ES cell

cardiac, skeletal, and smooth muscle myogenesis of mouse embryos was developed and used to investigate the effects of the disruption of the desmin gene on muscle cell differentiation. Wild-type, heterozygous, and homozygous cell lines with the mutated desmin allele were evaluated. Skeletal myogenesis was totally inhibited in desmin null mutant EBs, as manifested by the absence of myotube formation, contractility, and myoD, myogenin, myf5, and myosin heavy chain expression. Smooth muscle formation was also completely blocked in the absence of desmin. On the other hand, there were no obvious effects on cardiomyocyte differentiation in these desmin null mutant EBs. However, reduced desmin expression in EBs heterozygous for the desmin mutation leads to partial inhibition of cardiac muscle formation. These data suggest that in contrast to early cardiocyte differentiation, desmin is indispensable for skeletal and smooth muscle formation. ᭧

To expand the repertoire of transposable elements available for gene transfer in higher vertebrates, we investigated the properties of the Tol2 transposon in mammalian cells and in vivo. Tol2 is a member of the hAT family of DNA transposons and is derived from an active element found in the tyrosinase gene of medaka fish (Oryzias latipes). Tol2 can transpose in mouse ES cells, suggesting its potential use as a gene transfer reagent in higher vertebrates.

In vitro differentiation of embryonic stem (ES) cells is often used to study hematopoiesis. However, the differentiation pathway of lymphocytes, in particular natural killer (NK) cells, from ES cells is still unclear. Here, we used a multi-step in vitro ES cell differentiation system to study lymphocyte development from ES cells, and to characterize NK developmental intermediates. We generated embryoid bodies (EBs) from ES cells, isolated CD34 + EB cells and cultured them on OP9 stroma with a cocktail of cytokines to generate cells we termed ES-derived hematopoietic progenitors (ES-HPs). EB cell subsets, as well as ES-HPs derived from EBs, were tested for NK, T, B and myeloid lineage potentials using lineage specific cultures. ES-HPs derived from CD34 + EBs differentiated into NK cells when cultured on OP9 stroma with IL-2 and IL-15, and into T cells on Delta-like 1transduced OP9 (OP9-DL1) with IL-7 and Flt3-L. Among CD34 + EB cells, NK and T cell potentials were detected in a CD45 2 subset, whereas CD45 + EB cells had myeloid but not lymphoid potentials. Limiting dilution analysis of ES-HPs generated from CD34 + CD45 2 EB cells showed that CD45 + Mac-1 2 Ter119 2 ES-HPs are highly enriched for NK progenitors, but they also have T, B and myeloid potentials. We concluded that CD45 2 CD34 + EB cells have lymphoid potential, and they differentiate into more mature CD45 + Lin 2 hematopoietic progenitors that have lymphoid and myeloid potential. NK progenitors among ES-HPs are CD122 2 and they rapidly acquire CD122 as they differentiate along the NK lineage.

To investigate the molecular basis of PTENmediated tumor suppression, we introduced a null mutation into the mouse Pten gene by homologous recombination in embryonic stem (ES) cells. Pten ؊/؊ ES cells exhibited an increased growth rate and proliferated even in the absence of serum. ES cells lacking PTEN function also displayed advanced entry into S phase. This accelerated G 1 ͞S transition was accompanied by down-regulation of p27 KIP1 , a major inhibitor for G 1 cyclindependent kinases. Inactivation of PTEN in ES cells and in embryonic fibroblasts resulted in elevated levels of phosphatidylinositol 3,4,5,-trisphosphate, a product of phosphatidylinositol 3 kinase. Consequently, PTEN deficiency led to dosagedependent increases in phosphorylation and activation of Akt͞ protein kinase B, a well-characterized target of the phosphatidylinositol 3 kinase signaling pathway. Akt activation increased Bad phosphorylation and promoted Pten ؊/؊ cell survival. Our studies suggest that PTEN regulates the phosphatidylinositol 3,4,5,-trisphosphate and Akt signaling pathway and consequently modulates two critical cellular processes: cell cycle progression and cell survival.

originally isolated and maintained by coculture on a feeder layer of mitotically inactivated mouse embryo fibroblasts (Evans and Kaufman, 1981; Martin, 1981). The essential function of the fibroblast feeder layer is provision of the cytokine leukemia inhibitory factor (LIF). West Mains Road LIF null fibroblasts are deficient at supporting selfrenewal (Stewart et al., 1992) and LIF can replace the Edinburgh Scotland EH9 3JQ requirement for feeders in both routine propagation (Smith et al., 1988; Williams et al., 1988) and de novo derivation (Nichols et al., 1990) of mouse ES cells. LIF and related cytokines that engage the gp130 re-Summary ceptor provide the only molecularly defined pathway that will sustain long-term self-renewal of mouse ES The cytokine leukemia inhibitory factor (LIF) drives self-renewal of mouse embryonic stem (ES) cells by cells with retention of the cardinal attributes of undifferentiated phenotype, pluripotency, and embryo coloniza-activating the transcription factor STAT3. In serumfree cultures, however, LIF is insufficient to block neu-tion capacity (Smith, 2001b). LIF directs ES cell selfrenewal through activation of the latent transcription ral differentiation and maintain pluripotency. Here, we report that bone morphogenetic proteins (BMPs) act factor STAT3 (Matsuda et al., 1999; Niwa et al., 1998). Paradoxically gp130 cytokines also stimulate the prodif-in combination with LIF to sustain self-renewal and preserve multilineage differentiation, chimera coloni-ferentiative Ras-erk pathway (Burdon et al.,

The pluripotency and high proliferative capacity of embryonic stem (ES) cells (1-3) makes them an attractive source of different cell types for biomedical research and cell replacement therapies. A major prerequisite for these applications is the availability of a homogeneous population of the desired cell type. However, ES cell-derived material contains, for example, undifferentiated cells, which can cause tumor formation after transplantation into the brain (4). To avoid such unwanted side effects, effective purification of distinct types of cells needs to be developed. Here, we describe an immunoisolation procedure to purify neurons from in vitro differentiated mouse ES cells using an antibody against the neuronal cell adhesion molecule L1 (5, 6). Our procedure yields a pure population of differentiated neurons, which are electrically excitable and form excitatory, glutamatergic, and inhibitory GABAergic synapses. The ability to highly purify ES cell-derived neurons will boost their molecular characterization and the further exploration of their therapeutic potential.

cytokines that act through a common receptor complex comprising the LIF receptor subunit (LIF-R) and the signal transducer gp130. However, the findings that embryos lacking LIF-R or gp130 can develop beyond gastrulation argue for the existence of an alternative pathway(s) governing the maintenance of pluripotency in vivo. In order to define those factors that contribute to self-renewal in ES cell cultures, we have generated ES cells in which both copies of the lif gene are deleted. These cells showed a significantly reduced capacity for regeneration of stem cell colonies when induced to differentiate, confirming that LIF is the major endogenous regulatory cytokine in ES cell cultures. However, self-renewal was not abolished and undifferentiated ES cell colonies were still obtained in the complete absence of LIF. A differentiated, LIF-deficient, parietal endoderm-like cell line was derived and shown to support ES cell propagation via production of a soluble, macromolecular, trypsin-sensitive activity. This activity, which we name ES cell renewal factor (ESRF), is distinct from members of the IL-6/LIF family because (i) it is effective on ES cells lacking LIF-R; (ii) it is not blocked by anti-gp130 neutralizing antibodies; and (iii) it acts without activation of STAT3. ES cells propagated clonally using ESRF alone can contribute fully to chimaeras and engender germline transmission. These findings establish that ES cell pluripotency can be sustained via a LIF-R/gp130-independent, STAT-3 independent, signaling pathway. Operation of this pathway in vivo could play an important role in the regulation of pluripotency in the epiblast and account for the viability of lifr ؊/؊ and gp130 ؊/؊ embryos.

We previously reported the differentiation of mouse embryonic stem (ES) cells into retinal progenitors. However, these progenitors rarely differentiate into photoreceptors unless they are cultured with embryonic retinal tissues. Here we show the in vitro generation of putative rod and cone photoreceptors from mouse, monkey and human ES cells by stepwise treatments under defined culture conditions, in the absence of retinal tissues. With mouse ES cells, Crx + photoreceptor precursors were induced from Rx + retinal progenitors by treatment with a Notch signal inhibitor. Further application of fibroblast growth factors, Shh, taurine and retinoic acid yielded a greater number of rhodopsin + rod photoreceptors, in addition to default cone production. With monkey and human ES cells, feeder-and serum-free suspension culture combined with Wnt and Nodal inhibitors induced differentiation of Rx + or Mitf + retinal progenitors, which produced retinal pigment epithelial cells. Subsequent treatment with retinoic acid and taurine induced photoreceptor differentiation. These findings may facilitate the development of human ES cellbased transplantation therapies for retinal diseases. Various ocular diseases, including retinitis pigmentosa, cone dystrophy and age-related macular degeneration, are characterized by a loss of photoreceptor cells, leading to blindness 1. One promising therapeutic strategy is to transplant functional photoreceptor cells 2-6. In addition to replacing photoreceptors, it may be important to regenerate retinal pigment epithelial (RPE) cells 4,5,7 , which are essential for maintenance functions such as outer segment shedding, supplying nutrients to the retina and maintaining the blood-retinal barrier. However, a reliable, efficient method for producing large numbers of photoreceptors and RPE cells has not been devised. Specifically, a chemically defined culture method to efficiently generate photoreceptors from human ES cells would overcome a critical limitation for transplantation therapies. Many groups, including ours, have attempted to generate photoreceptors in vitro from iris tissues 8,9 , ciliary tissues 10 or ES cells 11-14. Compared with tissue stem cells, ES cells are superior because their potential for infinite proliferation would permit production of a sufficient number of cells for research and treatment 15,16. Retinal progenitors can be produced from ES cells in vitro 11-14 , but generation of photoreceptors from these progenitors remains inefficient unless the cells are cocultured with developing retinal tissues. For instance, in our previous report 13 , we induced neural retinal progenitors (B16%) from mouse ES cells using a serum-free floating culture of embryoid body-like aggregates (SFEB) system 17 combined with Dkk1, LeftyA, serum and activin treatment (SFEB/DLFA). However, in the absence of coculture with embryonic retinal tissues, differentiation of the retinal progenitors into photoreceptors was infrequent, suggesting

We have examined the gene structure and regulatory regions of octamer-binding transcription factor 3/4 (Oct 3/4), sex determining region Y box 2 (Sox2), signal transducer and activator of transcription 3 (Stat3), embryonal stem cell-specific gene 1 (ESG), Nanog homeobox (Nanog), and several other genes highly expressed in embryonic stem (ES) cells across different species. Our analysis showed that ES cell-expressed Ras (ERAS) was orthologous to a human pseudogene Harvey Ras (HRASP) and that the promoter and other regulatory sequences were highly divergent. No ortholog of (ES) cell-derived homeobox containing gene (Ehox) could be identified in human, and the closest paralogs PEPP gene subfamily 1 (PEPP1), PEPP2, and extraembryonic, spermatogenesis, homeobox 1 (Esx1) were not expressed by ES cells and shared little homology. The Sox2 promoter was the most conserved across species and the Oct3/4 promoter region showed significant homology particularly in the distal enhancer active in ES cells. Analysis suggested common and divergent pathways of regulation. Conserved Oct3/4 and Sox2 co-binding domains were identified in most ES expressed genes, highlighting the importance of this transcriptional pathway. Conserved fibroblast growth factor response element sites were identified in regulatory regions, suggesting a potential parallel pathway for regulation by FGFs. A central role of Stat3 activation in self-renewal and in a regulatory feedback loop was suggested by the identification of the conserved binding sites in most pathways. Although most pathways were evolutionarily conserved, promoters and genomic structure of the leukemia inhibitory factor (LIF) pathway components were divergent, likely explaining the differential requirement of LIF for human and rodent cells. Our analysis further suggested that the Nanog regulatory pathway was relatively independent of the LIF/Oct pathway and may interact with the Nodal/transforming growth factor-β pathway. These results provide a framework for examining the current reported differences between rodent and human ES cells and define targets for future perturbation studies.

Background: Induced pluripotent stem (iPS) cells have the capability to undergo self-renewal and differentiation into all somatic cell types. Since they can be produced through somatic cell reprogramming, which uses a defined set of transcription factors, iPS cells represent important sources of patient-specific cells for clinical applications. However, before these cells can be used in therapeutic designs, it is essential to understand their genetic stability.

Culturing pluripotent embryonic stem cells represents a unique model system for in vitro studies of embryo cell growth and differentiation, and represents a connection between in vitro and in vivo manipulation of genes. To further develop and refine stem cell technology for marine fish, we have established cultures of embryonic stem cells isolated from turbot blastulas. The pluripotent nature of our turbot-ES-like cells was supported by their morphology and elevated levels of alkaline phosphatase enzyme activity, their ability to remain undifferentiated for a prolonged culture period, their spontaneous differentiation potential in vitro and their ability to form embryoid bodies (EB) in response to changes in the extracellular environment. In addition, we show that turbot ES like cells express Oct-4 required for the maintenance of pluripotency of ES cells.

Human pluripotent stem cells (hPSCs) are a promising source of cells for applications in regenerative medicine. Directed differentiation of hPSCs into specialized cells such as spinal motoneurons 1 or midbrain dopamine (DA) neurons 2 has been achieved. However, the effective use of hPSCs for cell therapy has lagged behind. While mouse PSC-derived DA neurons have shown efficacy in models of Parkinson's disease (PD) 3, 4 , DA neurons from human PSCs generally display poor in vivo performance 5 . There are also considerable safety concerns for hPSCs related to their potential for teratoma formation or neural overgrowth 6, 7