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.     

诱导体外培养的骨髓间充质干细胞向心肌样细胞的分化

选用Wistar大鼠分离骨髓间充质干细胞作体外培养及鉴定其表达抗原CD44、CDw90;采用10μmol／L 5-氮胞苷诱导第1代的骨髓间充质干细胞,于诱导后2、4周进行免疫细胞化学反应检测α-横纹肌肌动蛋白、肌钙蛋白T。证实体外培养的第1代骨髓间充质干细胞经5-氮胞苷诱导可分化为心肌样细胞,为指导体外诱导的心肌细胞应用于。临床提供一定的理论依据和技术手段。     

诱导小鼠胚胎干细胞在体外分化为骨骼肌细胞的实验研究

小鼠胚胎干细胞是从胚泡未分化的内部细胞团中得到的干细胞,它在体外培养的环境中具有无限增殖、自我更新以及多向分化的特性。将小鼠胚胎干细胞在体外诱导分化为肌肉细胞,并且利用这些分化得来的肌肉细胞治疗肌肉退行性疾病,是干细胞研究领域的热点。该实验的目的在于筛选小鼠胚胎干细胞向骨骼肌细胞定向分化的实验条件,有效地将体外单层贴壁培养的小鼠胚胎干细胞诱导分化成骨骼肌细胞。最终发现,10-8mol/L维甲酸(retinoid acid,RA)+0.5%二甲基亚砜(dimethyl sulfoxide,DMSO)组诱导小鼠胚胎干细胞在体外分化成骨骼肌前体细胞的效率最高,分化得到的骨骼肌前体细胞经进一步纯化,能分化为多核的肌管。该实验为治疗肌肉退行性疾病提供了细胞来源,也为研究小鼠胚胎干细胞分化为骨骼肌细胞的机制提供了有利的条件。     

Adipose Stem Cells Promote Smooth Muscle Cells to Secrete Elastin in Rat Abdominal Aortic Aneurysm

Background

Abdominal aortic aneurysm (AAA) is a life-threatening disease and its prevalence rate increases with social aging. The degradation of elastic is an important factor in the formation of AAA.

Methods

Adipose derived stem cells (ADSCs) and bone marrow mesenchymal stem cells (BMSCs) were isolated from rats, and identified by Oil red O and alizarin red staining after adipogenesis and osteogenesis induction. In addition, ADSCs were also identified by flow cytometry with CD markers. AAA model in rats was established, and smooth muscle cells (SMCs) were isolated from AAA aortic wall and identified by immunohistochemistry. ADSCs or BMSCs were co-cultured with AAA aortic wall for in vitro experiment, and ADSCs were injected into AAA model for in vivo test. Then orcein staining was used for observing the morphology of elastic fiber, Western blot and real-time PCR were used respectively to detect the protein and gene expression of elastin, gelatinases spectrum analysis was used to detect the activity of matrix metalloproteinase-2 (MMP-2) and MMP-9.

Results

Lots of red lipid droplets were visible by Oil red O staining after adipogenesis induction, and black calcium nodules appeared by alizarin red staining after osteogenesis induction. The results of flow cytometry showed that ADSCs expressed CD44 and CD105, but exhibited negligible expression of CD31 and CD45. SMCs exhibited spindle-like morphology and α-actin protein was positive in cytoplasm. After co-cultured with ADSCs or BMSCs, the elastic fiber recovered normal winding shape, both the gene and protein expression of elastin increased, and the activity of MMP-2 decreased. The in vivo result was similar to that of in vitro.

Conclusions

ADSCs promote the expression of elastin in SMCs and contribute to the reconstruction of elastic fiber, which may provide new ideas for treating AAA.     

Attenuation of Hind-Limb Ischemia in Mice with Endothelial-Like Cells Derived from Different Sources of Human Stem Cells

Functional endothelial-like cells (EC) have been successfully derived from different cell sources and potentially used for treatment of cardiovascular diseases; however, their relative therapeutic efficacy remains unclear. We differentiated functional EC from human bone marrow mononuclear cells (BM-EC), human embryonic stem cells (hESC-EC) and human induced pluripotent stem cells (hiPSC-EC), and compared their in-vitro tube formation, migration and cytokine expression profiles, and in-vivo capacity to attenuate hind-limb ischemia in mice. Successful differentiation of BM-EC was only achieved in 1/6 patient with severe coronary artery disease. Nevertheless, BM-EC, hESC-EC and hiPSC-EC exhibited typical cobblestone morphology, had the ability of uptaking DiI-labeled acetylated low-density-lipoprotein, and binding of Ulex europaeus lectin. In-vitro functional assay demonstrated that hiPSC-EC and hESC-EC had similar capacity for tube formation and migration as human umbilical cord endothelial cells (HUVEC) and BM-EC (P>0.05). While increased expression of major angiogenic factors including epidermal growth factor, hepatocyte growth factor, vascular endothelial growth factor, placental growth factor and stromal derived factor-1 were observed in all EC cultures during hypoxia compared with normoxia (P<0.05), the magnitudes of cytokine up-regulation upon hypoxic were more dramatic in hiPSC-EC and hESC-EC (P<0.05). Compared with medium, transplanting BM-EC (n = 6), HUVEC (n = 6), hESC-EC (n = 8) or hiPSC-EC (n = 8) significantly attenuated severe hind-limb ischemia in mice via enhancement of neovascularization. In conclusion, functional EC can be generated from hECS and hiPSC with similar therapeutic efficacy for attenuation of severe hind-limb ischemia. Differentiation of functional BM-EC was more difficult to achieve in patients with cardiovascular diseases, and hESC-EC or iPSC-EC are readily available as “off-the-shelf” format for the treatment of tissue ischemia.     

Mechanisms of Propagation of Intercellular Calcium Waves in Arterial Smooth Muscle Cells

In rat mesenteric arteries, smooth muscle cells exhibit intercellular calcium waves in response to local phenylephrine stimulation. These waves have a velocity of ∼20 cells/s and a range of ∼80 cells. We analyze these waves in a theoretical model of a population of coupled smooth muscle cells, based on the hypothesis that the wave results from cell membrane depolarization propagation. We study the underlying mechanisms and highlight the importance of voltage-operated channels, calcium-induced calcium release, and chloride channels. Our model is in agreement with experimental observations, and we demonstrate that calcium waves presenting a velocity of ∼20 cells/s can be mediated by electrical coupling. The wave velocity is limited by the time needed for calcium influx through voltage-operated calcium channels and the subsequent calcium-induced calcium release, and not by the speed of the depolarization spreading. The waves are partially regenerated, but have a spatial limit in propagation. Moreover, the model predicts that a refractory period of calcium signaling may significantly affect the wave appearance.     

Human Decidua-Derived Mesenchymal Stem Cells Differentiate into Functional Alveolar Type II-Like Cells that Synthesize and Secrete Pulmonary Surfactant Complexes

Lung alveolar type II (ATII) cells are specialized in the synthesis and secretion of pulmonary surfactant, a lipid-protein complex that reduces surface tension to minimize the work of breathing. Surfactant synthesis, assembly and secretion are closely regulated and its impairment is associated with severe respiratory disorders. At present, well-established ATII cell culture models are not available. In this work, Decidua-derived Mesenchymal Stem Cells (DMSCs) have been differentiated into Alveolar Type II- Like Cells (ATII-LCs), which display membranous cytoplasmic organelles resembling lamellar bodies, the organelles involved in surfactant storage and secretion by native ATII cells, and accumulate disaturated phospholipid species, a surfactant hallmark. Expression of characteristic ATII cells markers was demonstrated in ATII-LCs at gene and protein level. Mimicking the response of ATII cells to secretagogues, ATII-LCs were able to exocytose lipid-rich assemblies, which displayed highly surface active capabilities, including faster interfacial adsorption kinetics than standard native surfactant, even in the presence of inhibitory agents. ATII-LCs could constitute a highly useful ex vivo model for the study of surfactant biogenesis and the mechanisms involved in protein processing and lipid trafficking, as well as the packing and storage of surfactant complexes.     

间充质干细胞与肿瘤的关系

最近的研究表明问充质干细胞(mesenchymal stem cells,MSCs)与多种肿瘤的发生发展有密切关系.MSCs对多种肿瘤具有趋向性,外源性(局部混合注射或静脉注射)MSCs可参与肿瘤间质的形成,同时MSCs的免疫抑制作用可以促进肿瘤在体内的生长.通过细胞因子介导或直接的细胞接触,MSCs与多种肿瘤细胞之间存在相互作用.MSCs可以抑制肿瘤细胞的的凋亡,促进肿瘤细胞的增殖及肿瘤的转移.由于MSCs易于分离、体外扩增及进行基因修饰,因此可以利用MSCs对肿瘤的趋向性,使MSCs携带抗肿瘤基因来实现对肿瘤的靶向治疗.     

Wnt3a诱导小鼠胚胎干细胞向心肌细胞分化的研究

目的：研究Wnt3a在诱导小鼠胚胎干细胞心肌细胞分化中的作用和原理。方法：设计不同浓度,不同成分的Wnt3a条件培养基对小鼠胚胎干细胞诱导分化,对分化细胞进行形态学鉴定,通过免疫细胞化学检测心肌肌钙蛋白-T（cTnT）的表达,通过RT．PCR检测肌球蛋白重链（ot．MHC）和肌球蛋白轻链（MLC．2v）的表达。结果：Wnt3a诱导小鼠胚胎干细胞分化为心肌样细胞,分化细胞具有自动收缩性,免疫细胞化学检测心肌肌钙蛋白．T（cTllT）表达阳性,RT．PCR检测肌球蛋白重链（d—MHC）和肌球蛋白轻链（MLC-2v）表达阳性。经典Wnt信号途径的抑制剂Frizzled一8／Fc,能够抑制Wnt3a的诱导分化作用。结论：Wnt3a通过经典Wnt信号途径诱导小鼠胚胎干细胞向心肌细胞分化。     

平滑肌细胞骨架结构及其信号调节途径

平滑肌细胞骨架是一个复杂的动态性网络,是细胞生命活动不可缺少的细胞结构。Rho通过活化其下游靶分子促进应力纤维的形成,其中Rho—associated coiled—coil kinase(ROCK)和Dial在该过程中起关键作用;PKC通过在细胞内不同定位的亚型使细胞骨架蛋白磷酸化,发挥其调节细胞骨架重构的作用。两条信号转导途径通过Src途径相互联系,共同参与细胞骨架动力学的调节。     

Reconstruction of Beagle Hemi-Mandibular Defects with Allogenic Mandibular Scaffolds and Autologous Mesenchymal Stem Cells

Objective

Massive bone allografts are frequently used in orthopedic reconstructive surgery, but carry a high failure rate of approximately 25%. We tested whether treatment of graft with mesenchymal stem cells (MSCs) can increase the integration of massive allografts (hemi-mandible) in a large animal model.

Methods

Thirty beagle dogs received surgical left-sided hemi-mandibular defects, and then divided into two equal groups. Bony defects of the control group were reconstructed using allografts only. Those of the experimental group were reconstructed using allogenic mandibular scaffold-loaded autologous MSCs. Beagles from each group were killed at4 (n = 4), 12 (n = 4), 24 (n = 4) or 48 weeks (n = 3) postoperatively. CT and micro-CT scans, histological analyses and the bone mineral density (BMD) of transplants were used to evaluate defect reconstruction outcomes.

Results

Gross and CT examinations showed that the autologous bone grafts had healed in both groups. At 48 weeks, the allogenic mandibular scaffolds of the experimental group had been completely replaced by new bone, which has a smaller surface area to that of the original allogenic scaffold, whereas the scaffold in control dogs remained the same size as the original allogenic scaffold throughout. At 12 weeks, the BMD of the experimental group was significantly higher than the control group (p<0.05), and all micro-architectural parameters were significantly different between groups (p<0.05). Histological analyses showed almost all transplanted allogeneic bone was replaced by new bone, principally fibrous ossification, in the experimental group, which differed from the control group where little new bone formed.

Conclusions

Our study demonstrated the feasibility of MSC-loaded allogenic mandibular scaffolds for the reconstruction of hemi-mandibular defects. Further studies are needed to test whether these results can be surpassed by the use of allogenic mandibular scaffolds loaded with a combination of MSCs and osteoinductive growth factors.     

食蟹猴胚胎干细胞向间充质前体细胞诱导分化及鉴定

间充质干细胞(mesenchymal stem cells,MSCs)可以诱导分化成脂肪、软骨、骨骼和骨骼肌细胞,并可作为骨骼、软骨或肌肉移植中的再生干细胞,广泛应用于细胞治疗和组织工程。胚胎干细胞(embryonic stem cells,ESCs)具有体外培养无限增殖和多向分化的特性,能被诱导分化为机体几乎所有的细胞类型。该研究通过无血清条件下诱导食蟹猴ESCs形成类胚体(embryoid bodies,EBs),然后在血清条件下贴壁分化EBs成间充质前体细胞(mesenchymal precursor cells,MPCs),再经过长期体外培养,纯化和扩增MPCs。结果显示,纯化后的MPCs具有MSCs生物学特征,并能在体外诱导分化成脂肪细胞和骨细胞。将这些细胞皮下注射给SCID小鼠,并未发现形成肿瘤,提示食蟹猴ESCs来源的MPCs具有一定的安全性。     

间充质干细胞表达趋化因子及受体的相关生物学功能

间充质干细胞(mesenchymal stem cells,MSCs)是一群存在于骨髓间质和其他组织间质的干细胞,表达CD34和CD133.近来研究发现,存在于骨髓的间充质干细胞除了能支持造血,向骨细胞、软骨细胞和脂肪细胞进行多向分化外,其分泌的趋化因子及其相关受体在MSCs的信号转导、维持内环境的稳定、损伤修复、免疫调节、支持造血等功能中也发挥了关键性的作用.     

Ecto-Mesenchymal Stem Cells from Facial Process: Potential for Muscle Regeneration

Ecto-mesenchymal stem cells (EMSCs) originate from the cranial neural crest and participate in the formation of tooth, salivary, and muscle in early development stage. The transplantation of EMSCs, a potential source of myoblast stem cell, might improve muscle regeneration. The purpose of this study was to explore whether EMSCs have the potential to differentiate and display a myogenic phenotype in vitro the in vitro. Here, we characterized the EMSCs isolated from the facial process, and p75 + EMSCs were collected by a FACS calibur flow cytometer. In vitro, p75 + EMSCs induced by DMSO can accumulate and fuse into multinucleated myotubes and further differentiate into the skeletal muscle cells in form of cell sheet. Functional myoblast phenotypes of p75 + EMSCs were found in vivo model of muscle injury. The remarkable ability of stem cells to regenerate skeletal muscle indicated their potential role in the cell therapy and tissue engineering of the skeletal muscle.     

An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug – clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy.     

Selective Development of Myogenic Mesenchymal Cells from Human Embryonic and Induced Pluripotent Stem Cells

Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are promising sources for the cell therapy of muscle diseases and can serve as powerful experimental tools for skeletal muscle research, provided an effective method to induce skeletal muscle cells is established. However, the current methods for myogenic differentiation from human ES cells are still inefficient for clinical use, while myogenic differentiation from human iPS cells remains to be accomplished. Here, we aimed to establish a practical differentiation method to induce skeletal myogenesis from both human ES and iPS cells. To accomplish this goal, we developed a novel stepwise culture method for the selective expansion of mesenchymal cells from cell aggregations called embryoid bodies. These mesenchymal cells, which were obtained by dissociation and re-cultivation of embryoid bodies, uniformly expressed CD56 and the mesenchymal markers CD73, CD105, CD166, and CD29, and finally differentiated into mature myotubes in vitro. Furthermore, these myogenic mesenchymal cells exhibited stable long-term engraftment in injured muscles of immunodeficient mice in vivo and were reactivated upon subsequent muscle damage, increasing in number to reconstruct damaged muscles. Our simple differentiation system facilitates further utilization of ES and iPS cells in both developmental and pathological muscle research and in serving as a practical donor source for cell therapy of muscle diseases.     

Mesenchymal Stem Cells Exhibit Regulated Exocytosis in Response to Chemerin and IGF

Mesenchymal stem cells (MSCs) play important roles in tissue repair and cancer progression. Our recent work suggests that some mesenchymal cells, notably myofibroblasts exhibit regulated exocytosis resembling that seen in neuroendocrine cells. We now report that MSCs also exhibit regulated exocytosis. Both a G-protein coupled receptor agonist, chemerin, and a receptor tyrosine kinase stimulant, IGF-II, evoked rapid increases in secretion of a marker protein, TGFβig-h3. The calcium ionophore, ionomycin, also rapidly increased secretion of TGFβig-h3 while inhibitors of translation (cycloheximide) or secretory protein transport (brefeldin A) had no effect, indicating secretion from preformed secretory vesicles. Inhibitors of the chemerin and IGF receptors specifically reduced the secretory response. Confocal microscopy of MSCs loaded with Fluo-4 revealed chemerin and IGF-II triggered intracellular Ca²⁺ oscillations requiring extracellular calcium. Immunocytochemistry showed co-localisation of TGFβig-h3 and MMP-2 to secretory vesicles, and transmission electron-microscopy showed dense-core secretory vesicles in proximity to the Golgi apparatus. Proteomic studies on the MSC secretome identified 64 proteins including TGFβig-h3 and MMP-2 that exhibited increased secretion in response to IGF-II treatment for 30min and western blot of selected proteins confirmed these data. Gene ontology analysis of proteins exhibiting regulated secretion indicated functions primarily associated with cell adhesion and in bioassays chemerin increased adhesion of MSCs and adhesion, proliferation and migration of myofibroblasts. Thus, MSCs exhibit regulated exocytosis that is compatible with an early role in tissue remodelling.     

Strategies to Rescue Mesenchymal Stem Cells (MSCs) and Dental Pulp Stem Cells (DPSCs) from NK Cell Mediated Cytotoxicity

Background

The aim of this paper is to study the function of allogeneic and autologous NK cells against Dental Pulp Stem Cells (DPSCs) and Mesenchymal Stem Cells (MSCs) and to determine the function of NK cells in a three way interaction with monocytes and stem cells.

Methodology/Principal Findings

We demonstrate here that freshly isolated untreated or IL-2 treated NK cells are potent inducers of cell death in DPSCs and MSCs, and that anti-CD16 antibody which induces functional split anergy and apoptosis in NK cells inhibits NK cell mediated lysis of DPSCs and MSCs. Monocytes co-cultured with either DPSCs or MSCs decrease lysis of stem cells by untreated or IL-2 treated NK cells. Monocytes also prevent NK cell apoptosis thereby raising the overall survival and function of NK cells, DPSCs or MSCs. Both total population of monocytes and those depleted of CD16⁺ subsets were able to prevent NK cell mediated lysis of MSCs and DPSCs, and to trigger an increased secretion of IFN-γ by IL-2 treated NK cells. Protection of stem cells from NK cell mediated lysis was also seen when monocytes were sorted out from stem cells before they were added to NK cells. However, this effect was not specific to monocytes since the addition of T and B cells to stem cells also protected stem cells from NK cell mediated lysis. NK cells were found to lyse monocytes, as well as T and B cells.

Conclusion/Significance

By increasing the release of IFN-γ and decreasing the cytotoxic function of NK cells monocytes are able to shield stem cells from killing by the NK cells, resulting in an increased protection and differentiation of stem cells. More importantly studies reported in this paper indicate that anti-CD16 antibody can be used to prevent NK cell induced rejection of stem cells.     

蜗轴螺旋动脉平滑肌的培养及其鉴定

目的:本研究通过联合两种常用的细胞培养方法,建立了一个蜗轴螺旋动脉平滑肌细胞原代培养的模型.方法:分离的蜗轴螺旋动脉的平滑肌来自于豚鼠.切碎血管组织并且将其放入37度的0.1%胰蛋白酶溶液中消化20分钟.消化后,将这些消化后的组织块在35-mm的培养皿中贴壁.运用这种培养方法,混杂的成纤维细胞通过与血管平滑肌细胞不同的贴壁能力,可在传代的时候被去除.在7-10天后,细胞从组织块中长出来.大约3周,细胞可长满培养皿.在第三代培养的血管平滑肌细胞中,纯的并且活性好的细胞可以被活得.经过形态学,免疫荧光化学和电镜对这种方法得到的血管平滑肌细胞进行了鉴别.结果:显示了典型的平滑肌的细胞的特点:形态学的"峰-谷"样的生长形态,免疫荧光化学显示这些细胞表达平滑肌细胞的特异性标志物(α-SM-actin和myosin.结论:通过本文研究方法得到的大量的纯的蜗轴螺旋动脉血管平滑肌细胞是一个很好的研究血管平滑肌细胞在内耳循环紊乱过程生理功能的一个体外模型.除此之外,还可以是一些作用于血管平滑肌药物评价的体外模型.