Physiological rationale for responsiveness of mouse embryonic stem cells to gp130 cytokines

Embryonic stem cells are established directly from the pluripotent epiblast of the preimplantation mouse embryo. Their derivation and propagation are dependent upon cytokine-stimulated activation of gp130 signal transduction. Embryonic stem cells maintain a close resemblance to epiblast in developmental potency and gene expression profile. The presumption of equivalence between embryonic stem cells and epiblast is challenged, however, by the finding that early embryogenesis can proceed in the absence of gp130. To explore this issue further, we have examined the capacity of gp130 mutant embryos to accommodate perturbation of normal developmental progression. Mouse embryos arrest at the late blastocyst stage when implantation is prevented. This process of diapause occurs naturally in lactating females or can be induced experimentally by removal of the ovaries. We report that gp130(-/-) embryos survive unimplanted in the uterus after ovariectomy but, in contrast to wild-type or heterozygous embryos, are subsequently unable to resume development. Inner cell masses explanted from gp130(-/-) delayed blastocysts produce only parietal endoderm, a derivative of the hypoblast. Intact mutant embryos show an absence of epiblast cells, and Hoechst staining and TUNEL analysis reveal a preceding increased incidence of cell death. These findings establish that gp130 signalling is essential for the prolonged maintenance of epiblast in vivo, which is commonly required of mouse embryos in the wild. We propose that the responsiveness of embryonic stem cells to gp130 signalling has its origin in this adaptive physiological function.     

The topographical regulation of embryonic stem cell differentiation

The potential use of pluripotent stem cells for tissue repair or replacement is now well recognized. While the ability of embryonic stem (ES) cells to differentiate into all cells of the body is undisputed, their use is currently restricted by our limited knowledge of the mechanisms controlling their differentiation. This review discusses recent work by ourselves and others investigating the intercellular signalling events that occur within aggregates of mouse ES cells. The work illustrates that the processes of ES cell differentiation, epithelialization and programmed cell death are dependent upon their location within the aggregates and coordinated by the extracellular matrix. Establishment of the mechanisms involved in these events is not only of use for the manipulation of ES cells themselves, but it also throws light on the ways in which differentiation is coordinated during embryogenesis.     

Regulation of bone marrow stromal cell differentiation by cytokines whose receptors share the gp130 Protein

The bone marrow stroma consists of a heterogeneous population of cells which participate in osteogenic, adipogenic, and hematopoietic events. The murine stromal cell line, BMS2, exhibits the adipocytic and osteoblastic phenotypes in vitro. BMS2 differentiation was examined in response to cytokines which share the gp130 signal transducing protein within their receptor complex. Four of the cytokines (interleukin 6, interleukin 11, leukemia inhibitory factor, and oncostatin M) inhibited hydrocortisone-induced adipocyte differentiation in a dose dependent manner based on lipid accumulation and lipoprotein lipase enzyme activity. Inhibition occurred only when the cytokines were present during the initial 24 h of the induction period; after 48 h, their effects were diminished. Likewise, these cytokines increased alkaline phosphatase enzyme activity twofold in preadipocyte BMS2 cells. Both leukemia inhibitory factor and oncostatin M induced early active gene expression in resting preadipocyte BMS2 cells and decreased the steady state mRNA level of a unique osteoblastic gene marker, osteocalcin. A fifth cytokine whose receptor complex shares the gp130 protein, ciliary neurotrophic factor, did not significantly regulate stromal cell differentiation when added by itself. However, with the addition of a missing component of its receptor complex, ciliary neurotrophic factor receptor α protein, this cytokine also inhibited BMS2 adipogenesis. Together, these data indicate that the cytokines whose receptors share the gp130 protein can modulate stromal cell commitment to the adipocyte and osteoblast differentiation pathways.     

A region encompassing the FERM domain of Jak1 is necessary for binding to the cytokine receptor gp130

Cardiac L-type Ca(2+) channel is facilitated by protein kinase A (PKA)-mediated phosphorylation. Here, we investigated the role of Ser(1901), a putative phosphorylation site in the carboxy-terminal of rat brain type-II alpha(1C) subunit (rbCII), in the PKA-mediated regulation. Forskolin (3 microM) enhanced Ca(2+) channel currents (I(Ca)) and shifted the activation curve to negative voltages, which were abolished by protein kinase inhibitor. Replacement of Ser(1901) of rbCII by Ala abolished the enhancement of I(Ca) by forskolin but not the shift of the activation curve. These results indicate that Ser(1901) is required for the PKA-mediated enhancement of I(Ca), and that the voltage-dependence of the activation of I(Ca) appears to be modulated via another PKA phosphorylation site.     

胚胎干细胞诱导分化的研究进展

胚胎干细胞(embryonic stem cell,ESC)因其具有自我更新能力和发育的多能性,成为当前医学研究的热点。ESC不但可以自发分化,而且在诱导因素作用下可以定向分化为某一种特定的成熟细胞。因此,ESC在移植医学、发育生物学等领域有着广阔的应用前景。本文对几种定向诱导ESC分化的策略进行了综述。     

Jak2 and Tyk2 are necessary for lineage-specific differentiation, but not for the maintenance of self-renewal of mouse embryonic stem cells

As the LIF-induced Jak1/STAT3 pathway has been reported to play a crucial role in self-renewal of mESCs, we sought to determine if Jak2, which is also expressed in mESCs, might also be involved in the pathway. By employing an RNAi strategy, we established both Jak2 and Jak2/Tyk2 knockdown mESC clones. Both Jak2 and Jak2/Tyk2 knockdown clones maintained the undifferentiated state as wild-type controls, even in a very low concentration of LIF. However, we observed not only faster onset of differentiation but also differential expression of tissue-specific lineage genes for ectodermal and mesodermal, but not endodermal origins from embryoid bodies generated from both types of knockdown clones compared to the wild-type. Furthermore, the reduced level of Jak2 caused differentiation of mESCs in the presence of LIF when the Wnt pathway was activated by LiCl treatment. Taken together, we demonstrated that Jak2 and Tyk2 are not involved in LIF-induced STAT3 pathway for self-renewal of mESCs, but play a role in early lineage decision of mESCs to various differentiated cell types.     

The UPD3 cytokine couples environmental challenge and intestinal stem cell division through modulation of JAK/STAT signaling in the stem cell microenvironment

In Drosophila, the replacement of spent enterocytes (ECs) relies on division of intestinal stem cells (ISCs) and differentiation of their progeny, the enteroblasts (EBs). Recent studies have revealed a role for JAK/STAT signaling in the modulation of the rate of ISC division in response to environmental challenge. Here, we demonstrate the critical role of the UPD3 cytokine in the JAK/STAT-dependent response to enteric infection. We show that upd3 expression is activated in ECs and in EBs that massively differentiate in response to challenge. We show that the UPD3 cytokine, which is secreted basally and accumulates at the basement membrane, is required for stimulation of JAK/STAT signaling in EBs and visceral muscles (VMs). We further show that stimulation of ISC division requires active JAK/STAT signaling in EBs and VMs, but apparently not in ISCs. Our results suggest that EBs and VMs modulate the rate of the EGFR-dependent ISC division through upd3-dependent production of the EGF ligands Spitz and Vein, respectively. This study therefore supports the notion that the production of the UPD3 cytokine in stem cell progeny (ECs and EBs) stimulates intestinal stem cell division through modulation of JAK/STAT signaling in the stem cell microenvironment (EBs and VMs).     

Self-renewal and differentiation capabilities are variable between human embryonic stem cell lines I3, I6 and BG01V

Background  

A unique and essential property of embryonic stem cells is the ability to self-renew and differentiate into multiple cell lineages. However, the possible differences in proliferation and differentiation capabilities among independently-derived human embryonic stem cells (hESCs) are not well known because of insufficient characterization. To address this question, a side-by-side comparison of 1) the ability to maintain an undifferentiated state and to self-renew under standard conditions; 2) the ability to spontaneously differentiate into three primary embryonic germ lineages in differentiating embryoid bodies; and 3) the responses to directed neural differentiation was made between three NIH registered hES cell lines I3 (TE03), I6 (TE06) and BG01V. Lines I3 and I6 possess normal XX and a normal XY karyotype while BG01V is a variant cell line with an abnormal karyotype derived from the karyotypically normal cell line BG01.     

Growth and differentiation of human embryonic stem cells for cardiac cell replacement therapy

Due to the limited proliferation capacity of cardiac cells, cell replacement therapy has been proposed to restore cardiac function in patients suffering from ischemic heart disease and congestive heart failure. However, this approach is challenged by an insufficient supply of appropriate cells. Because of their apparent indefinite replicative capacity and their cardiac differentiation potential, human embryonic stem cells (hESCs) are potential candidates as sources of cells for cell replacement therapy. Significant progress has been made in improving culture conditions of undifferentiated hESCs, and using various methods, several laboratories have reported the generation of contracting cardiomyocytes from hESCs in vitro. Application of these cardiomyocytes to the clinic, however, still requires substantial experimentation to show that 1) they are functional in vitro; 2) they are efficacious in animal models of cardiac injury and disease; 3) they are safe and effective in human conditions, and 4) a sufficient amount of cardiomyocytes with expected characteristics can be generated in a reproducible manner. Here we review and discuss current findings on growth and differentiation of hESCs, and on characterization, enrichment and transplantation of hESC-derived cardiomyocytes.