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We have previously identified multipotent neuroepithelial (NEP) stem cells and lineage-restricted,self-renewing precursor cells termed NRPs (neuron-restricted precursors) and GRPs (glial-restricted precursors) present in the developing rat spinal cord (A. Kalyani,K. Hobson,and M. S. Rao,1997,Dev. Biol. 186,202-223; M. S. Rao and M. Mayer-Proschel,1997,Dev. Biol. 188,48-63; M. Mayer-Proschel,A. J. Kalyani,T. Mujtaba,and M. S. Rao,1997,Neuron 19,773-785). We now show that cells identical to rat NEPs,NRPs,and GRPs are present in mouse neural tubes and that immunoselection against cell surface markers E-NCAM and A2B5 can be used to isolate NRPs and GRPs,respectively. Restricted precursors similar to NRPs and GRPs can also be isolated from mouse embryonic stem cells (ES cells). ES cell-derived NRPs are E-NCAM immunoreactive,undergo self-renewal in defined medium,and differentiate into multiple neuronal phenotypes in mass culture. ES cells also generate A2B5-immunoreactive cells that are similar to E9 NEP-cell-derived GRPs and can differentiate into oligodendrocytes and astrocytes. Thus,lineage restricted precursors can be generated in vitro from cultured ES cells and these restricted precursors resemble those derived from mouse neural tubes. These results demonstrate the utility of using ES cells as a source of late embryonic precursor cells. View Publication

OBJECTIVE: Various preparative protocols have been proposed for the acquisition and cultivation of mesenchymal stem cells (MSC). Whereas surface antigen markers have failed to precisely define this population,microarray analysis might provide a better tool for characterization of MSC. METHODS: In this study,we have analyzed global gene expression profiles of human MSC isolated from adipose tissue (AT),from umbilical cord blood (CB),and from bone marrow (BM) under two growth conditions and have compared them to terminally differentiated human fibroblasts (HS68). Profiles were compared using our Human Genome Microarray representing 51.144 different cDNA clones. RESULTS: Cultured with the appropriate conditions,osteogenic and adipogenic differentiation could be confirmed in all MSC preparations but not in fibroblasts. No phenotypic differences were observed by flow cytometry using a panel of 22 surface antigen markers. Whereas MSC derived from different donors using the same culture procedure yielded a consistent and reproducible gene expression profile,many genes were differentially expressed in MSC from different ontogenetic sources or from different culture conditions. Twenty-five genes were overlapping and upregulated in all MSC preparations from AT,CB,and BM as compared to HS68 fibroblasts. These genes included fibronectin,ECM2,glypican-4,ID1,NF1B,HOXA5,and HOXB6. Many genes upregulated in MSC are involved in extracellular matrix,morphogenesis,and development,whereas several inhibitors of the Wnt pathway (DKK1,DKK3,SFRP1) were highly expressed in fibroblasts. CONCLUSION: Our results have provided a foundation for a more reproducible and reliable quality control using genotypic analysis for defining MSC. View Publication

Although the source of embryonic stem (ES) cells presents ethical concerns,their use may lead to many clinical benefits if differentiated cell types can be derived from them and used to assemble functional organs. In pancreas,insulin is produced and secreted by specialized structures,islets of Langerhans. Diabetes,which affects 16 million people in the United States,results from abnormal function of pancreatic islets. We have generated cells expressing insulin and other pancreatic endocrine hormones from mouse ES cells. The cells self-assemble to form three-dimensional clusters similar in topology to normal pancreatic islets where pancreatic cell types are in close association with neurons. Glucose triggers insulin release from these cell clusters by mechanisms similar to those employed in vivo. When injected into diabetic mice,the insulin-producing cells undergo rapid vascularization and maintain a clustered,islet-like organization. View Publication

Synthetic materials that promote the growth or differentiation of cells have advanced the fields of tissue engineering and regenerative medicine. Most functional biomaterials are based on a handful of peptide sequences derived from protein ligands for cell surface receptors. Because few proteins possess short peptide sequences that alone can engage cell surface receptors,the repertoire of receptors that can be targeted with this approach is limited. Materials that bind diverse classes of receptors,however,may be needed to guide cell growth and differentiation. To provide access to such new materials,we utilized phage display to identify novel peptides that bind to the surface of pluripotent cells. Using human embryonal carcinoma (EC) cells as bait,approximately 3 x 10(4) potential cell-binding phage clones were isolated. The pool was narrowed using an enzyme-linked immunoassay: 370 clones were tested,and seven cell-binding peptides were identified. Of these,six sequences possess EC cell-binding ability. Specifically,when displayed by self-assembled monolayers (SAMs) of alkanethiols on gold,they mediate cell adhesion. The corresponding soluble peptides block this adhesion,indicating that the identified peptide sequences are specific. They also are functional. Synthetic surfaces displaying phage-derived peptides support growth of undifferentiated human embryonic stem (ES) cells. When these cells were cultured on SAMs presenting the sequence TVKHRPDALHPQ or LTTAPKLPKVTR in a chemically defined medium (mTeSR),they expressed markers of pluripotency at levels similar to those of cells cultured on Matrigel. Our results indicate that this screening strategy is a productive avenue for the generation of materials that control the growth and differentiation of cells. View Publication

Adipose tissue is an abundantly available source of proliferative and multipotent mesenchymal stem cells with promising potential for regenerative therapeutics. We previously demonstrated that both human and mouse adipose-derived stem cells (ASCs) can be reprogrammed into induced pluripotent stem cells (iPSCs) with efficiencies higher than those that have been reported for other cell types. The ASC-derived iPSCs can be generated in a feeder-independent manner,representing a unique model to study reprogramming and an important step toward establishing a safe,clinical grade of cells for therapeutic use. In this study,we provide a detailed protocol for isolation,preparation and transformation of ASCs from fat tissue into mouse iPSCs in feeder-free conditions and human iPSCs using feeder-dependent or feeder/xenobiotic-free processes. This protocol also describes how ASCs can be used as feeder cells for maintenance of other pluripotent stem cells. ASC derivation is rapid and can be completed in textless1 week,with mouse and human iPS reprogramming times averaging 1.5 and 2.5 weeks,respectively. View Publication

Here,we show that as human embryonic stem cells (ESCs) exit the pluripotent state,NANOG can play a key role in determining lineage outcome. It has previously been reported that BMPs induce differentiation of human ESCs into extraembryonic lineages. Here,we find that FGF2,acting through the MEK-ERK pathway,switches BMP4-induced human ESC differentiation outcome to mesendoderm,characterized by the uniform expression of T (brachyury) and other primitive streak markers. We also find that MEK-ERK signaling prolongs NANOG expression during BMP-induced differentiation,that forced NANOG expression results in FGF-independent BMP4 induction of mesendoderm,and that knockdown of NANOG greatly reduces T induction. Together,our results demonstrate that FGF2 signaling switches the outcome of BMP4-induced differentiation of human ESCs by maintaining NANOG levels through the MEK-ERK pathway. View Publication

Hutchinson-Gilford progeria syndrome (HGPS) is a rare and fatal human premature ageing disease,characterized by premature arteriosclerosis and degeneration of vascular smooth muscle cells (SMCs). HGPS is caused by a single point mutation in the lamin A (LMNA) gene,resulting in the generation of progerin,a truncated splicing mutant of lamin A. Accumulation of progerin leads to various ageing-associated nuclear defects including disorganization of nuclear lamina and loss of heterochromatin. Here we report the generation of induced pluripotent stem cells (iPSCs) from fibroblasts obtained from patients with HGPS. HGPS-iPSCs show absence of progerin,and more importantly,lack the nuclear envelope and epigenetic alterations normally associated with premature ageing. Upon differentiation of HGPS-iPSCs,progerin and its ageing-associated phenotypic consequences are restored. Specifically,directed differentiation of HGPS-iPSCs to SMCs leads to the appearance of premature senescence phenotypes associated with vascular ageing. Additionally,our studies identify DNA-dependent protein kinase catalytic subunit (DNAPKcs,also known as PRKDC) as a downstream target of progerin. The absence of nuclear DNAPK holoenzyme correlates with premature as well as physiological ageing. Because progerin also accumulates during physiological ageing,our results provide an in vitro iPSC-based model to study the pathogenesis of human premature and physiological vascular ageing. View Publication

Human embryonic stem (hES) cells have a potential use for the repair and regeneration of injured tissues. However,teratoma formation can be a major obstacle for hES-mediated cell therapy. Therefore,tracking the fate and function of transplanted hES cells with noninvasive imaging could be valuable for a better understanding of the biology and physiology of teratoma formation. In this study,hES cells were stably transduced with a double fusion reporter gene consisting of firefly luciferase and enhanced green fluorescent protein. Following bioluminescence imaging and histology,we demonstrated that engraftment of hES cells was followed by dramatically increasing signaling and led to teratoma formation confirmed by histology. Studies of the angiogenic processes within teratomas revealed that their vasculatures were derived from both differentiated hES cells and host. Moreover,FACS analysis showed that teratoma cells derived from hES cells expressed high levels of CD56 and SSEA-4,and the subcultured SSEA-4(+) cells showed a similar cell surface marker expression pattern when compared to undifferentiated hES cells. We report here for the first time that SSEA-4(+) cells derived from teratoma exhibited multipotency,retained their differentiation ability in vivo as confirmed by their differentiation into representative three germ layers. View Publication

Human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) are promising can- didate cell sources for regenerative medicine. However,despite the common ability of hiPSCs and hESCs to dif- ferentiate into all 3 germ layers,their functional equivalence at the single cell level remains to be demonstrated. Moreover,single cell heterogeneity amongst stem cell populations may underlie important cell fate decisions. Here,we used single cell analysis to resolve the gene expression profiles of 362 hiPSCs and hESCs for an array of 42 genes that characterize the pluripotent and differentiated states. Comparison between single hESCs and single hiPSCs revealed markedly more heterogeneity in gene expression levels in the hiPSCs,suggesting that hiPSCs occupy an alternate,less stable pluripotent state. hiPSCs also displayed slower growth kinetics and impaired directed differentiation as compared with hESCs. Our results suggest that caution should be exer- cised before assuming that hiPSCs occupy a pluripotent state equivalent to that of hESCs,particularly when producing differentiated cells for regenerative medicine aims. View Publication

With regard to human induced pluripotent stem cells (hiPSCs),in which adult cells are reprogrammed into embryonic-like cells using defined factors,their functional and transcriptional expression pattern during endothelial differentiation has yet to be characterized. In this study,hiPSCs and human embryonic stem cells (hESCs) were differentiated using the embryoid body method,and CD31(+) cells were sorted. Fluorescence activated cell sorting analysis of hiPSC-derived endothelial cells (hiPSC-ECs) and hESC-derived endothelial cells (hESC-ECs) demonstrated similar endothelial gene expression patterns. We showed functional vascular formation by hiPSC-ECs in a mouse Matrigel plug model. We compared the gene profiles of hiPSCs,hESCs,hiPSC-ECs,hESC-ECs,and human umbilical vein endothelial cells (HUVECs) using whole genome microarray. Our analysis demonstrates that gene expression variation of hiPSC-ECs and hESC-ECs contributes significantly to biological differences between hiPSC-ECs and hESC-ECs as well as to the distances" among hiPSCs� View Publication

BACKGROUND AND PURPOSE: Teratogenic substances induce adverse effects during the development of the embryo. Multilineage differentiation of human embryonic stem cells (hESCs) mimics the development of the embryo in vitro. Here,we propose a transcriptomic approach in hESCs for monitoring specific toxic effects of compounds as an alternative to traditional time-consuming and cost-intensive in vivo tests requiring large numbers of animals. This study was undertaken to explore the adverse effects of cytosine arabinoside (Ara-C) on randomly differentiated hESCs.backslashnbackslashnEXPERIMENTAL APPROACH: Human embryonic stem cells were used to investigate the effects of a developmental toxicant Ara-C. Sublethal concentrations of Ara-C were given for two time points,day 7 and day 14 during the differentiation. Gene expression was assessed with microarrays to determine the dysregulated transcripts in presence of Ara-C.backslashnbackslashnKEY RESULTS: Randomly differentiated hESCs were able to generate the multilineage markers. The low concentration of Ara-C (1 nM) induced the ectoderm and inhibited the mesoderm at day 14. The induction of ectodermal markers such as MAP2,TUBB III,PAX6,TH and NESTIN was observed with an inhibition of mesodermal markers such as HAND2,PITX2,GATA5,MYL4,TNNT2,COL1A1 and COL1A2. In addition,no induction of apoptosis was observed. Gene ontology revealed unique dysregulated biological process related to neuronal differentiation and mesoderm development. Pathway analysis showed the axon guidance pathway to be dysregulated.backslashnbackslashnCONCLUSIONS AND IMPLICATIONS: Our results suggest that hESCs in combination with toxicogenomics offer a sensitive in vitro developmental toxicity model as an alternative to traditional animal experiments. View Publication