mouse ES cells into Cdx2-expressing intestinal endoderm efficiently by co-culturing with M15, a mouse mesonephric cell line, and treatment with two chemical compounds. The chemical compounds used are BIO and DAPT. BIO is a Gsk3 inhibitor, that activate Wnt signaling pathway, and DAPT is a-secretase inhibitor that inhibit Notch signaling pathway. BIO and DAPT treatment yielded all representative cell lineages, enterocytes, goblet cells, enteroendocrine cells and paneth cells, to be derived from human ESCs. The protocol for human ESCs is principally very similar with that for the mouse ESCs, with some modifications.

Abstract 19803: TMEM88: A Novel Regulator of Cardiac Development Identified Through Chromatin Dynamics

Cardiac development is dependent on the biphasic activities of Wnt/β-catenin signaling where it is required for mesoderm development but must be blocked to specify the cardiogenic mesoderm. The mechanism mediating this inhibition of Wnt/β-catenin signaling in cardiac progenitor cells is not known. Methods and Results. RNA seq was used to analyze genes most highly induced in the cardiac progenitor cell during cardiac differentiation of human ES cells. Transmembrane protein 88 (TMEM88) was identified for further study. In line with previous work showing TMEM88 binds Disheveled by a C-terminal PDZ binding motif, we used transgenic hES cells to show that TMEM88 blocks Wnt/β-catenin signaling during cardiac differentiation. Chromatin dynamics showed marked suppression (H3K27me3) of this locus in the transition through mesoderm with a switch to an activated state (H3K4me3) in the cardiac progenitor; a signature resembling other genes involved in mediating cell fate changes such as transcription factors. By qPCR, TMEM88 is highly up-regulated prior to Nkx2.5 and Isl1 expression. In situ hybridization of developing zebrafish embryos showed expression of TMEM88 specifically in the cardiogenic ventro-lateral mesoderm overlapping the expression profile of cardiac transcription factors such as Nkx2.5 and MEF2c. Short hairpin RNA targeting the TMEM88 transcript was used to determine its requirement for cardiac differentiation of hES cells. Suppression of TMEM88 resulted in a failure to specify the cardiac lineage based on significantly reduced Nkx2.5, Tbx5, and cTnT expression. Linear regression analysis revealed a dose dependent effect of increased TMEM88 significantly correlating with higher cTnT expression. In vivo, morpholino knockdown of TMEM88 in zebrafish markedly alters heart development with some embryos failing to generate a heart. Phylogenetic analysis of TMEM88 orthologs identifies the expression of this gene only in vertebrate chordates. Conclusion. These data show that TMEM88 is required for cardiac development in vitro and in vivo acting mechanistically in a cell autonomous manner to inhibit Wnt/β-catenin signaling during the specification of the cardiac lineage in progenitor cells of the primordial heart.

Role of miRNA in differentiation of human ES cells to vascular endothelium

Role of miRNA in differentiation of human ES cells to vascular endothelium

Differentiating Human ES and Patient Derived IPS Cells to Hematopoietic Stem/Progenitor Cells.

AbstractAbstract 2628The ability to generate hematopoietic stem cells (HSC's) from patient derived induced pluripotent stem (iPS) cells, would enable the generation of an unlimited supply of HLA matched transplantable cells for the treatment of both hematological disorders and malignancies. The goal of this project is to find the molecular determinants of human ES and iPS cell differentiation to transplantable HSC's. We have developed an advanced ES/iPS-to-blood differentiation protocol that mimics embryonic development towards the hematopoietic lineage. Current progress has resulted in up to 84% of human CD45+ hematopoietic cells (mean 41 ± 16%, n = 7) and with significant numbers of hematopoietic progenitor cells (CD45+CD34+, 29 ± 4%, n = 6) as reported by FACS analysis. Moreover, the numbers of hematopoietic progenitor cells generated (mean 20 ± 1 for a keratinocyte derived iPS (kiPS)), as measured by colony forming unit assays were comparable to numbers obtained from fresh whole blood umbilical cord mononuclear cell isolates (mean 35 ± 2) on a per 10.000 CD45+ input cells. We now plan to overexpress key factors predicted to support the generation and maturation of bona fide HSC's with repopulating ability. For this we have generated a tet-regulatable lentiviral based gene expression system to, out of a pool of over 30 genes, express up to 5 cDNA's of interest simultaneously. By clonal selection of lenti-transduced ES and iPS cells we have generated novel pluripotent lines that carry the tet-transactivator gene (m2rtTA), capable of efficient differentiation into blood. FACS analysis of these lines show blood generation of 30.7% and 11.8% of cells positive for CD45 for the human ES line Hues3 and the kiPS line respectively, with progenitor levels (CD45+CD34+) at 8.8% and 20.2%. These values are similar in efficiency to that of untransduced sister lines from which they were derived. These lines strongly express genes of interest following lenti-viral transduction and the addition of Doxycyclin (as reported by GFP). It is our goal to show successful reconstitution with in vitro generated HSC's, and ultimately pave the way for therapeutically relevant HSC's for treating hematological malignancies. Disclosures:No relevant conflicts of interest to declare.

Differentiation of human ES and Parkinson's disease iPS cells into ventral midbrain dopaminergic neurons requires a high activity form of SHH, FGF8a and specific regionalization by retinoic acid

The cardinal motor symptoms of Parkinson's disease (PD) are caused by the vulnerability to dysfunction and degeneration of ventral midbrain (VM) dopaminergic (DA) neurons. A major limitation for experimental studies of current ES/iPS cell differentiation protocols is the lack of VM DA neurons with a stable phenotype as defined by an expression marker code of FOXA2/TH/β-tubulin. Here we demonstrate a combination of three modifications that were required to produce VM DA neurons. Firstly, early and specific exposure to 10 − 8 M (low dose) retinoic acid improved the regional identity of neural progenitor cells derived from human ES cells, PD or healthy subject-specific iPS cells. Secondly, a high activity form of human sonic hedgehog established a sizeable FOXA2 + neural progenitor cell population in vitro. Thirdly, early exposure to FGF8a, rather than Fgf8b, and WNT1 was required for robust differentiation of the FOXA2 + floor plate-like human neural progenitor cells into FOXA2 + DA neurons. FOXA2 + DA neurons were also generated when this protocol was adapted to feeder-free conditions. In summary, this new human ES and iPS cell differentiation protocol using FGF8a, WNT1, low dose retinoic acid and a high activity form of SHH can generate human VM DA neurons that are required for relevant new bioassays, drug discovery and cell based therapies for PD.

A conserved role of Shp2 in regulation of human and mouse ES cell differentiation

The establishment of human embryonic stem cell (hESC) lines has generated a great deal of excitement in the fields of stem cell biology and regenerative medicine. It has been hoped that hESCs and somatic stem cells could be potential sources of cells to regenerate or rejuvenate damaged tissues. Yet studies on the biology of hESCs have been hindered by difficulties in manipulating this type of cells in culture in vitro. This is largely due to lack of full understanding of the molecular and cellular basis for maintenance of hESC pluripotency. Since mESCs have received extensive attentions in the past two decades and relatively more information is available, characterization of hESCs has been naturally conducted by referencing mESC properties. Recent studies from a number of laboratories have suggested distinct biological properties and molecular signaling mechanisms between human and mouse embryonic stem cells (hESCs and mESCs). Here we report that Shp2, an intracellular tyrosine phosphatase with two SH2 domains, has a conserved role in promoting signals resulting in differentiation of both hESCs and mESCs. Homozygous ablation of Shp2 in mESCs resulted in impaired differentiation into all three germ layer cell lineages accompanied by improved self-renewal. siRNA-mediated Shp2 knockdown in hESCs resulted in a very similar phenotype of impaired differentiation. Furthermore, small molecule inhibitors for Shp2 enzyme suppressed both hESC and mESC differentiation in vitro. Shp2 modulates LIF and BMP signals flowing through Erk, Stat3 and Smad pathways in mESCs and also regulates FGF and BMP signaling through the Erk and Smad pathways in hESCs. These results reveal a common signaling mechanism shared by hESCs and mESCs via Shp2 modulation of overlapping and distinct pathways. Pharmaceutical intervention of Shp2 activity might be a practical means for amplification of hESCs in vitro.

Evolutionary Emergence of microRNAs in Human Embryonic Stem Cells

Human embryonic stem (hES) cells have unique abilities to divide indefinitely without differentiating and potential to differentiate into more than 200 cell types. These properties make hES cells an ideal model system for understanding early human development and for regenerative medicine. Molecular mechanisms including cellular signaling and transcriptional regulation play important roles in hES cell differentiation. However, very little information is available on posttranscriptional regulation of hES cell pluripotency, self-renewal, and early decisions about cell fate. microRNAs (miRNAs), 22-nt long non-coding small RNAs found in plants and animals, regulate gene expression by targeting mRNAs for cleavage or translation repression. In hES cells we found that 276 miRNAs were expressed; of these, a set of 30 miRNAs had significantly changed expression during differentiation. Using a representative example, miR-302b, we show that miRNAs in human ES cells assemble into a bona fide RISC that contains Ago2 and can specifically cleave perfectly matched target RNA. Our results demonstrate that human ES cell differentiation is accompanied by changes in the expression of a unique set of miRNAs, providing a glimpse of a new molecular circuitry that may regulate early development in humans. Chromosomes 19 and X contained 98 and 40 miRNA genes, respectively, indicating that majority of miRNA genes in hES cells were expressed from these two chromosomes. Strikingly, distribution analysis of miRNA gene loci across six species including dog, rat, mouse, rhesus, chimpanzee, and human showed that miRNA genes encoded in chromosome 19 were drastically increased in chimpanzees and humans while miRNA gene loci on other chrosmomes were decreased as compared with dog, rat, and mouse. Comparative genomic studies showed 99% conservation of chromosome 19 miRNA genes between chimpanzees and humans. Together, these findings reveal the evolutionary emergence, ∼5 million years ago, of miRNAs involved in regulating early human development. One could imagine that this burst of miRNA gene clusters at specific chromosomes was part of an evolutionary event during species divergence.

BMP and Wnt Induce Embryonic Blood Fate by Activation of the Cdx-Hox Pathway.

The hematopoietic morphogen BMP4 has been implicated in early blood development, however the precise role and the underlying genetic pathways that link BMP action to blood fate specification remain poorly understood. In this study, we have employed the in vitro system of murine embryonic stem (mES) cell differentiating into embryoid bodies (EBs) as a model to explore mesoderm induction and hematopoietic specification during mammalian embryogenesis. We show that BMP plays a role in blood fate specification in two sequential phases: initially, BMP acts as a dorsoventral patterning agent, inducing ventroposterior mesoderm; at a later phase, BMP is required for hematopoietic fate specification from mesoderm. In dissecting this second specification phase, we discovered a previously unsuspected coordination between BMP4 and Wnt3a signaling, and linked these morphogens directly to Cdx and Hox genes. Using chromatin immunoprecipitation assays performed on EB-derived cells, we detected the association of the Wnt signaling effector LEF1 with the proximal Cdx1 promoter. This association was modulated by BMP signaling. In promoter reporter assays both BMP4 and Wnt3a activated the Cdx1 promoter while Wnt inhibitors or point mutations in the LEF1 responsive elements of the Cdx1 promoter abrogated this response. Together, these results suggest that BMP4 activates the Cdx1 promoter via LEF1 and the Wnt pathway. In functional assays, inhibition of Wnt signaling strongly suppressed the formation of hematopoietic colony initiating cells, suggesting that activation of the Wnt-Cdx-Hox pathway is a central downstream component of the BMP dependent blood fate specification program. Using ES cell lines engineered for conditonal Cdx1 or Cdx4 gene overexpression, we demonstrate that ectopic activation of Cdx genes is sufficient to establish hematopoietic fate decisions in the absence of BMP signaling during mesoderm specification to blood. Finally, to explore the relevance of our findings in vivo, we studied the effects of conditional BMP overexpression on cdx4 expression and embryonic blood formation during zebrafish embryogenesis. Matching our results with EBs, we find that overexpression of bmp2b at midgastrulation expands the cdx4 expression domain and strongly enhances blood formation. Taken together our results reveal an interaction between the BMP and Wnt signaling pathways, upstream of the Cdx genes, that is critical for the induction of the embryonic blood cell program. Given the evolutionary conservation of the BMP-Wnt-Cdx-Hox effect on blood formation from fish to mice, we anticipate that similar pathways can be exploited to direct the differentiation of human ES cells into blood.

Artificial Hematopoietic Stem Cell Niche Sustains Growth and Differentiation of Human ES-Derived Early Hematopoietic Progenitors.

Using a novel Microplate Biomaterial Microarray (MBM™) technology, we have created an artificial hematopoietic stem cell niche that can sustain growth and differentiation of human embryonic stem cells-derived (hES) early hematopoietic progenitors. This hydrogel based ex-vivo niche allows uploading of human embryonal stem cells, human mesenchymal stem cells (MSC), genes (bcl-2 preventing apoptosis and HoxB4 enhancing hematopoiesis) and extracellular matrices to support growth and differentiation of human ES cells. These experiments were done using NIH-approved hES cell lines H1 and H9. Serum-free, feeder-free culture conditions were established and early hematopoietic progenitors grown using SCF, TPO, VEGF and IL-3 with high efficiency. At day 3–5 dual CD34+/CD31+ progenitors were identified, while on day 7–8 CD34+ hematopoietic progenitors were isolated, which formed typical hematopoietic colonies. These progenitors expressed genes related to early hematopoiesis, such as TAL1/SCL, FLT1, GATA2, GATA1, EPOR and TPOR. The early dual endothelio-hematopoietic progenitor (hemangioblast) expressed PECAM-1 and CD34 and showed typical blast-like morphology. Based on mathematical simulations, various micro-niches were designed to establish optimal differentiation conditions for this progenitor using IL-3, IL-6, TPO, EPO, VEGF, SFC, Flt-3 ligand and various extracellular matrices. Specific micro-niches were created for generation of CFU-E, BFU-E, CFU-GM, CFU-GEMM, CFU-M, CFU-G, and CFU-MK progenitors from human ES-derived hemangioblast. Kinetic uploading of TPO, EPO, SCF and VEGF created a niche-sustaining growth of ES-derived hemangioblast with high efficiency and low apoptosis rate. These niches used pulse -delivery of anti-apoptotic bcl-2 gene and hematopoiesis-enhancing Hoxb4 gene. The model of artifical niche sustaing growth and differentiation of human ES-derived hemangioblast was established. In the future, this system will allow optimized and upscaled generation of early hematopoietic progenitors from human ES cells, as a first step towards clinical applications of human embryonic stem cells. [Display omitted]

Neural Differentiation of Human ES Cells

Human embryonic stem cells (hESCs) may be converted into highly enriched cultures of neural precursors under defined culture conditions. The neural precursors can proliferate in culture for prolonged periods of time, and can differentiate in vitro into mature neurons, astrocytes, and oligodendrocytes. The neurons are functional and have normal electrophysiological properties. After transplantation to the developing rodent brain, the neural precursors migrate extensively into the host brain parenchyma, respond to host brain signals, and differentiate in a region-specific manner to progeny of the three neural lineages. The establishment of neuroectodermal precursors from hESCs allows the study of human neurogenesis in vitro and is an aid in drug discovery. In addition, the neural precursors may potentially serve as a platform for the development of specific functional neural cells for transplantation and gene therapy of neurological disorders. In this unit, we introduce methods for the derivation, propagation and characterization of hESC-derived neural precursors.