人胚胎干细胞向内皮祖细胞诱导分化及其应用研究进展

近年来,内皮细胞的应用价值不断提高,应用领域不断拓宽,但其来源有限,成为研究应用的主要障碍.胚胎干细胞在体外可分化为多种组织细胞系,有可能成为获取内皮细胞的另一来源.就人胚胎干细胞向内皮祖细胞分化、分离方法、相关分子机制及内皮祖细胞应用价值等进行阐述,以期能够引起更多的关注,推动其研究的进展.     

CD133+ hepatic stellate cells are progenitor cells

Hepatic stellate cells (HSC) play an important role in the development of liver fibrosis. Here, we report that HSC express the stem/progenitor cell marker CD133 and exhibit properties of progenitor cells. CD133+ HSC of rats were selected by specific antibodies and magnetic cell sorting. Selected cells displayed typical markers of HSC, endothelial progenitor cells (EPC), and monocytes. In cell culture, CD133+ HSC transformed into alpha-smooth muscle actin positive myofibroblast-like cells, whereas application of cytokines known to facilitate EPC differentiation into endothelial cells led to the formation of branched tube-like structures and induced expression of the endothelial cell markers endothelial nitric oxide synthase and vascular-endothelial cadherin. Moreover, cytokines that guide stem cells to develop hepatocytes led to the appearance of rotund cells and expression of the hepatocyte markers alpha-fetoprotein and albumin. It is concluded that CD133+ HSC are a not yet recognized progenitor cell compartment with characteristics of early EPC. Their potential to differentiate into endothelial or hepatocyte lineages suggests important functions of CD133+ HSC during liver regeneration.     

TGF-beta receptor kinase inhibitor enhances growth and integrity of embryonic stem cell-derived endothelial cells

Recent findings have shown that embryonic vascular progenitor cells are capable of differentiating into mural and endothelial cells. However, the molecular mechanisms that regulate their differentiation, proliferation, and endothelial sheet formation remain to be elucidated. Here, we show that members of the transforming growth factor (TGF)-beta superfamily play important roles during differentiation of vascular progenitor cells derived from mouse embryonic stem cells (ESCs) and from 8.5-days postcoitum embryos. TGF-beta and activin inhibited proliferation and sheet formation of endothelial cells. Interestingly, SB-431542, a synthetic molecule that inhibits the kinases of receptors for TGF-beta and activin, facilitated proliferation and sheet formation of ESC-derived endothelial cells. Moreover, SB-431542 up-regulated the expression of claudin-5, an endothelial specific component of tight junctions. These results suggest that endogenous TGF-beta/activin signals play important roles in regulating vascular growth and permeability.     

Mediator subunit MED1 modulates intranuclear dynamics of the thyroid hormone receptor

The thyroid hormone receptors (TRs) mediate thyroid hormone (T₃)-dependent gene expression. The nuclear import and export signals that direct TR shuttling are well characterized, but little is known about factors modulating nuclear retention. We used fluorescence-based nucleocytoplasmic scoring and fluorescence recovery after photobleaching in transfected cells to investigate whether Mediator subunits MED1 and MED13 play a role in nuclear retention of TR. When MED1 was overexpressed, there was a striking shift towards a greater nuclear localization of TRβ1 and the oncoprotein v-ErbA, subtypes with cytosolic populations at steady-state, and TRβ1 intranuclear mobility was reduced. For TRα1, there was no observable change in its predominantly nuclear distribution pattern or mobility. Consistent with a role for MED1 in nuclear retention, the cytosolic TRα1 and TRβ1 population were significantly greater in MED1^−/− cells, compared with MED1^+/+ cells. Exposure to T₃ and epidermal growth factor, which induces MED1 phosphorylation, also altered TR intranuclear dynamics. Overexpression of miR-208a, which downregulates MED13, led to a more cytosolic distribution of nuclear-localized TRα1; however, overexpression of MED13 had no effect on TRβ1 localization. The known binding site of MED1 overlaps with a transactivation domain and nuclear export signal in helix 12 of TR's ligand-binding domain (LBD). Coimmunoprecipitation assays demonstrated that TR's LBD interacts directly with exportins 5 and 7, suggesting that binding of exportins and MED1 to TR may be mutually exclusive. Collectively, our data provide evidence that MED1 promotes nuclear retention of TR, and highlight the dual functionality of helix 12 in TR transactivation and nuclear export.     

BMP4 regulates vascular progenitor development in human embryonic stem cells through a smad‐dependent pathway

The signals that direct pluripotent stem cell differentiation into lineage‐specific cells remain largely unknown. Here, we investigated the roles of BMP on vascular progenitor development from human embryonic stem cells (hESCs). In a serum‐free condition, hESCs sequentially differentiated into CD34+CD31?, CD34+CD31+, and then CD34?CD31+ cells during vascular cell development. CD34+CD31+ cells contained vascular progenitor population that gives rise to endothelial cells and smooth muscle cells. BMP4 promoted hESC differentiation into CD34+CD31+ cells at an early stage. In contrast, TGFβ suppressed BMP4‐induced CD34+CD31+ cell development, and promoted CD34+CD31? cells that failed to give rise to either endothelial or smooth muscle cells. The BMP‐Smad inhibitor, dorsomorphin, inhibited phosphorylation of Smad1/5/8, and blocked hESC differentiation to CD34+CD31+ progenitor cells, suggesting that BMP Smad‐dependent signaling is critical for CD34+CD31+ vascular progenitor development. Our findings provide new insight into how pluripotent hESCs differentiate into vascular cells. J. Cell. Biochem. 109: 363–374, 2010. © 2009 Wiley‐Liss, Inc.     

三维联合培养牙髓细胞和血管内皮祖细胞促进成牙本质向/成骨向分化

目的:探讨三维联合培养牙髓细胞(dental pulp cells, DPCs)和血管内皮祖细胞(endothelial progenitor cells, EPCs)对成牙本质向/成骨向分化的影响。方法:取单独培养DPCs及联合培养的DPCs和EPCs进行三维培养后成牙本质向/成骨向诱导,使用茜素红染色及半定量分析、RT-PCR和细胞免疫荧光检测成牙本质向/成骨向分化能力。采用SPSS 23.0统计软件对数据进行统计学分析。结果:茜素红染色显示联合培养组和单独培养组之间未见显著差异。RT-PCR和细胞免疫荧光显示成牙本质向/成骨向相关基因m RNAs和蛋白表达水平联合培养组显著高于单独培养组。结论:三维联合培养的DPCs和EPCs促进成牙本质向/成骨向分化,为牙髓再生提供可能实验依据。     

Tumor skewing of CD34+ cell differentiation from a dendritic cell pathway into endothelial cells

Patients and animals bearing tumors have increased levels of CD34⁺ progenitor cells, which are capable of developing into dendritic cells. However, addition of medium conditioned by murine Lewis lung carcinoma cells increases the cellularity of the CD34⁺ cell cultures and redirects their differentiation into endothelial cells. The resulting cells resemble endothelial cells phenotypically as well as functionally by their capacity to reorganize into cord structures. Mechanisms by which tumors induced the increased cellularity and skewing toward endothelial cells were examined. Tumor-derived VEGF contributed to the increase in cellularity, but not to the redirection of differentiation. Differentiation into endothelial cells was blocked with sTie-2, suggesting tumor-derived angiopoietins in skewing differentiation. These studies show the capacity of tumors to skew progenitor cell development toward endothelial cells and define the mediators that contribute to endothelial cell development.     

Notch signaling in blood vessels: from morphogenesis to homeostasis

Notch signaling is an evolutionarily conserved intercellular signaling pathway that plays numerous crucial roles in vascular development and physiology. Compelling evidence indicates that Notch signaling is vital for vascular morphogenesis including arterial and venous differentiation and endothelial tip and stalk cell specification during sprouting angiogenesis and also vessel maturation featured by mural cell differentiation and recruitment. Notch signaling is also required for vascular homeostasis in adults by keeping quiescent phalanx cells from re-entering cell cycle and by modulating the behavior of endothelial progenitor cells. We will summarize recent advances of Notch pathway in vascular biology with special emphasis on the underlying molecular mechanisms.     

Differentiation of circulating endothelial progenitor cells to a cardiomyogenic phenotype depends on E-cadherin

Progenitor cells may contribute to cardiac regeneration. Here, we investigated the role of cadherins and integrins for differentiation of human adult circulating endothelial progenitor cells (EPCs) into cardiomyocytes (CM) in a co-culture system. N- and E-cadherin were expressed in EPCs and were localized at the interface between EPCs and CM. Incubation of a blocking antibody against E-cadherin reduced the expression of CM marker protein in EPCs. Blocking antibodies against N- or P-cadherin or the beta1- and beta2-integrins were not effective. These data suggested that cell-to-cell communication mediated by E-cadherin contributes to the acquirement of a cardiomyogenic phenotype of human endothelial progenitor cells.     

Identification of stage-specific markers during differentiation of hair cells from mouse inner ear stem cells or progenitor cells in vitro

The induction of inner ear hair cells from stem cells or progenitor cells in the inner ear proceeds through a committed inner ear sensory progenitor cell stage prior to hair cell differentiation. To increase the efficacy of inducing inner ear hair cell differentiation from the stem cells or progenitor cells, it is essential to identify comprehensive markers for the stem cells/progenitor cells from the inner ear, the committed inner ear sensory progenitor cells and the differentiating hair cells to optimize induction conditions. Here, we report that we efficiently isolated and expanded the stem cells or progenitor cells from postnatal mouse cochleae, and induced the generation of inner ear progenitor cells and subsequent differentiation of hair cells. We profiled the gene expression of the stem cells or progenitor cells, the inner ear progenitor cells, and hair cells using aRNA microarray analysis. The pathway and gene ontology (GO) analysis of differentially expressed genes was performed. Analysis of genes exclusively detected in one particular cellular population revealed 30, 38, and 31 genes specific for inner ear stem cells, inner ear progenitor cells, and hair cells, respectively. We further examined the expression of these genes in vivo and determined that Gdf10+Ccdc121, Tmprss9+Orm1, and Chrna9+Espnl are marker genes specific for inner ear stem cells, inner ear progenitor cells, and differentiating hair cells, respectively. The identification of these marker genes will likely help the effort to increase the efficacy of hair cell induction from the stem cells or progenitor cells.     

低氧通过上调Wnt5a促进人胚胎干细胞向生血内皮细胞分化

胚胎的早期发育是在低氧条件下进行的,低氧环境在胚胎血管发生及造血发育中起着重要作用,低氧条件能促进胚胎干细胞在体外向内皮细胞和造血细胞的分化,但低氧条件对造血细胞产生的具体作用及相应机制尚不清楚．本研究利用人Es细胞向造血祖细胞定向分化体系,发现低氧环境可以促进CD31＋TIE2＋造血内皮祖细胞的产生,2天后造血内皮祖细胞开始表达终生造血基因．进一步研究发现,低氧能够上调Wnt5a的表达,干涉Wnt5a能够抑制低氧环境对生血内皮细胞分化的促进作用．在正常氧环境下加入Wnt5a产生促进生血内皮细胞分化的效应,该效应与低氧处理促进生血内皮细胞产生的作用相似．本研究首次证明了低氧通过上调Wnt5a的表达促进人Es细胞向生血内皮细胞的分化,为ES细胞向生血内皮细胞的分化及造血祖细胞分化的研究提供了新的线索．     

Activated Notch1 alters differentiation of embryonic stem cells into mesodermal cell lineages at multiple stages of development

Signals of Notch transmembrane receptors function to regulate a wide variety of developmental cell fates. Here we investigate the role of Notch signaling in the development of mesodermal cell types by expressing a tamoxifen-inducible, activated form of Notch1 in embryonic stem cells (ESC). For differentiation of ESC into first mesodermal progenitor cells and then endothelial, mural, cardiac muscle and hematopoietic cells, the OP9 stroma co-culture system was used. Timed activation of Notch signaling by the addition of tamoxifen at various stages during differentiation of ESC into mesodermal cell lineages results in profound alterations in the generation of all of these cells. Differentiation of ESC into Flk1(+) mesodermal cells is inhibited by activated Notch. When Notch signaling is activated in mesodermal cells, generation of cardiac muscle, endothelial and hematopoietic cells is inhibited, favoring the generation of mural cells. Activation of Notch signaling in hematopoietic cells reduces colony formation and maintenance of hematopoiesis. These data suggest that Notch signaling plays a regulatory role in mesodermal development, cardiomyogenesis, the balanced generation of endothelial versus mural cells of blood vessels and hematopoietic development.