Intestinal lineage commitment of embryonic stem cells

Generating lineage-committed intestinal stem cells from embryonic stem cells (ESCs) could provide a tractable experimental system for understanding intestinal differentiation pathways and may ultimately provide cells for regenerating damaged intestinal tissue. We tested a two-step differentiation procedure in which ESCs were first cultured with activin A to favor formation of definitive endoderm, and then treated with fibroblast-conditioned medium with or without Wnt3A. The definitive endoderm expressed a number of genes associated with gut-tube development through mouse embryonic day 8.5 (Sox17, Foxa2, and Gata4 expressed and Id2 silent). The intestinal stem cell marker Lgr5 gene was also activated in the endodermal cells, whereas the Msi1, Ephb2, and Dcamkl1 intestinal stem cell markers were not. Exposure of the endoderm to fibroblast-conditioned medium with Wnt3A resulted in the activation of Id2, the remaining intestinal stem cell markers and the later gut markers Cdx2, Fabp2, and Muc2. Interestingly, genes associated with distal gut-associated mesoderm (Foxf2, Hlx, and Hoxd8) were also simulated by Wnt3A. The two-step differentiation protocol generated gut bodies with crypt-like structures that included regions of Lgr5-expressing proliferating cells and regions of cell differentiation. These gut bodies also had a smooth muscle component and some underwent peristaltic movement. The ability of the definitive endoderm to differentiate into intestinal epithelium was supported by the vivo engraftment of these cells into mouse colonic mucosa. These findings demonstrate that definitive endoderm derived from ESCs can carry out intestinal cell differentiation pathways and may provide cells to restore damaged intestinal tissue.     

Notch signaling activation in human embryonic stem cells is required for embryonic, but not trophoblastic, lineage commitment

The Notch signaling pathway plays important roles in cell-fate determination during embryonic development and adult life. In this study, we focus on the role of Notch signaling in governing cell-fate choices in human embryonic stem cells (hESCs). Using genetic and pharmacological approaches, we achieved both blockade and conditional activation of Notch signaling in several hESC lines. We report here that activation of Notch signaling is required for undifferentiated hESCs to form the progeny of all three embryonic germ layers, but not trophoblast cells. In addition, transient Notch signaling pathway activation enhanced generation of hematopoietic cells from committed hESCs. These new insights into the roles of Notch in hESC-fate determination may help to efficiently direct hESC differentiation into therapeutically relevant cell types.     

Phospholipase Cdelta1 is required for skin stem cell lineage commitment

Phosphoinositide-specific phospholipase C (PLC) is a key enzyme in phosphoinositide turnover and is involved in a variety of physiological functions. Here we report that PLCdelta(1)-deficient mice undergo progressive hair loss in the first postnatal hair cycle. Epidermal hyperplasia was observed, and many hairs in the skin of PLCdelta(1)-deficient mice failed to penetrate the epidermis and became zigzagged owing to occlusion of the hair canal. Two major downstream signals of PLC, calcium elevation and protein kinase C activation, were impaired in the keratinocytes and skin of PLCdelta(1)-deficient mice. In addition, many cysts that had remarkable similarities to interfollicular epidermis, as well as hyperplasia of sebaceous glands, were observed. Furthermore, PLCdelta(1)-deficient mice developed spontaneous skin tumors that had characteristics of both interfollicular epidermis and sebaceous glands. From these results, we conclude that PLCdelta(1) is required for skin stem cell lineage commitment.     

Glutathione peroxidase-1 is required for self-renewal of murine embryonic stem cells

Embryonic stem (ES) cells are pluripotent cells that are capable of giving rise to any type of cells in the body and possess unlimited self-renewal potential. However, the exact regulatory mechanisms that govern the self-renewal ability of ES cells remain elusive. To understand the immediate early events during ES cell differentiation, we performed a proteomics study and analyzed the proteomic difference in murine ES cells before and after a 6-h spontaneous differentiation. We found that the expression level of glutathione peroxidase-1 (GPx-1), an antioxidant enzyme, is dramatically decreased upon the differentiation. Both knockdown of GPx-1 expression with shRNA and inhibiting GPx-1 activity by inhibitor led to the differentiation of ES cells. Furthermore, we showed that during early differentiation, the quick degradation of GPx-1 was mediated by proteasome. Thus, our data indicated that GPx-1 is a key regulator of self-renewal of murine embryonic stem cells.     

ERG is required for the differentiation of embryonic stem cells along the endothelial lineage

Background

During skeletogenesis, protein levels of β-catenin in the canonical Wnt signaling pathway determine lineage commitment of skeletal precursor cells to osteoblasts and chondrocytes. Adenomatous polyposis coli (Apc) is a key controller of β-catenin turnover by down-regulating intracellular levels of β-catenin.

Results

To investigate whether Apc is involved in lineage commitment of skeletal precursor cells, we generated conditional knockout mice lacking functional Apc in Col2a1-expressing cells. In contrast to other models in which an oncogenic variant of β-catenin was used, our approach resulted in the accumulation of wild type β-catenin protein due to functional loss of Apc. Conditional homozygous Apc mutant mice died perinatally showing greatly impaired skeletogenesis. All endochondral bones were misshaped and lacked structural integrity. Lack of functional Apc resulted in a pleiotropic skeletal cell phenotype. The majority of the precursor cells lacking Apc failed to differentiate into chondrocytes or osteoblasts. However, skeletal precursor cells in the proximal ribs were able to escape the noxious effect of functional loss of Apc resulting in formation of highly active osteoblasts. Inactivation of Apc in chondrocytes was associated with dedifferentiation of these cells.

Conclusion

Our data indicate that a tight Apc-mediated control of β-catenin levels is essential for differentiation of skeletal precursors as well as for the maintenance of a chondrocytic phenotype in a spatio-temporal regulated manner.     

Transcriptional analysis of early lineage commitment in human embryonic stem cells

Background  

The mechanisms responsible for the maintenance of pluripotency in human embryonic stem cells, and those that drive their commitment into particular differentiation lineages, are poorly understood. In fact, even our knowledge of the phenotype of hESC is limited, because the immunological and molecular criteria presently used to define this phenotype describe the properties of a heterogeneous population of cells.     

DeltaNp63 is essential for epidermal commitment of embryonic stem cells

In vivo studies have demonstrated that p63 plays complex and pivotal roles in pluristratified squamous epithelial development, but its precise function and the nature of the isoform involved remain controversial. Here, we investigate the role of p63 in epithelial differentiation, using an in vitro ES cell model that mimics the early embryonic steps of epidermal development. We show that the DeltaNp63 isoform is activated soon after treatment with BMP-4, a morphogen required to commit differentiating ES cells from a neuroectodermal to an ectodermal cell fate. DeltaNp63 gene expression remains high during epithelial development. P63 loss of function drastically prevents ectodermal cells to commit to the K5/K14-positive stratified epithelial pathway while gain of function experiments show that DeltaNp63 allows this commitment. Interestingly, other epithelial cell fates are not affected, allowing the production of K5/K18-positive epithelial cells. Therefore, our results demonstrate that DeltaNp63 may be dispensable for some epithelial differentiation, but is necessary for the commitment of ES cells into K5/K14-positive squamous stratified epithelial cells.     

DNA synthesis is not required for the commitment of murine erythroleukemia cells

We have assessed the relationship between DNA synthesis and the differentiation of MEL cells induced by DMSO. Under conditions where the rate of incorporation of ³H-deoxyadenosine into DNA was inhibited by 99%, the rate at which MEL cells become committed to terminal erythroid differentiation was identical to that of a culture treated with inducer alone. We conclude that commitment of MEL cells does not require concomitant DNA synthesis.     

Fourier transform infrared microspectroscopy identifies early lineage commitment in differentiating human embryonic stem cells

Human ESCs (hESCs) are a valuable tool for the study of early human development and represent a source of normal differentiated cells for pharmaceutical and biotechnology applications and ultimately for cell replacement therapies. For all applications, it will be necessary to develop assays to validate the efficacy of hESC differentiation. We explored the capacity for FTIR spectroscopy, a technique that rapidly characterises cellular macromolecular composition, to discriminate mesendoderm or ectoderm committed cells from undifferentiated hESCs. Distinct infrared spectroscopic “signatures” readily distinguished hESCs from these early differentiated progeny, with bioinformatic models able to correctly classify over 97% of spectra. These data identify a role for FTIR spectroscopy as a new modality to complement conventional analyses of hESCs and their derivatives. FTIR spectroscopy has the potential to provide low-cost, automatable measurements for the quality control of stem and differentiated cells to be used in industry and regenerative medicine.     

Beta-catenin signaling is required for neural differentiation of embryonic stem cells

Culture of embryonic stem (ES) cells at high density inhibits both beta-catenin signaling and neural differentiation. ES cell density does not influence beta-catenin expression, but a greater proportion of beta-catenin is targeted for degradation in high-density cultures. Moreover, in high-density cultures, beta-catenin is preferentially localized to the membrane further reducing beta-catenin signaling. Increasing beta-catenin signaling by treatment with Wnt3a-conditioned medium, by overexpression of beta-catenin, or by overexpression of a dominant-negative form of E-cadherin promotes neurogenesis. Furthermore, beta-catenin signaling is sufficient to induce neurogenesis in high-density cultures even in the absence of retinoic acid (RA), although RA potentiates the effects of beta-catenin. By contrast, RA does not induce neurogenesis in high-density cultures in the absence of beta-catenin signaling. Truncation of the armadillo domain of beta-catenin, but not the C terminus or the N terminus, eliminates its proneural effects. The proneural effects of beta-catenin reflect enhanced lineage commitment rather than proliferation of neural progenitor cells. Neurons induced by beta-catenin overexpression either alone or in association with RA express the caudal neuronal marker Hoxc4. However, RA treatment inhibits the beta-catenin-mediated generation of tyrosine hydroxylase-positive neurons, suggesting that not all of the effects of RA are dependent upon beta-catenin signaling. These observations suggest that beta-catenin signaling promotes neural lineage commitment by ES cells, and that beta-catenin signaling may be a necessary co-factor for RA-mediated neuronal differentiation. Further, enhancement of beta-catenin signaling with RA treatment significantly increases the numbers of neurons generated from ES cells, thus suggesting a method for obtaining large numbers of neural species for possible use in for ES cell transplantation.     

Cardiac commitment of primate embryonic stem cells

Primate nonhuman and human embryonic stem (ES) cells provide a powerful model of early cardiogenesis. Furthermore, engineering of cardiac progenitors or cardiomyocytes from ES cells offers a tool for drug screening in toxicology or to search for molecules to improve and scale up the process of cardiac differentiation using high-throughput screening technology, as well as a source of cell therapy of heart failure. Spontaneous differentiation of ES cells into cardiomyocytes is, however, limited. Herein, we describe a simple protocol to commit both rhesus and human ES cells toward a cardiac lineage and to sort out early cardiac progenitors. Primate ES cells are challenged for 4 d with the cardiogenic morphogen bone morphogenetic protein 2 (BMP2) and sorted out using anti-SSEA-1 antibody-conjugated magnetic beads. Cardiac progenitor cells can be generated and isolated in 4 d using this protocol.     

Cdk1 is required for the self-renewal of mouse embryonic stem cells

Cyclin-dependent kinase 1 (Cdk1) is indispensible for the early development of the embryo. However, its role in maintaining the undifferentiated state of the embryonic stem (ES) cells remains unknown. In this study, we dissected the function of Cdk1 in mouse ES cells by RNA-interference and gene expression analyses. Cdk1 expression is tightly correlated with the undifferentiated state of the ES cells. Upon differentiation, Cdk1 expression reduced drastically. Cdk1 knock-down by RNA interference resulted in the loss of proliferation and colony formation potential of the ES cells. Consequentially, expression of self-renewal genes was reduced while differentiation markers such as Cdx2 were induced. Our results suggest a role for Cdk1 in maintaining the unique undifferentiated and self-renewing state of the mouse ES cells.     

p53 is required for etoposide-induced apoptosis of human embryonic stem cells

The molecular mechanisms controlling DNA-damage-induced apoptosis of human embryonic stem cells (hESC) are poorly understood. Here we investigate the role of p53 in etoposide-induced apoptosis. We show that p53 is constitutively expressed at high levels in the cytoplasm of hESC. Etoposide treatment results in a rapid and extensive induction of apoptosis and leads to a further increase in p53 and PUMA expression as well as Bax processing. p53 both translocates to the nucleus and associates with the mitochondria, accompanied by colocalization of Bax with Mcl1. hESC stably transduced with p53 shRNA display 80% reduction of endogenous p53 and exhibit an 80% reduction in etoposide-induced apoptosis accompanied by constitutive downregulation of Bax and an attenuated upregulation of PUMA. Our data further show that undifferentiated hESC that express Oct4 are much more sensitive to etoposide-induced apoptosis than their more differentiated progeny. Our study demonstrates that p53 is required for etoposide-induced apoptosis of hESC and reveals, at least in part, the molecular mechanism of DNA-damage-induced apoptosis in hESC.     

Regulation of developmental competence and commitment towards the definitive endoderm lineage in human embryonic stem cells

Human embryonic stem cells (hESCs) can self-renew and become all three germ layers. Nodal/Activin signaling specifies developmental status in hESCs: moderate Nodal/Activin signaling maintains pluripotency, while enhancement and inhibition promote definitive endoderm (DE) and neuroectoderm (NE) development, respectively. However, how modulation of Nodal/Activin signaling influences developmental competence and commitment toward specific lineages is still unclear. Here, we showed that enhancement of Nodal/Activin signaling for 4 days was necessary and sufficient to upregulate DE markers, while it diminished the upregulation of NE markers by inhibition of Nodal/Activin signaling. This suggests that after 4 days of enhanced Nodal/Activin signaling, hESCs are committed to the DE lineage and have lost competence toward the NE lineage. In contrast, inhibition of Nodal/Activin signaling using LY364947 for 2 days was sufficient to impair competence toward the DE lineage, although cells were still able to activate LEFTY1 and NODAL, direct targets of Nodal/Activin signaling. Expression analyses indicated that the levels of pluripotency regulators NANOG and POU5F1 were significantly diminished by 2 days of LY364947 treatment, although the expression of NANOG, but not POU5F1, was restored immediately upon Activin A treatment. Thus, downregulation of POU5F1 coincided with the abrogation of DE competence caused by inhibition of Nodal/Activin signaling.     

Protein tyrosine phosphatase 1B (PTP1B) is required for cardiac lineage differentiation of mouse embryonic stem cells

Protein tyrosine phosphatase 1B (PTP1B) has been shown to regulate multiple cellular events such as differentiation, cell growth, and proliferation; however, the role of PTP1B in differentiation of embryonic stem (ES) cells into cardiomyocytes remains unexplored. In the present study, we investigated the effects of PTP1B inhibition on differentiation of ES cells into cardiomyocytes. PTP1B mRNA and protein levels were increased during the differentiation of ES cells into cardiomyocytes. Accordingly, a stable ES cell line expressing PTP1B shRNA was established. In vitro, the number and size of spontaneously beating embryoid bodies were significantly decreased in PTP1B-knockdown cells, compared with the control cells. Decreased expression of cardiac-specific markers Nkx2-5, MHC-α, cTnT, and CX43, as assessed by real-time PCR analysis, was further confirmed by immunocytochemistry of the markers. The results also showed that PTP1B inhibition induced apoptosis in both differentiated and undifferentiated ES cells, as presented by increasing the level of cleaved caspase-3, cytochrome C, and cleaved PARP. Further analyses revealed that PTP1B inhibition did not change proliferation and pluripotency of undifferentiated ES cells. Taken together, the data presented here suggest that PTP1B is essential for proper differentiation of ES cells into cardiomyocytes.