Commitment of mouse embryonic stem cells to the adipocyte lineage requires retinoic acid receptor beta and active GSK3

Key events leading to terminal differentiation of preadipocytes into adipocytes have been identified in recent years. However, signaling pathways involved in the decision of stem cells to follow the adipogenic lineage have not yet been characterized. We have previously shown that differentiating mouse embryonic stem (mES) cells give rise to functional adipocytes upon an early treatment with retinoic acid (RA). The goal of this work was to identify regulators of RA-induced commitment of mES cells to the adipocyte lineage. First, we investigated the role of RA receptor (RAR) isotypes in the induction of mES cell adipogenesis. Using synthetic retinoids selective of RAR isotypes, we show that RARbeta activation is both sufficient and necessary to trigger commitment of mES cells to adipocytes. Then, we performed a small-scale drug screening to find signaling pathways involved in RARbeta-induced mES cell adipogenesis. We show that pharmacological inhibitors of glycogen synthase kinase (GSK) 3, completely inhibit RARbeta-induced adipogenesis in mES cells. This finding uncovers the requirement of active GSK3 in RARbeta-induced commitment of mES cells toward the adipocyte lineage. Finally, we investigated the role of the Wnt pathway, in which GSK3 is a critical negative regulator, in adipocyte commitment by analyzing Wnt pathway activity in RA- and RARbeta-induced mES cell adipogenesis. Our results suggest that although RARbeta and active GSK3 are required for RA-induced adipogenesis, they might be acting through a Wnt pathway-independent mechanism.     

The influence of Leucine-rich amelogenin peptide on MSC fate by inducing Wnt10b expression

Amelogenin is the most abundant protein of the enamel organic matrix and is a structural protein indispensable for enamel formation. One of the amelogenin splicing isoforms, Leucine-rich Amelogenin Peptide (LRAP) induces osteogenesis in various cell types. Previously, we demonstrated that LRAP activates the canonical Wnt signaling pathway to induce osteogenic differentiation of mouse ES cells through the concerted regulation of Wnt agonists and antagonists. There is a reciprocal relationship between osteogenic and adipogenic differentiation in bone marrow mesenchymal stem cells (BMMSCs). Wnt10b-mediated activation of canonical Wnt signaling has been shown to regulate mesenchymal stem cell fate. Using the bipotential bone marrow stromal cell line ST2, we have demonstrated that LRAP activates the canonical Wnt/β-catenin signaling pathway. A specific Wnt inhibitor sFRP-1 abolishes the effect of LRAP on the stimulation of osteogenesis and the inhibition of adipogenesis of ST2 cells. LRAP treatment elevates the Wnt10b expression level whereas Wnt10b knockdown by siRNA abrogates the effect of LRAP. We show here that LRAP promotes osteogenesis of mesenchymal stem cells at the expense of adipogenesis through upregulating Wnt10b expression to activate Wnt signaling.     

Embryonic stem cell-derived adipogenesis

Key events leading to terminal differentiation of preadipocytes into adipocytes have been characterized in the recent years. However, master genes that commit progression from multipotent mesenchymal stem cell to the adipoblast stage of development have not yet been identified. The use of embryonic stem (ES) cells as a route to study early events in adipogenesis and to characterize factors involved in the decision of stem cells to follow the adipogenic pathway is described in this paper. The capacity of lif-/- and lifr-/- ES cells to undergo adipocyte differentiation is reported as an application of mutant ES cells to study gene function during the development of adipose cells.     

Hedgehog信号通路与淋巴细胞及干细胞分化

Hedgehog(Hh)蛋白是一种广泛分布的分泌蛋白,在机体的分化发育成熟中起着非常重要的作用.Hh蛋白通过与其膜受体结合从而激活胞内一系列的信号因子,包括第二信使Set-Thr蛋白激酶Fu、Fu抑制剂Su(Fu)、激酶样蛋白Cos-2和核转录因子Ci.Hh通路在淋巴细胞的发生发展过程中占重要地位,主要在T、B淋巴细胞...     

Wnt Signaling and the Control of Human Stem Cell Fate

Wnt signaling determines major developmental processes in the embryonic state and regulates maintenance, self-renewal and differentiation of adult mammalian tissue stem cells. Both β-catenin dependent and independent Wnt pathways exist, and both affect stem cell fate in developing and adult tissues. In this review, we debate the response to Wnt signal activation in embryonic stem cells and human, adult stem cells of mesenchymal, hematopoetic, intestinal, gastric, epidermal, mammary and neural lineages, and discuss the need for Wnt signaling in these cell types. Due to the vital actions of Wnt signaling in developmental and maintenance processes, deregulation of the pathway can culminate into a broad spectrum of developmental and genetic diseases, including cancer. The way in which Wnt signals can feed tumors and maintain cancer stem stells is discussed as well. Manipulation of Wnt signals both in vivo and in vitro thus carries potential for therapeutic approaches such as tissue engineering for regenerative medicine and anti-cancer treatment. Although many questions remain regarding the complete Wnt signal cell-type specific response and interplay of Wnt signaling with pathways such as BMP, Hedgehog and Notch, we hereby provide an overview of current knowledge on Wnt signaling and its control over human stem cell fate.     

Hedgehog信号通道与相关干细胞增殖与分化的研究进展

背景：Hedgehog蛋白为成形素蛋白,其相关信号通道参与胚胎发育和成体组织再生。 目的：对Hedgehog信号通道相关干细胞的增殖及分化研究近况进行综述。 方法：应用计算机检索CNKI和PubMed数据库中2000-01/2010-12关于Hedgehog信号通路的文章,在标题和摘要中以“hedgehog信号通道,细胞增殖,细胞分化,信号调控”或“Hedgehog signaling pathway,cell proliferation,cell differentiation,signal pathway controlling”为检索词进行检索,选择关于Hedgehog信号通道对各种干细胞及成体器官作用内容的文章,初检到169篇,根据纳入标准选择27篇进行综述。 结果与结论：具有多种分化潜能的干细胞发育成具有特定生物学功能的组织细胞是受到干细胞自身或外在的、近程或远程的信号调控的,其中细胞之间的相互信息传递在细胞发育分化过程中扮演着重要角色。Hedgehog 信号通路起到维持细胞增殖、分化、凋亡之间的平衡,其能够调节相邻细胞之间的分子差异,对细胞分化命运起决定性作用。     

A real-time PCR approach to evaluate adipogenic potential of amniotic fluid-derived human mesenchymal stem cells

Regulation of adipocyte differentiation is an important process in the control of adipose tissue development. So far, adipogenesis has been investigated through the use of various experimental models. In this work, we used human mesenchymal stem cells (hMSCs) obtained from amniotic fluid (AF) as an alternative model more representative of what naturally happens in vivo. In our opinion, these hMSCs are still not influenced by differentiation stimuli and could act in a way more correspondent to the physiological process of adipogenesis, representing also an ethically acceptable alternative to totipotent human embryonic stem cells (ES). Adipocyte differentiation was monitorated following the expressions of key genes. We measured the expression levels of PPARgamma2, PPARgamma-C1alpha, UCP-1, adipsin, and leptin genes using quantitative real-time PCR. We tested our experimental model with two different media. Understanding in vivo adipogenesis mechanisms will shed light on the pathophysiology of many diseases.     

调控骨髓间充质干细胞成骨分化的Wnt/β-catenin信号通路及相关因素

背景:骨髓间充质干细胞诱导成骨而抑制成脂分化是骨质疏松症防治的关键,也是骨组织修复工程种子细胞的来源,Wnt信号通路对骨形成起着重要作用.目的:综述Wnt/β-catenin信号通路调控骨髓间充质干细胞成骨分化的相关因素及分子机制.方法:应用计算机检索CNKI、PubMed及万方医学数据库建库至2020年2月发表的相关...     

Metaplastic change in mesenchymal stem cells induced by activated ras oncogene.

3T3 T murine mesenchymal stem cells have the potential to differentiate into a variety of different cell types even though they show a predilection to undergo adipocyte differentiation in vitro. The possibility that the activated c-Ha-ras (EJras) oncogene might influence the pathway of differentiation of these stem cells is investigated in the current study. Activated ras oncogene was transfected and stably expressed in 3T3 T cells; assays then were performed to determine its effect on differentiation. The results show that all EJras-transfected cell lines lose their ability to differentiate to adipocytes and instead differentiate into cells that express many characteristics of macrophages. Such cells contain numerous cytoplasmic granules, extensive nonspecific esterase activity, and anchorage-independent growth. The modulation of differentiation pathway from an adipocyte lineage to a macrophagelike cell lineage does not result from the transforming effect of EJras, because a nontransformed cell clone that expresses p21EJras protein also exhibits this modified differentiation pathway. These data suggest that the EJras oncogene specifically modulates the differentiation pathway of 3T3 T mesenchymal stem cells. This experimental system should therefore provide an excellent model to evaluate the mechanistic role of EJras in the process of metaplasia.     

Duration and magnitude of extracellular signal-regulated protein kinase phosphorylation determine adipogenesis or osteogenesis in human bone marrow-derived stem cells

Purpose

Imbalances between osteogenic and adipogenic differentiation leads to diseases such as osteoporosis. The aim of our study was to demonstrate the differences in extracellular signal-regulated kinase (ERK) phosphorylation during both adipogenesis and osteogenesis of human bone marrow-derived stem cells (BMSCs).

Materials and Methods

Using troglitazone, GW9662 and U0126, we investigated their role in hBMSC differentiation to adipogenic and osteogenic fates.

Results

ERK1/2 inhibition by U0126 suppressed proliferator-activated receptor (PPAR)γ expression and lipid accumulation, while it decreased the mRNA expression of adipogenic genes (lipoprotein lipase, PPARγ, and adipocyte protein) and osteogenic genes (type I collagen and osteopontin). ERK phosphorylation was transient and decreased during adipogenesis, whereas it occurred steadily during osteogenesis. Troglitazone, a PPARγ agonist, induced adipogenesis by inhibiting ERK phosphorylation even in an osteogenic medium, suggesting that ERK signaling needs to be shut off in order to proceed with adipose cell commitment. Cell proliferation was greatly increased in osteogenesis but was not changed during adipogenesis, indicating that ERK might play different roles in cellular proliferation and differentiation between the two committed cell types.

Conclusion

The duration and magnitude of ERK activation might be a crucial factor for the balance between adipogenesis and osteogenesis in human bone marrow-derived stem cells.     

Mediator MED23 plays opposing roles in directing smooth muscle cell and adipocyte differentiation

The Mediator complex functions as a control center, orchestrating diverse signaling, gene activities, and biological processes. However, how Mediator subunits determine distinct cell fates remains to be fully elucidated. Here, we show that Mediator MED23 controls the cell fate preference that directs differentiation into smooth muscle cells (SMCs) or adipocytes. Med23 deficiency facilitates SMC differentiation but represses adipocyte differentiation from the multipotent mesenchymal stem cells. Gene profiling revealed that the presence or absence of Med23 oppositely regulates two sets of genes: the RhoA/MAL targeted cytoskeleton/SMC genes and the Ras/ELK1 targeted growth/adipogenic genes. Mechanistically, MED23 favors ELK1–SRF binding to SMC gene promoters for repression, whereas the lack of MED23 favors MAL–SRF binding to SMC gene promoters for activation. Remarkably, the effect of MED23 on SMC differentiation can be recapitulated in zebrafish embryogenesis. Collectively, our data demonstrate the dual, opposing roles for MED23 in regulating the cytoskeleton/SMC and growth/adipogenic gene programs, suggesting its “Ying-Yang” function in directing adipogenesis versus SMC differentiation.     

Lentiviral vector gene transfer is limited by the proteasome at postentry steps in various types of stem cells

The isolation of human embryonic and somatic stem cells of different types has made it possible to design novel gene and cell replacement therapies. Vectors derived from retro/lentiviruses are used to stably introduce genes into stem cells and their progeny. However, the permissivity to retroviral infection varies among cell types. We previously showed that hematopoietic stem cells are poorly permissive to human immunodeficiency virus (HIV)-derived vectors and that pharmacological inhibition of the proteasome strongly enhances gene transfer. Here we report that the proteasome limits lentiviral gene transfer in all stem cell types tested, including embryonic, mesenchymal, and neural, of both human and mouse origin. Remarkably, this inhibitory activity was sharply reduced upon differentiation of the stem cells, suggesting that it represents a novel feature of the stem cell/immature progenitor phenotype. Proteasome-mediated inhibition was specific for lentiviral vectors and occurred at a postentry infection step. It was not mediated by activation of nuclear factor-kappaB, a major signaling pathway modulated by the proteasome, and did not correlate with high proteasome activity. Interaction of the virion core with cyclophilin A was required to maximize the effect of proteasome inhibitor on the infection pathway. These findings are relevant to uncover new mediators of HIV gene transfer and help in designing more effective protocols for the genetic modification of stem cells. Disclosure of potential conflicts of interest is found at the end of this article.     

Mediation of osteogenic differentiation of human mesenchymal stem cells on titanium surfaces by a Wnt-integrin feedback loop

Peri-implant bone formation depends on the ability of mesenchymal cells to colonize the implant surface and differentiate into osteoblasts. Human mesenchymal stem cells (HMSCs) undergo osteoblastic differentiation on microstructured titanium (Ti) surfaces in the absence of exogenous factors, but the mechanisms are unknown. Wnt proteins are associated with an osteoblast phenotype, but how Wnt signaling regulates HMSC differentiation on microstructured Ti surfaces is not known. HMSCs were cultured on tissue culture polystyrene or Ti (PT [Sa = 0.33 μm, θ = 96°], SLA [Sa = 2.5 μm, θ = 132°], modSLA [hydrophilic-SLA]). Expression of calcium-dependent Wnt ligand WNT5A increased and canonical Wnt pathway ligands decreased on microstructured Ti in a time-dependent manner. Treatment of HMSCs with canonical ligand Wnt3a preserved the mesenchymal phenotype on smooth surfaces. Treatment with Wnt5a increased osteoblastic differentiation. Expression of integrins ITGA1, ITGA2, and ITGAV increased over time and correlated with increased WNT5A expression. Treatment of HMSCs with Wnt5a, but not Wnt3a, increased integrin expression. Regulation of integrin expression due to surface roughness and energy was ablated in WNT5A-knockdown HMSCs. This indicates that surface properties regulate stem cell fate and induce osteoblast differentiation via the Wnt calcium-dependent pathway. Wnt5a enhances osteogenesis through a positive feedback with integrins and local factor regulation, particularly though BMP signaling.