Regulation of protein phosphatase 2A methylation by LCMT1 and PME-1 plays a critical role in differentiation of neuroblastoma cells

Neuritic alterations are a major feature of many neurodegenerative disorders. Methylation of protein phosphatase 2A (PP2A) catalytic C subunit by the leucine carboxyl methyltransferase (LCMT1), and demethylation by the protein phosphatase methylesterase 1, is a critical PP2A regulatory mechanism. It modulates the formation of PP2A holoenzymes containing the Bα subunit, which dephosphorylate key neuronal cytoskeletal proteins, including tau. Significantly, we have reported that LCMT1, methylated C and Bα expression levels are down-regulated in Alzheimer disease-affected brain regions. In this study, we show that enhanced expression of LCMT1 in cultured N2a neuroblastoma cells, which increases endogenous methylated C and Bα levels, induces changes in F-actin organization. It promotes serum-independent neuritogenesis and development of extended tau-positive processes upon N2a cell differentiation. These stimulatory effects can be abrogated by LCMT1 knockdown and S-adenosylhomocysteine, an inhibitor of methylation reactions. Expression of protein phosphatase methylesterase 1 and the methylation-site L309Δ C subunit mutant, which decrease intracellular methylated C and Bα levels, block N2a cell differentiation and LCMT1-mediated neurite formation. Lastly, inducible and non-inducible knockdown of Bα in N2a cells inhibit process outgrowth. Altogether, our results establish a novel mechanistic link between PP2A methylation and development of neurite-like processes.     

Hematopoietic differentiation of embryonic stem cells

This review is focused on methods that are used to derive hematopoietic cells from embryonic stem cells (ESCs). One of the strategies that have been recently used to achieve this goal is an approach of mimicking the hematopoietic niche in vitro by using hematopoiesis-supportive feeder cells, cocktails of soluble hematopoietic growth factors and a variety of matrices. While there is clear evidence that it is possible to derive hematopoietic stem cells (HSCs) and subsequently committed hematopoietic progenitors and mature cells from ESCs, there remains the need to address multiple issues including the efficiency of HSCs derivation in vitro and their proper functionality.     

Gene profiling on mixed embryonic stem cell populations reveals a biphasic role for beta-catenin in osteogenic differentiation

The differentiation of embryonic stem cells (ESCs) into osteoblasts is enhanced to 60% when exposed to vitamin D3 (VD3) but leaves a remainder of one half of the cell population unidentified. To increase differentiation outcome, the known osteoinducers retinoic acid (RA) and bone morphogenetic protein-2 (BMP-2) were evaluated. Initial studies using RA and BMP-2 during early osteogenesis in addition to VD3 increased osteogenic yield in the case of RA, but surprisingly decreased osteogenesis when BMP-2 was administered together with VD3 or RA. This paper describes a comprehensive microarray study examining the gene expression profile of differentiating osteoblasts in these mixed ESC populations. In addition to five other families of signaling molecules (insulin growth factors, prostaglandin, follistatin, TGFbeta2, and Wnt molecules), we identified an endogenous expression pattern for BMPs and RA that differed from our previous exogenous administration of these molecules. By mimicking the change in expression of the RA and BMP-2 families with exogenous supplementation at the correct time, it was then possible to increase the number of ESC-derived osteoblasts to 90%. This effect was mediated through alteration in beta-catenin (CatnB) expression levels and nuclear CatnB activity, both of which are modulated by VD3, RA, and BMP-2. Our results suggest that blockage of CatnB activity by VD3 and RA is opposed by induction of CatnB activity through BMP-2 when administered together. Hence, osteoinduction, in vitro, is an intricate process involving both temporal and quantitative changes in gene expression and CatnB activity.     

胚胎干细胞向造血系统的分化

胚胎干细胞是指从囊胚期的内细胞团中分离出来的尚未分化的胚胎细胞,可分化形成各种组织类型。在合适的条件下,胚胎干细胞可发育成造血干细胞及各类成熟血细胞,为造血干细胞移植及血细胞输注开辟了新的来源,同时也为造血发生及造血调控研究提供了有效可靠的模型。本文将综述ES细胞向造血系统分化的诱导条件、调控机制及应用前景。     

A regulatory role of Wnt signaling pathway in the hematopoietic differentiation of murine embryonic stem cells

One of the most important issues in stem cell research is to understand the regulatory mechanisms responsible for their differentiation. An extensive understanding of mechanism underlying the process of differentiation is crucial in order to prompt stem cells to perform a particular function after differentiation. To elucidate the molecular mechanisms responsible for the hematopoietic differentiation of embryonic stem cells (ESCs), we investigated murine ES cells for the presence of hematopoietic lineage markers as well as Wnt signaling pathway during treatments with different cytokines alone or in combination with another. Here we report that Wnt/beta-catenin signaling is down-regulated in hematopoietic differentiation of murine ES cells. We also found that differentiation induced by the interleukin-3, interleukin-6, and erythropoietin combinations resulted in high expression of CD3e, CD11b, CD45R/B220, Ly-6G, and TER-119 in differentiated ES cells. A high expression of beta-catenin was observed in two undifferentiated ES cell lines. Gene and protein expression analysis revealed that the members downstream of Wnt in this signaling pathway including beta-catenin, GSK-3beta, Axin, and TCF4 were significantly down-regulated as ES cells differentiated into hematopoietic progenitors. Our results show that the Wnt/beta-catenin signaling pathway plays a role in the hematopoietic differentiation of murine ESCs and also may support beta-catenin as a crucial factor in the maintenance of ES cells in their undifferentiated state.     

Trophoblast differentiation of human embryonic stem cells

Molecular mechanisms regulating human trophoblast differentiation remain poorly understood due to difficulties in obtaining primary tissues from very early developmental stages in humans. Therefore, the use of human embryonic stem cells (hESCs) as a source for generating trophoblast tissues is of significant interest. Trophoblast-like cells have been obtained through treatment of hESCs with bone morphogenetic protein (BMP) or inhibitors of activin/nodal/transforming growth factor-β signaling, or through protocols involving formation of embryoid bodies (EBs); however, there is controversy over whether hESC-derived cells are indeed analogous to true trophoblasts found in vivo. In this review, we provide an overview of previously described efforts to obtain trophoblasts from hESCs. We also discuss the merits and limitations of hESCs as a source of trophoblast derivatives.     

Neural differentiation of human embryonic stem cells

Availability of human embryonic stem cells (hESC) has enhanced human neural differentiation research. The derivation of neural progenitor (NP) cells from hESC facilitates the interrogation of human embryonic development through the generation of neuronal subtypes and supporting glial cells. These cells will likely lead to novel drug screening and cell therapy uses. This review will discuss the current status of derivation, maintenance and further differentiation of NP cells with special emphasis on the cellular signaling involved in these processes. The derivation process affects the yield and homogeneity of the NP cells. Then when exposed to the correct environmental signaling cues, NP cells can follow a unique and robust temporal cell differentiation process forming numerous phenotypes.     

Neuronal differentiation of NTE-deficient embryonic stem cells

Organophosphates induce neurological disorders. One of the enzymes inhibited by these compounds is neuropathy target esterase (NTE). In vitro, inhibition of NTE activity by organophosphates is correlated with inhibition of neurite initiation and reduction of neurite length, supporting the hypothesis that organophosphate-induced neurological disorders are caused by inhibition of NTE activity. However, there is no direct evidence for the involvement of NTE in organophosphate-induced impairment of neurites in vitro. To examine the role of NTE, we have generated NTE-deficient mouse embryonic stem cells. These cells can differentiate into neuron-like cells. Although NTE-deficient cells exhibited a delay in neurite initiation in vitro, both the proportion of neuron-like cells which initiated neurites and the elongation of these neurites occurred at the normal rate. These results demonstrate that NTE activity is not required for neurite initiation or elongation per se, but is essential for the optimal rate of neurite initiation.     

Reversible permeabilization of the mitochondrial membrane promotes human cardiomyocyte differentiation from embryonic stem cells

Cell death and differentiation appear to share similar cellular features. In this study, we aimed to investigate whether differentiation and mitochondrial cell death use a common pathway. We assessed the hallmarks of apoptosis during cardiomyocyte differentiation of human embryonic stem cells and found remarkable changes in P53, reactive oxygen species, apoptotic protease-activating factor 1, poly[ADP-ribose]polymerase 1, cellular adenosine triphosphate, and mitochondrial complex I activity. Furthermore, we observed reversible mitochondrial membrane permeabilization during cardiomyocyte differentiation accompanied by reversible loss of mitochondrial membrane potential, and these changes coincided with the fluctuating patterns of cytosolic cytochrome c accumulation and subsequent caspase-9 and -3/7 activation. Moreover, the use of apoptosis inhibitors (BCL2-associated X protein [BAX] inhibitor and caspase-3/7 inhibitor) during differentiation impaired cardiomyocyte development, resulting in substantial downregulation of T, MESP1, NKX2.5, and α-MHC. Additionally, although the expression of specific differentiation markers (T, MESP1, NKX2.5, MEF2C, GATA4, and SOX17) was enhanced in doxorubicin-induced human embryonic stem cells, the stemness-specific markers (OCT4 and NANOG) showed significant downregulation. With increasing doxorubicin concentration (0.03–0.6 µM; IC₅₀ = 0.5 µM), we observed a marked increase in the expression of mesoderm and endoderm markers. In summary, we suggest that reversible mitochondrial outer membrane permeabilization promotes cardiomyocyte differentiation through an attenuated mitochondria-mediated apoptosis-like pathway.     

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.     

Chromatin in pluripotent embryonic stem cells and differentiation

Embryonic stem (ES) cells are unique in that they are pluripotent and have the ability to self-renew. The molecular mechanisms that underlie these two fundamental properties are largely unknown. We discuss how unique properties of chromatin in ES cells contribute to the maintenance of pluripotency and the determination of differentiation properties.     

Effect of dual-specificity protein phosphatase 5 on pluripotency maintenance and differentiation of mouse embryonic stem cells

The MAPK/Erk signaling pathway is considered as a key regulator of the pluripotency and differentiation of embryonic stem (ES) cells, while dual-specificity protein phosphatases (DUSPs) are negative regulators of MAPK. Although DUSPs are potential embryogenesis regulators, their functions in the regulation of ES cell differentiation have not been demonstrated. The present study revealed that Dusp5 was expressed in mouse ES (mES) cells and that its expression was correlated with the undifferentiated state of these cells. Exogenous Dusp5 expression enhanced mES cell clonogenicity and suppressed mES cell differentiation by maintaining Nanog expression via the inhibition of the Erk pathway. Following Dusp5 knockdown, Nanog and Oct4 expression was significantly attenuated and the Erk signaling pathway was activated. Additionally, EBs derived from Dusp5 knockdown mES cells (KDEBs) exhibited a weak adherence capability, very little outgrowth, and a reduction in the number of epithelial-like cells. The expression of Gata6 (an endodermal marker) and Flk1 and Twist1 (mesodermal markers) was inhibited in KDEBs, which indicated that Dusp5 influenced the differentiation of these germ layers during EB development. Collectively, this study suggested that Dusp5 plays an important role in the maintenance of pluripotency in mES cells, and that Dusp5 may be required for EB development.     

Neural differentiation of murine embryonic stem cells ES

Pluripotent murine embryonic stem (ES) cells can differentiate into all cell types both in vivo and in vitro. Based on their capability to proliferate and differentiate, these ES cells appear as a very promising tool for cell therapy. The understanding of the molecular mechanisms underlying the neural differentiation of the ES cells is a pre-requisite for selecting adequately the cells and conditions which will be able to correctly repair damaged brain and restore altered cognitive functions. Different methods allow obtaining neural cells from ES cells. Most of the techniques differentiate ES cells by treating embryoid bodies in order to keep an embryonic organization. More recent techniques, based on conditioned media, induce a direct differentiation of ES cells into neural cells, without going through the step of embryonic bodies. Beyond the fact that these techniques allow obtaining large numbers of neural precursors and more differentiated neural cells, these approaches also provide valuable information on the process of differentiation of ES cells into neural cells. Indeed, sequential studies of this process of differentiation have revealed that globally ES cells differentiating into neural cells in vitro recapitulate the molecular events governing the in vivo differentiation of neural cells. Altogether these data suggest that murine ES cells remain a highly valuable tool to obtain large amounts of precursor and differentiated neural cells as well as to get a better understanding of the mechanisms of neural differentiation, prior to a potential move towards the use of human ES cells in therapy.     

Osteoblastic differentiation of monkey embryonic stem cells in vitro

Monkey embryonic stem (ES) cell is a useful tool for preclinical studies of regenerative medicine. In this paper, we investigated whether monkey ES cells can be differentiated into osteoblasts in vitro using factors known to promote osteogenesis. We prepared embryoid bodies (EB) in the presence of retinoic acid (RA) and subsequently differentiated in the medium containing either dexamethasone (DEX) or bone morphogenetic protein (BMP)-2 in addition to osteogenic supplements (OS), specifically ascorbic acid and beta-glycerophosphate. RA treatment during EB formation induced osteoblastic marker genes, such as collagen type 1, osteopontin, and Cbfa1. For the expression of osteocalcin, however, cultivation with medium containing either DEX or BMP-2 in addition to OS was required. These results showed that osteoblasts could be derived from monkey ES cells in vitro and BMP-2 + OS was effective to induce calcification.     

Protocols for cardiac differentiation of embryonic stem cells

Both mouse 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. Spontaneous differentiation of ES cells into cardiomyocytes is however limited. Herein, I described simple protocols to commit both mouse and human ES cells toward a cardiac lineage and in turn to improve the process of in vitro differentiation.     

Hepatic differentiation of murine embryonic stem cells.

Murine embryonic stem (ES) cells can replicate indefinitely in culture and can give rise to all tissues, including the germline, when reimplanted into a murine blastocyst. ES cells can also be differentiated in vitro into a wide range of cell types. We have utilized a liver-specific marker to demonstrate that murine ES cells can differentiate into hepatocytes in vitro. We have used ES cells carrying a gene trap vector insertion (I.114) into an ankyrin repeat-containing gene (Gtar) that we have previously shown provides an exclusive beta-galactosidase marker for the early differentiation of hepatocytes in vivo. beta-Galactosidase-positive cells were differentiated from I.114 ES cells in vitro. The identity of these cells was confirmed by the expression of the proteins alpha-fetoprotein, albumin, and transferrin and by the fact that they have an ultrastructural appearance consistent with that of embryonic hepatocytes. We propose that this model system of hepatic differentiation in vitro could be used to define factors that are involved in specification of the hepatocyte lineage. In addition, human ES cells have recently been derived and it has been proposed that they may provide a source of differentiated cell types for cell replacement therapies in the treatment of a variety of diseases.