An improved protocol that induces human embryonic stem cells to differentiate into neural cells in vitro

Human embryonic stem (ES) cells have the capacity for self-renewal and are able to differentiate into any cell type. However, obtaining high-efficient neural differentiation from human ES cells remains a challenge. This study describes an improved 4-stage protocol to induce a human ES cell line derived from a Chinese population to differentiate into neural cells. At the first stage, embryonic bodies (EBs) were formed in a chemically-defined neural inducing medium rather than in traditional serum or serum-replacement medium. At the second stage, rosette-like structures were formed. At the third stage, the rosette-like structures were manually selected rather than enzymatically digested to form floating neurospheres. At the fourth stage, the neurospheres were further differentiated into neurons. The results show that, at the second stage, the rate of the formation of rosette-like structures from EBs induced by noggin was 88+/-6.32%, higher than that of retinoic acid 55+/-5.27%. Immunocytochemistry staining was used to confirm the neural identity of the cells. These results show a major improvement in obtaining efficient neural differentiation of human ES cells.     

WNT/beta-catenin pathway up-regulates Stat3 and converges on LIF to prevent differentiation of mouse embryonic stem cells

Embryonic stem (ES) cells rely on growth factors provided by feeder cells or exogenously to maintain their pluripotency. In order to identify such factors, we have established sub-lines of STO feeder cells which exhibit variable ability in supporting ES cell self-renewal. Functional screening identifies WNT5A and WNT6 as STO cell-produced factors that potently inhibit ES cell differentiation in a serum-dependent manner. Furthermore, direct activation of beta-catenin without disturbing the upstream components of the WNT/beta-catenin pathway fully recapitulates the effect of WNTs on ES cells. Importantly, the WNT/beta-catenin pathway up-regulates the mRNA for Stat3, a known regulator of ES cell self-renewal in the mouse. Finally, LIF is able to mimic the serum effect to act synergistically with WNT proteins to inhibit ES cell differentiation. Therefore, our study reveals part of the molecular mechanisms by which the WNT/beta-catenin pathway acts to prevent ES cell differentiation through convergence on the LIF/JAK-STAT pathway at the level of STAT3.     

BMP4 induces primitive endoderm but not trophectoderm in monkey embryonic stem cells

Monkey embryonic stem (ES) cells share similar characteristics to human ES cells and provide a primate model of allotransplantation, which allows to validate efficacy and safety of cell transplantation therapy in regenerative medicine. Bone morphogenetic protein 4 (BMP4) is known to promote trophoblast differentiation in human ES cells in contrast to mouse ES cells where BMP4 synergistically maintains self-renewal with leukemia inhibitory factor (LIF), which represents a significant difference in signal transduction of self-renewal and differentiation between murine and human ES cells. As the similarity of the differentiation mechanism between monkey and human ES cells is of critical importance for their use as a primate model system, we investigated whether BMP4 induces trophoblast differentiation in monkey ES cells. Interestingly, BMP4 did not induce trophoblast differentiation, but instead induced primitive endoderm differentiation. Prominent downregulation of Sox2, which plays a pivotal role not only in pluripotency but also placenta development, was observed in cells treated with BMP4. In addition, upregulation of Hand1, Cdx2, and chorionic gonadotropin beta (CG-beta), which are markers of trophoblast, was not observed. In contrast, BMP4 induced significant upregulation of Gata6, Gata4, and LamininB1, suggesting differentiation into the primitive endoderm, visceral endoderm, and parietal endoderm, respectively. The threshold of BMP4 activity was estimated as about 10 ng/mL. These findings suggest that BMP4 induced differentiation into the primitive endoderm lineage but not into trophoblast in monkey ES cells.     

Inhibition of Caspase-mediated Anoikis Is Critical for Basic Fibroblast Growth Factor-sustained Culture of Human Pluripotent Stem Cells

Apoptosis and proliferation are two dynamically and tightly regulated processes that together maintain the homeostasis of renewable tissues. Anoikis is a subtype of apoptosis induced by detachment of adherent cells from the extracellular matrix. By using the defined mTeSR1 medium and collecting freshly detached cells, we found here that human pluripotent stem (PS) cells including embryonic stem (ES) cells and induced pluripotent stem cells are subject to constant anoikis in culture, which is escalated in the absence of basic fibroblast growth factor (bFGF). Withdrawal of bFGF also promotes apoptosis and differentiation of the remaining adherent cells without affecting their cell cycle progression. Insulin-like growth factor 2 (IGF2) has previously been reported to act downstream of FGF signaling to support self-renewal of human ES cells. However, we found that IGF2 cannot substitute bFGF in the TeSR1-supported culture, although endogenous IGF signaling is required to sustain self-renewal of human ES cells. On the other hand, all of the bFGF withdrawal effects observed here can be markedly prevented by the caspase inhibitor z-VAD-FMK. We further demonstrated that the bFGF-repressed anoikis is dependent on activation of ERK and AKT and associated with inhibition of Bcl-2-interacting mediator of cell death and the caspase-ROCK1-myosin signaling. Anoikis is independent of pre-detachment apoptosis and differentiation of the cells. Because previous studies of human PS cells have been focused on attached cells, our findings revealed a neglected role of bFGF in sustaining self-renewal of human PS cells: preventing them from anoikis via inhibition of caspase activation.     

Potential of embryonic stem cells

Embryonic stem (ES) cells are pluripotent cell lines established from undifferentiated embryonic cells characterized by nearly unlimited self-renewal and differentiation capacity. During differentiation in vitro, ES cells were found to be able to develop into specialized somatic cells types and to recapitulate processes of early embryonic development. These properties allow to use ES cells as model system for studying early embryonic development by gain- or loss-of-function approaches, or to investigate the effects of drugs and environmental factors on differentiation and cell function in embryotoxicity and pharmacology. Now, ES cells derived of human blastocysts may be used for the generation of somatic precursor or differentiated cells in cell and tissue therapy. The review presents data of mouse ES cell differentiation and gives an outlook on future perspectives and problems of using human ES cells in regenerative medicine.     

Retinoic acid directs neuronal differentiation of human pluripotent stem cell lines in a non-cell-autonomous manner

Differentiation of human embryonic stem (ES) cells and embryonal carcinoma (EC) cells provides an in vitro model to study the process of neuronal differentiation. Retinoic acid (RA) is frequently used to promote neural differentiation of pluripotent cells under a wide variety of culture conditions. Through systematic comparison of differentiation conditions we demonstrate that RA induced neuronal differentiation of human ES and EC cells requires prolonged RA exposure and intercellular communication mediated by high cell density. These parameters are necessary for the up-regulation of neural gene expression (SOX2, PAX6 and NeuroD1) and the eventual appearance of neurons. Forced over-expression of neither SOX2 nor NEUROD1 was sufficient to overcome the density dependency of neuronal differentiation. Furthermore, inhibition of GSK3β activity blocked the ability of RA to direct cell differentiation along the neural lineage, suggesting a role for appropriately regulated WNT signalling. These data indicate that RA mediated neuronal differentiation of human EC and ES cell lines is not a cell autonomous program but comprises of a multi-staged program that requires intercellular input.     

Notch promotes neural lineage entry by pluripotent embryonic stem cells

A central challenge in embryonic stem (ES) cell biology is to understand how to impose direction on primary lineage commitment. In basal culture conditions, the majority of ES cells convert asynchronously into neural cells. However, many cells resist differentiation and others adopt nonneural fates. Mosaic activation of the neural reporter Sox-green fluorescent protein suggests regulation by cell-cell interactions. We detected expression of Notch receptors and ligands in mouse ES cells and investigated the role of this pathway. Genetic manipulation to activate Notch constitutively does not alter the stem cell phenotype. However, upon withdrawal of self-renewal stimuli, differentiation is directed rapidly and exclusively into the neural lineage. Conversely, pharmacological or genetic interference with Notch signalling suppresses the neural fate choice. Notch promotion of neural commitment requires parallel signalling through the fibroblast growth factor receptor. Stromal cells expressing Notch ligand stimulate neural specification of human ES cells, indicating that this is a conserved pathway in pluripotent stem cells. These findings define an unexpected and decisive role for Notch in ES cell fate determination. Limiting activation of endogenous Notch results in heterogeneous lineage commitment. Manipulation of Notch signalling is therefore likely to be a key factor in taking command of ES cell lineage choice.     

Requirement of B-Raf,C-Raf,and A-Raf for the growth and survival of mouse embryonic stem cells

Extracellular signal-regulated kinases (ERKs) have been implicated to be dispensable for self-renewal of mouse embryonic stem (ES) cells, and simultaneous inhibition of both ERK signaling and glycogen synthase kinase 3 (GSK3) not only allows mouse ES cells to self-renew independent of extracellular stimuli but also enables more efficient derivation of naïve ES cells from mouse and rat strains. Interestingly, some ERKs stay active in mouse ES cells which are maintained in regular medium containing leukemia inhibitory factor (LIF) and bone morphogenetic protein (BMP). Yet, the upstream signaling for ERK activation and their roles in mouse ES cells, other than promoting or priming differentiation, have not been determined. Here we found that mouse ES cells express three forms of Raf kinases, A-Raf, B-Raf, and C-Raf. Knocking-down each single Raf member failed to affect the sustained ERK activity, neither did A-Raf and B-Raf double knockdown or B-Raf and C-Raf double knockdown change it in ES cells. Interestingly, B-Raf and C-Raf double knockdown, not A-Raf and B-Raf knockdown, inhibited the maximal ERK activation induced by LIF, concomitant with the slower growth of ES cells. On the other hand, A-Raf, B-Raf, and C-Raf triple knockdown markedly inhibited both the maximal and sustained ERK activity in ES cells. Moreover, Raf triple knockdown, similar to the treatment of U-0126, an MEK inhibitor, significantly inhibited the survival and proliferation of ES cells, thereby compromising the colony propagation of mouse ES cells. In summary, our data demonstrate that all three Raf members are required for ERK activation in mouse ES cells and are involved in growth and survival of mouse ES cells.     

Surface marker antigens in the characterization of human embryonic stem cells

The use of cell surface antigens to characterise embryonic stem (ES) cells, and to monitor their differentiation, has had a long history, stretching back to the early studies of differentiation antigens in the haematopoietic system, and their application to teratocarcinomas and embryonal carcinoma (EC) cells in the laboratory mouse. A wide series of such antigens, which include both glycolipids and glycoproteins are now extensively used in studies of human ES cells. Many of these were first identified using both mouse and human EC cells, although the cell surface antigen phenotype of human EC and ES cells has proved to be significantly different from that of murine EC and ES cells.     

Differential effects on cell motility, embryonic stem cell self-renewal and senescence by diverse Src kinase family inhibitors

The Src family of non-receptor tyrosine kinases (SFKs) has been shown to play an intricate role in embryonic stem (ES) cell maintenance. In the present study we have focused on the underlying molecular mechanisms responsible for the vastly different effects induced by various commonly used SFK inhibitors. We show that several diverse cell types, including fibroblasts completely lacking SFKs, cannot undergo mitosis in response to SU6656 and that this is caused by an unselective inhibition of Aurora kinases. In contrast, PP2 and PD173952 block motility immediately upon exposure and forces cells to grow in dense colonies. The subsequent halt in proliferation of fibroblast and epithelial cells in the center of the colonies approximately 24 h post-treatment appears to be caused by cell-to-cell contact inhibition rather than a direct effect of SFK kinase inhibition. Interestingly, in addition to generating more homogenous and dense ES cell cultures, without any diverse effect on proliferation, PP2 and PD173652 also promote ES cell self-renewal by reducing the small amount of spontaneous differentiation typically observed under standard ES cell culture conditions. These effects could not be mirrored by the use of Gleevec, a potent inhibitor of c-Abl and PDGFR kinases that are also inhibited by PP2.     

wnt3a but not wnt11 supports self-renewal of embryonic stem cells

wnt proteins (wnts) promote both differentiation of midbrain dopaminergic cells and self-renewal of haematopoietic stem cells. Mouse embryonic stem (ES) cells can be maintained and self-renew on mouse feeder cell layers or in media containing leukemia inhibitory factor (LIF). However, the effects of wnts on ES cells self-renewal and differentiation are not clearly understood. In the present study, we found that conditioned medium prepared from L cells expressing wnt3a can replace feeder cell layers and medium containing LIF in maintaining ES cells in the proliferation without differentiation (self-renewal) state. By contrast, conditioned medium from NIH3T3 cells expressing wnt11 did not. Alkaline phosphatase staining and compact colony formation were used as criteria of cells being in the undifferentiated state. ES cells maintained in medium conditioned by Wnt3a expressing cells underwent freezing and thawing while maintaining properties seen with LIF maintained ES cells. Purified wnt3a did not maintain self-renewal of ES cells for prolonged intervals. Thus, other factors in the medium conditioned by wnt3a expressing cells may have contributed to maintenance of ES cells in a self-renewal state. Pluripotency of ES cells was determined with the use of embryoid bodies in vitro. PD98059, a MEK specific inhibitor, promoted the growth of undifferentiated ES cells maintained in conditioned medium from wnt3a expressing cells. By contrast, the P38 MAPK inhibitor SB230580 did not, suggesting a role for the MEK pathway in self-renewal and differentiation of ES cells maintained in the wnt3a cell conditioned medium. Thus, our results show that conditioned medium from wnt3a but not wnt11 expressing cells can maintain ES cells in self-renewal and in a pluripotent state.     

The CDK inhibitor p27 enhances neural differentiation in pluripotent NTERA2 human EC cells, but does not permit differentiation of 2102Ep nullipotent human EC cells

Embryonal carcinoma (EC) cells, the stem cells of teratocarcinomas, are the malignant counterparts of pluripotent embryonic stem (ES) cells, but commonly exhibit a reduced ability to differentiate, presumably because of continual selection for genetic changes that alter the balance between self-renewal, differentiation and apoptosis in favour of self-renewal. To explore the nature of the genetic changes that promote nullipotency, we have compared two human EC cell lines, a 'nullipotent' line, 2102Ep, and a 'pluripotent' line, NTERA2. A hybrid derived by fusion of these cells differentiates in response to retinoic acid but, unlike the parental NTERA2 line, does not form terminally differentiated neurons. This implies that the nullipotent EC cell line, 2102Ep, differs in expression of at least two functions in comparison with the NTERA2 pluripotent line, one affecting commitment to differentiation, and one affecting terminal neural differentiation. We have now investigated the possible role of the CDK inhibitor, p27kip1 (p27) in commitment and terminal differentiation. In NTERA2, but not in 2102Ep cells, retinoic acid induces up-regulation of p27 expression, suggesting that 2102Ep cells lack this capacity. However, constitutive expression of a p27 transgene does not overcome the block to differentiation in the 2102Ep parental cells; commitment to differentiation must be blocked elsewhere. On the other hand, constitutive over-expression of p27 from a transgene enhances the neural differentiation of NTERA2 cells. Our results suggest that p27 plays a role in terminal neuronal differentiation of human EC cells, but not in their initial commitment to differentiation, and that other factors, possibly Cyclin D2, specifically limit its ability to promote neural differentiation.