基因谱系示踪揭示小鼠Tbx18+肾脏间质祖细胞分化为输尿管平滑肌

本文旨在研究Tbx18+肾脏间质祖细胞分化为输尿管平滑肌细胞的命运及转录因子Tbx18在小鼠输尿管平滑肌发育形成中起到的作用．实验建立Tbx18:Cre/R26REYFP和Tbx18:Cre/R26RLacZ两种谱系示踪系统和Tbx18:Cre/Cre 敲除模型．该示踪模型通过cre重组酶的表达能有效地示踪Tbx18+肾脏间质祖细胞在泌尿系统的发育命运．通过免疫荧光染色和X-gal染色,同时发现Tbx18+肾脏间质祖细胞可分化为输尿管平滑肌细胞,但不分化为输尿管移行上皮细胞．在Tbx18:Cre/Cre基因突变模型中,泌尿系统出现明显的肾积水和输尿管积水,肾盏、肾盂扩张,输尿管明显缩短和扩张．实验结果揭示,Tbx18+ 肾脏间质祖细胞可以分化为输尿管平滑肌细胞,且转录因子Tbx18在哺乳动物输尿管平滑肌的发育中起到重要的作用．     

Tbx18 is essential for normal development of vasculature network and glomerular mesangium in the mammalian kidney

Tbx18 has been shown to be essential for ureteral development. However, it remains unclear whether it plays a direct role in kidney development. Here we addressed this by focusing on examining the pattern and contribution of Tbx18+ cells in the kidney and its role in kidney vascular development. Expression studies and genetic lineage tracing revealed that Tbx18 is expressed in renal capsule, vascular smooth muscle cells and pericytes and glomerular mesangial cells in the kidney and that Tbx18-expressing progenitors contribute to these cell types. Examination of Tbx18^−/− kidneys revealed large reduction in vasculature density and dilation of glomerular capillary loops. While SMA+ cells were reduced in the mutant, PDGFRβ+ cells were seen in early capillary loop renal corpuscles in the mutant, but fewer than in the controls, and further development of the mesangium failed. Analysis of kidney explants cultured from E12.5 excluded the possibility that the defects observed in the mutant were caused by ureter obstruction. Reduced proliferation in glomerular tuft and increased apoptosis in perivascular mesenchyme were observed in Tbx18^−/− kidneys. Thus, our analyses have identified a novel role of Tbx18 in kidney vasculature development.     

Muscle-derived stem cells differentiate into functional smooth muscle cells for ureter tissue engineering: An experimental study

We assessed the ability of muscle-derived stem cells (MDSC) to differentiate into smooth muscle cells (SMC) and their potential to promote the regeneration of smooth muscle with a vessel extracellular matrix (VECM) for tissue engineering of the ureter. MDSC were isolated, proliferated, and identified by flow cytometry. SMC phenotype differentiation was induced with a smooth muscle induction medium. Gene expression was evaluated by real-time quantitative polymerase chain reaction (PCR) and Western blot studies. The VECM was obtained by a decellularization process, and cytotoxic effects were evaluated by exposing the induced cells to a VECM extract. The induced cells were seeded onto VECM in vitro for 1 week, and then the compound grafts were used for ureter reconstitution in vivo. The grafts were obtained for histological studies at 2, 4, 8, and 16 weeks post-operation. Intravenous urography was used to evaluate renal function and ureteral patency. Flow cytometry demonstrated that the MDSC expressed Sca-1 and desmin, but did not express CD45. After induction, SMC phenotype gene expression was confirmed in the induced cells by real-time quantitative PCR and Western blot studies. VECM exhibited a nontoxic effect on the induced cells in vitro. At 16 weeks post-operation, a histological evaluation showed that multilayered urothelium and organized muscle fiber bundles had formed in the grafts. Intravenous urography demonstrated no evidence of ureteral stricture or hydroureteronephrosis. These results demonstrate that MDSC can be induced into SMC and that this was useful for promoting regeneration of smooth muscles for ureter tissue engineering.     

Canonical Wnt signaling regulates smooth muscle precursor development in the mouse ureter

Smooth muscle cells (SMCs) are a key component of many visceral organs, including the ureter, yet the molecular pathways that regulate their development from mesenchymal precursors are insufficiently understood. Here, we identified epithelial Wnt7b and Wnt9b as possible ligands of Fzd1-mediated β-catenin (Ctnnb1)-dependent (canonical) Wnt signaling in the adjacent undifferentiated ureteric mesenchyme. Mice with a conditional deletion of Ctnnb1 in the ureteric mesenchyme exhibited hydroureter and hydronephrosis at newborn stages due to functional obstruction of the ureter. Histological analysis revealed that the layer of undifferentiated mesenchymal cells directly adjacent to the ureteric epithelium did not undergo characteristic cell shape changes, exhibited reduced proliferation and failed to differentiate into SMCs. Molecular markers for prospective SMCs were lost, whereas markers of the outer layer of the ureteric mesenchyme fated to become adventitial fibroblasts were expanded to the inner layer. Conditional misexpression of a stabilized form of Ctnnb1 in the prospective ureteric mesenchyme resulted in the formation of a large domain of cells that exhibited histological and molecular features of prospective SMCs and differentiated along this lineage. Our analysis suggests that Wnt signals from the ureteric epithelium pattern the ureteric mesenchyme in a radial fashion by suppressing adventitial fibroblast differentiation and initiating smooth muscle precursor development in the innermost layer of mesenchymal cells.     

Sonic hedgehog regulates proliferation and differentiation of mesenchymal cells in the mouse metanephric kidney

Signaling by the ureteric bud epithelium is essential for survival, proliferation and differentiation of the metanephric mesenchyme during kidney development. Most studies that have addressed ureteric signaling have focused on the proximal, branching, ureteric epithelium. We demonstrate that sonic hedgehog is expressed in the ureteric epithelium of the distal, non-branching medullary collecting ducts and continues into the epithelium of the ureter -- the urinary outflow tract that connects the kidney with the bladder. Upregulation of patched 1, the sonic hedgehog receptor and a downstream target gene of the signaling pathway in the mesenchyme surrounding the distal collecting ducts and the ureter suggests that sonic hedgehog acts as a paracrine signal. In vivo and in vitro analyses demonstrate that sonic hedgehog promotes mesenchymal cell proliferation, regulates the timing of differentiation of smooth muscle progenitor cells, and sets the pattern of mesenchymal differentiation through its dose-dependent inhibition of smooth muscle formation. In addition, we also show that bone morphogenetic protein 4 is a downstream target gene of sonic hedgehog signaling in kidney stroma and ureteral mesenchyme, but does not mediate the effects of sonic hedgehog in the control of mesenchymal proliferation.     

MicroRNA-145-based differentiation of human mesenchymal stem cells to smooth muscle cells

Objectives

To investigate the role of microRNA-145, that regulates gene expression of genes related to differentiation, proliferation and the phenotype of smooth muscle cells (SMCs), in the differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) to SMCs.

Results

Real-time PCR analysis indicated significant upregulation of SMC markers, including SM-α-actin, calponin, caldesmon and SMMHC, in SMCs compared to hBM-MSCs. Conversely, Krüppel-like factor 4, the direct target of microRNA-145 and the suppressor of smooth muscle differentiation, was suppressed in hBM-MSC-derived SMCs. Western blot analysis and immunocytochemistry also confirmed that the introduction of microRNA-145 into hBM-MSCs induced mature contractile SMCs. The functionality of hBM-MSC-derived SMCs was assessed by proliferation assay using PDGF-BB and contractility assay using carbachol. The results showed that the produced SMCs contracted in response to carbachol stimulation.

Conclusion

Overexpression of microRNA-145 in undifferentiated hBM-MSCs results in functionally mature contractile SMCs that can be used in drug discovery and cell therapy in SMC disorders such as vascular disease.

     

Sodium Butyrate Promotes the Differentiation of Rat Bone Marrow Mesenchymal Stem Cells to Smooth Muscle Cells through Histone Acetylation

Establishing an effective method to improve stem cell differentiation is crucial in stem cell transplantation. Here we aimed to explore whether and how sodium butyrate (NaB) induces rat bone marrow mesenchymal stem cells (MSCs) to differentiate into bladder smooth muscle cells (SMCs). We found that NaB significantly suppressed MSC proliferation and promoted MSCs differentiation into SMCs, as evidenced by the enhanced expression of SMC specific genes in the MSCs. Co-culturing the MSCs with SMCs in a transwell system promoted the differentiation of MSCs into SMCs. NaB again promoted MSC differentiation in this system. Furthermore, NaB enhanced the acetylation of SMC gene-associated H3K9 and H4, and decreased the expression of HDAC2 and down-regulated the recruitment of HDAC2 to the promoter regions of SMC specific genes. Finally, we found that NaB significantly promoted MSC depolarization and increased the intracellular calcium level of MSCs upon carbachol stimulation. These results demonstrated that NaB effectively promotes MSC differentiation into SMCs, possibly by the marked inhibition of HDAC2 expression and disassociation of HDAC2 recruitment to SMC specific genes in MSCs, which further induces high levels of H3K9^ace and H4^ace and the enhanced expression of target genes, and this strategy could potentially be applied in clinical tissue engineering and cell transplantation.     

Activation of PPAR-α induces cell cycle arrest and inhibits transforming growth factor-β1 induction of smooth muscle cell phenotype in 10T1/2 mesenchymal cells

Transforming growth factor-β1 (TGF-β1) regulates the cell cycle and the differentiation of mesenchymal cells into smooth muscle cells (SMCs). However, the precise intracellular signaling pathways involved in these processes have not been fully clarified. It has also been shown that there is an increase in TGF-β1 expression in human atherosclerotic plaques. Furthermore, peroxisome proliferator-activated receptors (PPARs) and their agonists have recently gained more attention in the study of the pathogenesis of atherosclerosis. In this study, we examined the role of PPARs in the TGF-β1-mediated cell cycle control and SMC phenotypic modulation of C3H10T1/2 (10T1/2) mesenchymal cells. The results showed the following: (1) the PI3K/Akt/p70S6K signaling cascade is involved in TGF-β1-induced differentiation of 10T1/2 cells into cells with a SMC phenotype. (2) PPAR-α agonists (i.e., WY14,643 and clofibrate), but not a PPAR-δ/β agonist (GW501516) or PPAR-γ agonist (troglitazone), inhibit TGF-β1-induced SMC markers and the DNA binding activity of serum response factor (SRF) in 10T1/2 cells. (3) WY14,643 and clofibrate inhibit the TGF-β1 activation of the Smad3/Akt/P70S6K signaling cascade. (4) TGF-β1-induced cell cycle arrest at the G₀/G₁ phases is mediated by Smad3 in 10T1/2 cells. (5) The PPAR-α-mediated 10T1/2 cell cycle arrest at the G₀/G₁ phases is TGF-β receptor independent. These results suggest that PPAR-α mediates cell cycle control and TGF-β1-induced SMC phenotypic changes in 10T1/2 cells.     

An Atypical Canonical Bone Morphogenetic Protein (BMP) Signaling Pathway Regulates Msh Homeobox 1 (Msx1) Expression during Odontogenesis

Bone morphogenetic protein (BMP) signaling plays an essential role in early tooth development, evidenced by disruption of BMP signaling leading to an early arrested tooth development. Despite being a central mediator of BMP canonical signaling pathway, inactivation of Smad4 in dental mesenchyme does not result in early developmental defects. In the current study, we investigated the mechanism of receptor-activated Smads (R-Smads) and Smad4 in the regulation of the odontogenic gene Msx1 expression in the dental mesenchyme. We showed that the canonical BMP signaling is not operating in the early developing tooth, as assessed by failed activation of the BRE-Gal transgenic allele and the absence of phospho-(p)Smad1/5/8-Smad4 complexes. The absence of pSmad1/5/8-Smad4 complex appeared to be the consequence of saturation of Smad4 by pSmad2/3 in the dental mesenchyme as knockdown of Smad2/3 or overexpression of Smad4 led to the formation of pSmad1/5/8-Smad4 complexes and activation of canonical BMP signaling in dental mesenchymal cells. We showed that Smad1/5 but not Smad4 are required for BMP-induced expression of Msx1 in dental mesenchymal cells. We further presented evidence that in the absence of Smad4, BMPs are still able to induce pSmad1/5/8 nuclear translocation and their binding to the Msx1 promoter directly in dental mesenchymal cells. Our results demonstrate the functional operation of an atypical canonical BMP signaling (Smad4-independent and Smad1/5/8-dependent) pathway in the dental mesenchyme during early odontogenesis, which may have general implication in the development of other organs.     

Tbx18通过下调EMT信号分子参与调控心脏发育

转录因子Tbx18(Tbx18)在小鼠胚胎心外膜上皮细胞表达并调控心外膜上皮细胞向心系细胞分化.上皮间充质转化(EMT)过程是器官发育和形成的重要机制.为阐述Tbx18通过调控下游EMT关键信号分子参与心外膜上皮细胞分化和心脏发育,本研究运用Tbx18-Cre/Rosa26R-EYFP双杂合基因敲入小鼠和免疫荧光共聚焦,证实Tbx18+心系细胞和EMT关键信号分子Snail1、Smad、Slug、Twist在发育后期胚鼠心外膜和心外膜下间充质发生共聚焦.同时还发现,Tbx18在胚鼠不同发育阶段的表达模式和Tbx18+心系细胞内上述EMT关键信号分子的表达模式相似.Tbx18和EMT关键信号分子在发育心脏存在相似的时空表达模式,因此,它们之间可能存在相互调控作用.运用Tbx18突变技术揭示了Tbx18突变型胚鼠心脏EMT关键信号分子表达水平均较野生型显著下调,直接证实了上述4个EMT信号分子是Tbx18的可能靶点.理解Tbx18参与心脏发育的下游靶点有助于改善成年心脏损伤后的再生修复.