骨髓间充质干细胞向肝细胞分化的相关细胞因子及信号通路的研究进展

骨髓干细胞包括造血干细胞（HSCs）和间充质干细胞（MSCs）,骨髓间充质干细胞（BMSCs）是一类具有自我更新、增殖和多向分化能力的细胞,具有不对称分裂和无限增殖的特点。在肝细胞生长因子（HGF）的作用下,BMSCs可以分化为肝细胞,参与诱导这一分化过程的相关信号通路包括NF-kB信号通路、Notch信号通路、MAPK信号通路、Wnt信号通路和STAT3信号通路。文章主要就BMSCs分化为肝细胞的相关信号通路进行了综述。     

Bone marrow-derived mesenchymal stem cells already express specific neural proteins before any differentiation

Bone marrow mesenchymal stem cells (MSC) are multipotent cells. To explain their plasticity, we postulated that undifferentiated MSC may express proteins from other tissues such as neuronal tissues. MSC are obtained by two different approaches: plastic adhesion or negative depletion (RosetteSep and magnetic beads CD45/glycophorin A). MSC are evaluated through FACS analysis using a panel of antibodies (SH2, SH3, CD14, CD33, CD34, CD45, etc.). To confirm the multipotentiality in vitro, we have differentiated MSC into adipocytes, chondrocytes, osteocytes, and neuronal/glial cells using specific induction media. We have evaluated neuronal and glial proteins (Nestin, Tuj-I, betaIII Tubulin, tyrosine hydroxylase [TH], MAP-2, and GFAP) by using flow cytometry, Western blots, and RT-PCR. We found that MSC constituently express native immature neuronal proteins such as Nestin and Tuj-1. After only five passages, MSC can already express more mature neuronal or glial proteins, such as TH, MAP-2, and GFAP, without any specific induction. We noticed an increase in the expression of more mature neuronal/glial proteins (TH, MAP-2, and GFAP) after exposure to neural induction medium, thus confirming the differentiation of MSC into neurons and astrocytes. The constitutive expression of Nestin or Tuj-1 by MSC suggests that these cells are "multidifferentiated" cells and thus can retain the ability for neuronal differentiation, enhancing their potentiality to be employed in the treatment of neurological diseases.     

Culture and neural differentiation of rat bone marrow mesenchymal stem cells in vitro

Adult bone marrow mesenchymal stem cells (MSCs) can differentiate into several types of mesenchymal cells, including osteocytes, chondrocytes, and adipocytes, but can also differentiate into non-mesenchymal cells, such as neural cells, under appropriate experimental conditions. Until now, many protocols for inducing neuro-differentiation in MSCs in vitro have been reported. But due to the differences in MSCs' isolation and culture conditions, the results of previous studies lacked consistency and comparability. In this study, we induced differentiation into neural phenotype in the same MSCs population by three different treatments: beta-mercaptoethanol, serum-free medium and co-cultivation with fetal mouse brain astrocytes. In all of the three treatments, MSCs could express neural markers such as NeuN or GFAP, associating with remarkable morphological modifications. But these treatments led to neural phenotype in a non-identical manner. In serum-free medium, MSCs mainly differentiated into neuron-like cells, expressing neuronal marker NeuN, and BME can promote this process. Differently, after co-culturing with astrocytes, MSCs leaned to differentiate into GFAP(+) cells. These data confirmed that MSCs can exhibit plastic neuro-differentiational potential in vitro, depending on the protocols of inducement.     

Real-time quantitative RT-PCR analysis of human bone marrow stromal cells during osteogenic differentiation in vitro

We developed and used real-time RT-PCR assays to investigate how the expression of typical osteoblast-related genes by human bone marrow stromal cells (BMSC) is regulated by (i) the culture time in medium inducing osteogenic differentiation and (ii) the previous expansion in medium enhancing cell osteogenic commitment. BMSC from six healthy donors were expanded in medium without (CTR) or with fibroblast growth factor-2 and dexamethasone (FGF/Dex; these factors are known to increase BMSC osteogenic commitment) and further cultivated for up to 20 days with ascorbic acid, beta-glycerophosphate and dexamethasone (these factors are typically used to induce BMSC osteogenic differentiation). Despite a high variability in the gene expression levels among different individuals, we identified the following statistically significant patterns. The mRNA levels of bone morphogenetic protein-2 (BMP-2), bone sialo protein-II (BSP), osteopontin (OP) and to a lower extent cbfa-1 increased with culture time in osteogenic medium (OM), both in CTR- and FGF/Dex-expanded BMSC, unlike levels of alkaline phosphatase, collagen type I, osteocalcin, and osteonectin. After 20 days culture in OM, BMP-2, BSP, and OP were more expressed in FGF/Dex than in CTR-expanded BMSC (mRNA levels were, respectively, 9.5-, 14.9-, and 5.8-fold higher), unlike all the other investigated genes. Analysis of single-colony-derived strains of BMSC further revealed that after 20 days culture in OM, only a subset of FGF/Dex-expanded clones expressed higher mRNA levels of BMP-2, BSP, and OP than CTR-expanded clones. In conclusion, we provide evidence that mRNA levels of BMP-2, BSP, and OP, quantified using real-time RT-PCR, can be used as markers to monitor the extent of BMSC osteogenic differentiation in vitro; using those markers, we further demonstrated that only a few subpopulations of BMSC display enhanced osteogenic differentiation following FGF/Dex expansion.     

Gangliosides are involved in neural differentiation of human dental pulp-derived stem cells

Human dental pulp-derived stem cells (hDPSCs) have been considered alternative sources of adult stem cells because of their potential to differentiate into multiple cell lineages. This study investigated the possible role of gangliosides in the neural differentiation of hDPSCs. When hDPSCs were cultured under neural differentiation conditions, expression of neural cell marker genes such as Nestin, MAP-2, and NeuN was detected. Immunostaining and high-performance thin-layer chromatography analysis showed that an increase in ganglioside biosynthesis was associated with neural differentiation of hDPSCs. Specifically, a significant increase in GD3 and GD1a expression was observed during neural differentiation. To confirm the role of gangliosides in neural differentiation, ganglioside biosynthesis was inhibited in hDPSCs by knockdown of UDP-glucose ceramide glucosyltransferase (Ugcg), which prevented differentiation into neural cells. These results suggest that gangliosides may play a role in the neural differentiation process of hDPSCs.     

骨髓间充质干细胞向神经细胞分化中Tau蛋白的表达

目的：观察骨髓间充质干细胞（MSCs）分化为神经细胞过程中,神经元微管相关蛋白Tau及其磷酸化位点pSer202的表达和含量的差异,探讨Tau蛋白在此过程中的作用。方法：使用EGF和bFGF联合诱导第4、第8和第12代的MSCs向神经细胞分化;14d后,免疫细胞化学法检测Tau蛋白和pSer202的表达;ELISA法分析各代细胞Tau蛋白含量。结果：第4、第8和第12代未诱导组Tau蛋白阳性细胞均〈6％;诱导14d后,各代MSCs在形态上均分化为类似神经元样细胞,Tau蛋白阳性细胞率较未诱导组显著升高（P〈0．05）,但各代之间无显著性,而pSer202在各代MSCs未诱导组和诱导组中均未见表达。ELISA法检测发现Tau蛋白含量在诱导过程中呈上升趋势,14d时各代细胞分化后的Tau蛋白升高程度无显著性差异。结论：MSCs向神经细胞分化过程中Tau蛋白表达量增加且可能尚未发生磷酸化,将有助于神经细胞的正常分化和突触形成。     

Expression of Pdx-1 in bone marrow mesenchymal stem cells promotes differentiation of islet-like cells in vitro

Bone marrow mesenchymal stem cells (BMSCs) have the ability of self-renewal and multi-directional differentiation. Recent reports showed that BMSCs could differentiate into endocrine cells of pancreas. However, the differentiation is not efficient enough to produce insulin-producing cells for the future therapeutic use. Pdx-1 is a crucial regulator for pancreatic development. Therefore we constructed a eukaryotic expression vector containing Pdx-1 to determine the effect of Pdx-1 expression on differentiation of BMSCs in vitro. The results showed that BMSCs could self-assemble to form functional pancreatic islet-like structures after differentiation in vitro. The proportion of insulin-producing cells differentiated from Pdx-1⁺BMSCs was 28.23%±2.56%, higher than that from BMSCs transfected with vacant vector and Pdx-1 ⁻ BMSCs (7.23%±1.56% and 4.08%±2.69% respectively) by flow cytometry. Immunocytochemical examination also testified the expression of multiple β-cells-specific genes such as insulin, glucagons, somatostatin in differentiated BMSCs. The results also revealed that the expressions of genes mentioned above in Pdx-1⁺BMSCs were higher than that in Pdx-1⁻BMSCs, which was confirmed by Western blotting analysis and RT-PCR. Glucose-induced insulin secretion from Pdx-1⁺BMSCs in 5mmol/L and 25mmol/L glocuse was (56.61±4.82) μU/mL and (115.29±2.56) μU/mL respectively, which were much higher than those from Pdx-1⁻BMSCs((25.53±6.49) μU/mL and (53.26±7.56) μU/mL respectively). Grafted animals were able to maintain their body weight and survive for relatively longer periods of time than hyperglycemic sham-grafted controls, which demonstrated an overall beneficial effect of the grafted cells on the health of the animals. These findings thus suggested that exogenous expression of Pdx-1 should provide a promising approach for efficiently producing islet-like cells from BMSCs for the future therapeutic use in diabetic patients.     

Cholinergic neuronal differentiation of bone marrow mesenchymal stem cells in rhesus monkeys

The purpose of the present study was to determine the best cholinergic neuronal differentiation method of rhesus monkey bone marrow mesenchymal stem cells(BMSCs).Four methods were used to induce differentiation,and the groups were assigned accordingly:basal inducing group(culture media,bFGF,and forskolin);SHH inducing group(SHH,inducing group);RA inducing group(RA,basal inducing group);and SHH+RA inducing group(SHH,RA,and basal inducing group).All groups displayed neuronal morphology and increased expressio...     

鼠骨髓间充质干细胞的分离培养及定向诱导分化为内皮样细胞

目的：探讨体外大鼠骨髓间充质干细胞（rBMMSCs）的分离培养和血管内皮生长因子（VEGF）、碱性成纤维细胞生长因子（bFGF）对其定向诱导为内皮样细胞（ELCs）的可行性。方法：采用Percoll（1.073g/ml）分离液分离骨髓单个核细胞,用含10%胎牛血清（FBS）的LG-DMEM培养基贴壁纯化培养,倒置显微镜、免疫细胞化学法、流式细胞仪、MTT法、透射电镜（TEM）联合对rBMMSCs形态、表型、生长曲线、细胞周期以及超微结构进行鉴定;诱导后的细胞,采用倒置显微镜观察细胞形态,免疫细胞化学法检测CD31、CD144（VE-cadherin）和CD34表达以及摄取Dil-ac-LDL、结合FITC-UEA-1的功能特点。结果：rBMMSCs呈长梭形,漩涡状排列。细胞生长曲线显示潜伏期、对数生长期和平台期,符合干细胞的生长规律。透射电镜结果表明：rBMMSCs有两种不同的形态结构,其中体积较小、核质比大、胞质内细胞器稀少者为处于未分化或分化较低状态的幼稚型rBMMSCs。细胞周期分析显示：第4代细胞G0/G1期为95.67%,表明绝大部分细胞处于非增殖状态;诱导后的部分细胞形态可见类似ELCs改变,表达血管内皮细胞（ECs）特异性表面标志CD31、CD34和CD144,具有摄取Dil-ac-LDL以及结合FITC-UEA-1的功能特点。结论：采用Percoll密度梯度离心与贴壁培养相结合的方法所培养的rBMMSCs在体外具有定向诱导分化为ELCs的潜能,可能成为血管组织工程理想的种子细胞来源。     

Expression of Pdx-1 in bone marrow mesenchymal stem cells promotes differentiation of islet-like cells in vitro

Bone marrow mesenchymal stem cells(BMSCs) have the ability of self-renewal and multi-directional differentiation.Recent reports showed that BMSCs could differentiate into endocrine cells of pancreas.However,the differentiation is not efficient enough to produce insulin-producing cells for the future therapeutic use.Pdx-1 is a crucial regulator for pancreatic development.Therefore we constructed a eukaryotic expression vector containing Pdx-1 to determine the effect of Pdx-1 ex-pression on differentiation of BMSCs in vitro.The results showed that BMSCs could self-assemble to form functional pancreatic islet-like structures after differentiation in vitro.The proportion of insu-lin-producing cells differentiated from Pdx-1 BMSCs was 28.23%±2.56%,higher than that from BMSCs transfected with vacant vector and Pdx-1-BMSCs(7.23%±1.56% and 4.08%±2.69% respec-tively) by flow cytometry.Immunocytochemical examination also testified the expression of multiple β-cells-specific genes such as insulin,glucagons,somatostatin in differentiated BMSCs.The results also revealed that the expressions of genes mentioned above in Pdx-1 BMSCs were higher than that in Pdx-1-BMSCs,which was confirmed by Western blotting analysis and RT-PCR.Glucose-induced insulin secretion from Pdx-1 BMSCs in 5mmol/L and 25mmol/L glocuse was(56.61±4.82) μU/mL and(115.29±2.56) μU/mL respectively,which were much higher than those from Pdx-1-BMSCs((25.53±6.49) μU/mL and(53.26±7.56) μU/mL respectively) .Grafted animals were able to maintain their body weight and survive for relatively longer periods of time than hyperglycemic sham-grafted controls,which demonstrated an overall beneficial effect of the grafted cells on the health of the animals.These findings thus suggested that exogenous expression of Pdx-1 should provide a promising approach for efficiently producing islet-like cells from BMSCs for the future therapeutic use in diabetic patients.     

Optimization of an effective directed differentiation medium for differentiating mouse bone marrow mesenchymal stem cells into hepatocytes in vitro

We have used a uniform design to explore the most effective directed differentiation medium (MEDDM) for differentiating mouse bone marrow mesenchymal stem cells (mMSCs) into hepatocytes. Our methods involved arranging eight differentiation medium groups following uniform design. Flow cytometry was used to evaluate the percentage of ALB+ and CK18+ cells in each group. Factors and their concentrations in the MEDDMs were then identified. The MEDDMs were evaluated by their ability to differentiate mMSCs into hepatocytes by RNA and protein expressions and synthesis functions. FGF at 35 ng/ml and OSM at 30 ng/ml in the medium yielded the highest percentage of ALB+ and CK18+ cells. During directed differentiation using MEDDMs, ALB, CK18, TTR, AFP mRNAs were expressed. ALB and CK18 proteins were detected in the cells. The differentiated cells produced albumin and urea in a time dependent manner. Uniform design was adequate for choosing the MEDDM of mMSCs. MEDDM containing 35 ng/ml FGF and 30 ng/ml OSM was effective in differentiating mMSCs into hepatocytes.     

Ectopic osteogenesis and chondrogenesis of bone marrow stromal stem cells in alginate system

In orthopedics, the regeneration and repair of cartilage or bone defects after trauma, cancer, or metabolic disorders is still a major clinical challenge. Through developmental plasticity, bone marrow mesenchymal stem cells (BMSSCs) are important seed cells for the musculoskeletal tissue engineering approach. The present study sought to determine the ectopic osteogenic and chondrogenic ability of BMSSCs in combination with a scaffolding material made from alginate gel. After isolation from the bone marrow of BALB/C mice, BMSSCs were expanded in vitro and induced to chondrogenesis or osteogenesis for 14 days, respectively. Subsequently, these induced cells were seeded into alginate gel, and the constructs implanted into BALB/C nude mice subcutaneously for up to 8 weeks. In the histological analysis, the transmission electron microscopy of the retrieved specimens at various intervals showed obvious trends of ectopic cartilage or bone formation along with the alteration of the cellular phenotype. Simultaneously, the results of the immunohistochemical staining and RT-PCR both confirmed the expression of specific extracellular matrix (ECM) markers for cartilaginous tissue, such as collagen type II (Col-II), SOX9, and aggrecan, or alternatively, markers for osteoid tissue, such as osteopontin (OPN), osteocalcin (OCN), and collagen type I (Col-I). During subcutaneous implantation, the elevating production of ECM and the initiation of the characteristic structure were closely correlated with the increase of time. In contrast, there was an apparent degradation and resorption of the scaffolding material in blank controls, but with no newly formed tissues. Finally, the constructs that were made of non-induced BMSSCs nearly disappeared during the 8 weeks after implantation. Therefore, it is suggested that alginate gel, which is combined with BMSSCs undergoing differentiation into skeletal lineages, may represent a useful strategy for the clinical reconstruction of bone and cartilage defects.     

人骨髓间充质干细胞在成年大鼠脑内的迁移及分化

骨髓间充质干细胞 (mesenchymalstemcells,MSCs)是目前备受关注的一类具有多向分化潜能的组织干细胞 ,体外可以分化为骨、软骨、脂肪等多种细胞。因此 ,MSCs是细胞治疗和基因治疗的种子细胞之一。为了探索MSCs的迁移和分化趋势 ,为帕金森病 (Parkinsondisease,PD)的干细胞治疗提供理论和实验依据 ,本实验将体外扩增并转染增强型绿色荧光蛋白 (enhancedgreenfluorescentprotein ,EGFP)的人骨髓MSCs注入PD大鼠脑内纹状体 ,观察了人骨髓MSCs在大鼠脑内的存活、迁移、分化以及注射MSCs前后大鼠的行为变化。结果表明 ,人骨髓MSCs在大鼠脑内可存活较长时间 ( 10周以上 ) ;随着时间的延长 ,MSCs迁移范围扩大 ,分布于纹状体、胼胝体、皮质以及脑内血管壁 ;免疫组化法检测证实MSCs在大鼠脑内表达人神经丝蛋白 (neurofilament,NF)、神经元特异性烯醇化酶 (neuron specificeno lase,NSE)以及胶质原纤维酸性蛋白 ( glialfibrillaryacidprotein ,GFAP) ;PD大鼠的异常行为有所缓解 ,转圈数由 8 86±2 0 9r/min下降到 4 87± 2 0 6r/min ,统计学分析P <0 0 5为差异显著。以上观察结果表明 ,骨髓MSCs有望成为治疗PD的种子细胞     

溶血磷脂酸促进离体大鼠胚胎神经干细胞的增殖及其向胆碱能神经元分化

溶血磷脂酸（1ysophosphatidic acid,LPA）是一种细胞外磷脂信号。本研究用[^3H]-胸腺嘧啶掺入法、免疫细胞化学和Western blot等技术,观察了LPA对体外培养的大鼠胚胎神经干细胞（neural stem cells,NSCs）的增殖以及向MAF2标记的一般神经元和ChAT标记的胆碱能神经元的分化的影响。结果显示：（1）在特殊的无血清培养基中加入低浓度的LPA（0．01-1．0μmol/L）后,NSCs对【^3H】-胸腺嘧啶的摄入呈剂量依赖性增加,表明LPA对NSCs有显著的促增殖作用;（2）在培养基中加入胎牛血清以诱导NSCs的分化,发现低浓度的LPA增加MAF2阳性和ChAT阳性神经元的比例,0．1μmol／L LPA引起的增加达到峰值;（3）Western blot分析显示LPA促进了MAP2和ChAT的表达;（4）在诱导NSCs出现分化早期,用倒置显微镜观察到低浓度的LPA明显促进细胞突起的生长和细胞的迁移。以上结果表明,低浓度LPA在一定范围内可以促进NSCs的增殖、并分化为一般的MAP2阳性神经元和特殊的胆碱能神经元,而且LPA可以促进在分化早期出现的神经元或神经胶质细胞前体细胞的迁移和突起生长。     

circRNA Acbd6 promotes neural stem cell differentiation into cholinergic neurons via the miR-320-5p-Osbpl2 axis

Neural stem cells (NSCs) persist in the dentate gyrus of the hippocampus into adulthood and are essential for both neurogenesis and neural circuit integration. Exosomes have also been shown to play vital roles in regulating biological processes of receptor cells as a medium for cell-to-cell communication signaling molecules. The precise molecular mechanisms of exosome-mediated signaling, however, remain largely unknown. Here, we found that exosomes produced by denervated hippocampi following fimbria–fornix transection could promote the differentiation of hippocampal neural precursor cells into cholinergic neurons in coculture with NSCs. Furthermore, we found that 14 circular RNAs (circRNAs) were upregulated in hippocampal exosomes after fimbria–fornix transection using high-throughput RNA-Seq technology. We further characterized the function and mechanism by which the upregulated circRNA Acbd6 (acyl-CoA-binding domain–containing 6) promoted the differentiation of NSCs into cholinergic neurons using RT–quantitative PCR, Western blot, ELISA, flow cytometry, immunohistochemistry, and immunofluorescence assay. By luciferase reporter assay, we demonstrated that circAcbd6 functioned as an endogenous miR-320-5p sponge to inhibit miR-320-5p activity, resulting in increased oxysterol-binding protein–related protein 2 expression with subsequent facilitation of NSC differentiation. Taken together, our results suggest that circAcbd6 promotes differentiation of NSCs into cholinergic neurons via miR-320-5p/oxysterol-binding protein–related protein 2 axis, which contribute important insights to our understanding of how circRNAs regulate neurogenesis.