单轴、双轴循环拉力对小鼠肌腱源性干细胞肌腱样分化的影响

目的 探讨单轴、双轴循环拉力对小鼠肌腱源性干细胞(TDSCs)分化的影响,为临床肌腱损伤后的康复治疗提供理论基础。方法 取6~8周龄C57BL/6小鼠10只,无菌条件下暴露双侧后腿至脚掌,显微镜下解剖收集小鼠髌腱和跟腱组织块,体外分离培养细胞,观察第3代细胞的形态特点。(1)取传代至第3代的细胞,采用流式细胞术检测间充质干细胞标志物(CD44、CD90、Sca-1)、内皮细胞标志物(CD34、Flk-1)、造血细胞标志物(CD45),鉴定细胞是否符合TDSCs特点。(2)取传代至第3代的TDSCs进行成骨细胞、软骨细胞和脂肪细胞分化培养,分别采用茜素红、油红和阿尔新蓝染料对培养的三系细胞进行染色,鉴定细胞是否具有多向分化潜能。(3)取传代至第3代的TDSCs接种到硅胶底培养皿上,分为双轴循环拉力组、单轴循环拉力组、对照组3组。双轴循环拉力组细胞使用Flexcell^􀆿 FX-4000TM柔性基底拉伸加载系统,单轴循环拉力组细胞使用自制拉伸力生物反应器,对照组细胞无拉力。双轴循环拉力组、单轴循环拉力组施加机械负荷组的参数均设置为0.25 Hz、6%的循环拉力,在培养期间进行机械负荷加载,每天加载8 h,共加载6 d。第6天机械负荷刺激结束后,收集3组细胞进行实时荧光定量PCR (qPCR),检测肌腱、成骨、脂肪和软骨相关转录因子的表达。结果 显微镜下观察第3代TDSCs形态一致,呈梭形纤维状。(1)流式细胞技术检测结果显示,间充质干细胞标志物CD44、CD90和Sca-1表达阳性、内皮细胞标志物CD34和Flk-1表达阴性、造血细胞标志物CD45表达阴性,符合TDSCs标记鉴定特点。(2)三系分化细胞检测结果显示,提取的细胞成功分化为成骨细胞、脂肪细胞和软骨细胞,验证了提取的细胞具有向成骨细胞、软骨细胞和脂肪细胞分化的潜能。(3)对照组、单轴循环拉力组、双轴循环拉力组3组间比较,肌腱、成骨、软骨、脂肪相关转录因子的相对表达量差异均有统计学意义(P值均＜0.05)。组间两两比较:单轴循环拉力组与对照组比较,肌腱、成骨相关转录因子以及脂肪相关转录因子PPARγ的相对表达均增高,软骨相关转录因子的相对表达均降低,差异均有统计学意义(P值均＜0.05),而脂肪相关转录因子CEB/P的相对表达差异无统计学意义(P＞0.05);双轴循环拉力组与对照组比较,肌腱相关转录因子Scx、Mohawk的相对表达降低,成骨相关转录因子Runx2的相对表达增高、碱性磷酸酶(ALP)的相对表达降低,软骨相关转录因子Sox9相对表达增高、Col2a1的相对表达降低,脂肪相关转录因子的相对表达均增高,差异均有统计学意义(P值均＜0.05);单轴循环拉力组与双轴循环拉力组比较,双轴循环拉力组肌腱相关转录因子Scx、Mohawk、Col1a1的相对表达均降低,成骨相关转录因子ALP的相对表达降低,软骨、脂肪相关转录因子的相对表达均增高,差异均有统计学意义(P值均＜0.05)。结论 单轴循环拉力诱导TDSCs向肌腱细胞、成骨细胞分化,而双轴循环拉力诱导TDSCs向成骨细胞、脂肪细胞、软骨细胞分化。单轴循环拉力的作用下可以促进体外TDSCs向肌腱细胞分化,有利于肌腱组织的再生和损伤后的修复,为临床肌腱损伤后的康复治疗提供了理论依据。     

Comparison of proliferative and multilineage differentiation potential of human mesenchymal stem cells derived from umbilical cord and bone marrow

Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a high proliferative potential and the capacity to differentiate into an osteogenic phenotype. HUCPVCs have thus been considered a possible extra-embryonic mesenchymal stem cell (MSC) source for cell-based therapies. To assess this potential, we compared HUCPVCs to the "gold standard" bone marrow mesenchymal stromal cells (BMSCs) with respect to their proliferation, differentiation, and transfection capacities. HUCPVCs showed a higher proliferative potential than BMSCs and were capable of osteogenic, chondrogenic, and adipogenic differentiation. Interestingly, osteogenic differentiation of HUCPVCs proceeded more rapidly than BMSCs. Additionally, HUCPVCs expressed higher levels of CD146, a putative MSC marker, relative to BMSCs. HUCPVCs showed comparable transfection efficiency as BMSCs using a nucleofection method but were more amenable to transfection with liposomal methods (FuGENE). Gene array analysis showed that HUCPVCs also expressed Wnt signaling pathway genes that have been implicated in the regulation of MSCs. The similar characteristics between HUCPVCs and MSCs support the applicability of HUCPVCs for cell-based therapies. Disclosure of potential conflicts of interest is found at the end of this article.     

In Vitro Mesenchymal Trilineage Differentiation and Extracellular Matrix Production by Adipose and Bone Marrow Derived Adult Equine Multipotent Stromal Cells on a Collagen Scaffold

Directed differentiation of adult multipotent stromal cells (MSC) is critical for effective treatment strategies. This study was designed to evaluate the capability of equine MSC from bone marrow (BMSC) and adipose tissue (ASC) on a type I collagen (COLI) scaffold to undergo chondrogenic, osteogenic and adipogenic differentiation and form extracellular matrix (ECM) in vitro. Following determination of surface antigen expression, MSC were loaded into scaffolds in a perfusion bioreactor and loading efficiency was quantified. Cell-scaffold constructs were assessed after loading and 7, 14 and 21 days of culture in stromal or induction medium. Cell number was determined with DNA content, cell viability and spatial uniformity with confocal laser microscopy and cell phenotype and matrix production with light and scanning electron microscopy and mRNA levels. The MSC were positive for CD29 (>90 %), CD44 (>99 %), and CD105 (>60 %). Loading efficiencies were >70 %. The ASC and BMSC cell numbers on scaffolds were affected by culture in induction medium differently. Viable cells remained uniformly distributed in scaffolds for up to 21 days and could be directed to differentiate or to maintain an MSC phenotype. Micro- and ultrastructure showed lineage-specific cell and ECM changes. Lineage-specific mRNA levels differed between ASC and BMSC with induction and changed with time. Based on these results, equine ASC and BMSC differentiate into chondrogenic, osteogenic and adipogenic lineages and form ECM similarly on COLI scaffolds. The collected data supports the potential for equine MSC-COLI constructs to support diverse equine tissue formation for controlled biological studies.     

The effect of matrix stiffness on the differentiation of mesenchymal stem cells in response to TGF-β

Bone marrow mesenchymal stem cells (MSCs) are a valuable cell source for tissue engineering and regenerative medicine. Transforming growth factor β (TGF-β) can promote MSC differentiation into either smooth muscle cells (SMCs) or chondrogenic cells. Here we showed that the stiffness of cell adhesion substrates modulated these differential effects. MSCs on soft substrates had less spreading, fewer stress fibers and lower proliferation rate than MSCs on stiff substrates. MSCs on stiff substrates had higher expression of SMC markers α-actin and calponin-1; in contrast, MSCs on soft substrates had a higher expression of chondrogenic marker collagen-II and adipogenic marker lipoprotein lipase (LPL). TGF-β increased SMC marker expression on stiff substrates. However, TGF-β increased chondrogenic marker expression and suppressed adipogenic marker expression on soft substrates, while adipogenic medium and soft substrates induced adipogenic differentiation effectively. Rho GTPase was involved in the expression of all aforementioned lineage markers, but did not account for the differential effects of substrate stiffness. In addition, soft substrates did not significantly affect Rho activity, but inhibited Rho-induced stress fiber formation and α-actin assembly. Further analysis showed that MSCs on soft substrates had weaker cell adhesion, and that the suppression of cell adhesion strength mimicked the effects of soft substrates on the lineage marker expression. These results provide insights of how substrate stiffness differentially regulates stem cell differentiation, and have significant implications for the design of biomaterials with appropriate mechanical property for tissue regeneration.     

体外扩增过程中人骨髓间充质干细胞的增殖与分化规律

目的：系统考察体外扩增过程中人骨髓间充质干细胞（MSC）的增殖与分化规律，为MSC任组织修复以及细胞治疗中的应用提供参考、方法：以全骨髓贴壁法分离成人肋骨骨髓MSC，在相同条件下分别考察各代细胞形态、生长、表面标记、细胞周期、成骨、成软骨及成脂肪能力的变化情况。结果：随代次增加，MSC增殖能力、成骨、成脂肪能力均有所下降，而成软骨能力无明显降低；成骨、成软骨及成脂肪能乃均保持到细胞衰老。存扩增过程中，MSC始终保持较高的纯度，CD29、CD44、CD105的阳性率均在90％以上，CD14、CD34和CD45的阳性率均在4％以下、结论：在体外培养过程中MSC干细胞特性逐渐丢失，其中向骨、脂肪方向的分化潜能较软骨方向更易失去；而多向分化能力的保持较之自我更新能力更为持久。MSC在7代以前可作为基础研究及临床应用的良好对象。     

Characterization of human adipose-derived stem cells and expression of chondrogenic genes during induction of cartilage differentiation

OBJECTIVES:

Understanding the changes in chondrogenic gene expression that are involved in the differentiation of human adipose-derived stem cells to chondrogenic cells is important prior to using this approach for cartilage repair. The aims of the study were to characterize human adipose-derived stem cells and to examine chondrogenic gene expression after one, two, and three weeks of induction.

MATERIALS AND METHODS:

Human adipose-derived stem cells at passage 4 were evaluated by flow cytometry to examine the expression of surface markers. These adipose-derived stem cells were tested for adipogenic and osteogenic differentiation capacity. Ribonucleic acid was extracted from the cells for quantitative polymerase chain reaction analysis to determine the expression levels of chondrogenic genes after chondrogenic induction.

RESULTS:

Human adipose-derived stem cells were strongly positive for the mesenchymal markers CD90, CD73, CD44, CD9, and histocompatibility antigen and successfully differentiated into adipogenic and osteogenic lineages. The human adipose-derived stem cells aggregated and formed a dense matrix after chondrogenic induction. The expression of chondrogenic genes (collagen type II, aggrecan core protein, collagen type XI, COMP, and ELASTIN) was significantly higher after the first week of induction. However, a significantly elevated expression of collagen type X was observed after three weeks of chondrogenic induction.

CONCLUSION:

Human adipose-derived stem cells retain stem cell characteristics after expansion in culture to passage 4 and serve as a feasible source of cells for cartilage regeneration. Chondrogenesis in human adipose-derived stem cells was most prominent after one week of chondrogenic induction.     

小鼠骨髓间充质干细胞生物学特性和体外诱导分化

目的研究小鼠骨髓间充质干细胞的生物学性状和多系分化潜能。方法取Balb／c小鼠骨髓单个核细胞在低糖的培养液中培养出贴壁生长的细胞,进行形态学观察、细胞周期和免疫表型分析;在不同的因子作用下诱导向成骨细胞、软骨细胞,脂肪细胞分化,并检测诱导后细胞相应的基因表达。结果小鼠骨髓间充质干细胞贴壁生长后形态较均一,增殖能力随着传代逐渐增强,但从第8代后增殖能力明显减退。细胞表达CD29,CD38,CD44,CD106等标记,但CD34和H-2k表达阴性。在不同的诱导培养体系里间充质干细胞能分化为成骨细胞、软骨细胞和脂肪细胞,相应的骨钙蛋白基因,Ⅱ型胶原基因,脂蛋白脂酶基因表达都明显增强。结论从小鼠骨髓可以分离培养出间充质干细胞,在体外有效扩增和诱导分化。表明可以以小鼠为模型研究间充质干细胞在组织工程、细胞移植、基因治疗等领域的运用。     

小鼠间充质干细胞三系分化中SOX9与Ⅱ型胶原mRNA的定量

背景：研究表明,软骨中的主要成分Ⅱ型胶原的基因-Col2a1在软骨细胞中的表达与SOX9 的浓度呈剂量依赖正相关关系。 目的：通过成骨、成软骨、成脂肪诱导干细胞分化,分析3种分化过程及不同时期的SOX9与Ⅱ型胶原 mRNA含量的变化,探讨SOX9在不同时空分布的表达规律及与Ⅱ型胶原的相关关系。 方法：取4周龄昆明小鼠骨髓间充质细胞,体外培养得到间充质干细胞并传达至第3代,对间充质干细胞进行流式细胞仪鉴定细胞表型,共分3组每组设3个时间段,通过成骨、成软骨、成脂肪3种诱导培养液对3组细胞进行诱导,另设不进行诱导的细胞作为对照组。分别在诱导3,7,14 d后收集提取细胞的总RNA,通过RT-PCR进行SOX9与Ⅱ型胶原的mRNA定量检测,同时对诱导后的细胞进行染色、免疫荧光染色,观察其分化状态及相关统计分析。 结果与结论：第3代骨髓间充质干细胞生长良好,流式细胞仪鉴定细胞表型证实为干细胞,对诱导后细胞进行染色、免疫荧光染色结果证实细胞分化为骨、软骨、脂肪细胞。经RT-PCR检测,在3组诱导分化细胞中SOX9 mRNA含量由高到低分别是成软骨、成骨、成脂肪,Ⅱ型胶原 mRNA含量由高到低分别是成软骨、成脂肪、成骨。在成软骨分化中SOX9在3,7 d表达不断升高,14 d呈下降趋势。Ⅱ型胶原在3,7,14 d均逐渐升高。在成骨分化中SOX9 mRNA含量随着时间推移而增加,而Ⅱ型胶原则随着时间推移而不断降低。在成脂肪分化中SOX9 mRNA表达与对照组比较差异无显著性意义(P > 0.05);而Ⅱ型胶原的表达没有规律可循,时间点的延伸及检测未观察到。结果提示,SOX9在软骨分化中作用优于成骨、成脂肪组,且软骨分化中SOX9与Ⅱ型胶原存在相关性,可能在软骨分化的早期Ⅱ型胶原随着SOX9的变化而变化;且软骨分化和成骨分化过程中SOX9可能起到了一个互相协调促进平衡的关键作用。     

Stage-dependent effect of TGF-beta1 on chondrogenic differentiation of human embryonic stem cells

Transforming growth factor-beta (TGF-beta) is known to be a potent inducer of stem cell chondrogenic differentiation. Transforming growth factor-beta/activin/nodal-signaling pathway has also been shown to be involved in maintaining the pluripotency of embryonic stem cells (ESCs). In this study, the effect of TGF-beta1 in chondrogenic differentiation of ESCs was examined both with undifferentiated ESCs that bypassed classical embryoid body (EB) formation, and on 5-day EB-derived cells. The effect of TGF-beta1 was compared to cells differentiated in serum-free chondrogenic basal medium without growth factor supplement. Analysis by real-time polymerase chain reaction (PCR), type II collagen enzyme-linked immunosorbent assay, sulfated glycoaminoglycan quantification and fluorescence immunostaining demonstrated substantial chondrogenic differentiation of ESCs regardless of EB formation in the absence of the growth factor. Addition of TGF-beta1 significantly inhibited chondrogenic gene expression and collagen deposition with a more potent effect on the cells that bypassed EB formation. Our study using a TGF-beta/activin/nodal-signaling inhibitor suggested that TGF-beta inhibited early chondrogenic induction but was required at the later stage of differentiation, which was also reflected in the enhancing effect of TGF-beta1 on chondrogenic development at later time points in EB-derived cells. Analysis of the pluripotency markers demonstrated sustained Oct4 and Nanog expression in the presence of TGF-beta1 with Oct4-positive cells detected in subpopulations of the differentiated culture. Our results suggest that TGF-beta1 suppresses ESC chondrogenic induction and the degree of suppression is dependent on the differentiation-stage of the ESC. Transforming growth factor-beta signaling, however, is required for functional chondrogenic development of ESC. Our finding that TGF-beta can sustain an undifferentiated population of human ESCs within the differentiation culture suggests that caution should be exercised when using this growth factor as an ESC chondrogenic inducer and highlights the importance of a selection protocol for chondroprogenitor cells to avoid possible teratoma formation in vivo.     

Characterization of long-term in vitro culture-related alterations of human tonsil-derived mesenchymal stem cells: role for CCN1 in replicative senescence-associated increase in osteogenic differentiation

Although mesenchymal stem cells (MSC) isolated from bone marrow and adipose tissues are known to be subjected to in vitro culture-related alterations in their stem cell properties, such data have not been reported in human tonsil-derived MSC (T-MSC). Here, we investigated the culture-related changes of phenotypes, the senescence, and the differentiation potential of T-MSC. T-MSC were serially passaged by a standard protocol, and their characteristics were assessed, including MSC-specific surface antigen profiles, the senescence, and the differentiation potentials into adipocytes, chondrocytes and osteocytes. Up to at least passage 15, we found no alterations in either MSC-specific surface marker, CD14, CD34, CD45, CD73 and CD90, or the mRNA expression of embryonic stem cell gene markers, Nanog, Oct4-A and Sox-2. However, the expression of CD146, recently identified another MSC marker, dramatically decreased with increasing passages from ∼ 23% at passage 3 to ∼ 1% at passage 15. The average doubling time increased significantly from ∼ 38 h at passage 10 to ∼ 46 h at passage 15. From passage 10, the cell size increased slightly and SA-β-gal staining was evident. Both Alizarin Red S staining and osteocalcin expression showed that the osteogenic differentiation potential increased up to passage 10 and decreased thereafter. However, the adipogenic and chondrogenic differentiation potential decreased passage-dependently from the start, as evidenced by staining of Oil Red O and Alcian Blue, respectively. Consistent with a passage-dependent osteogenic differentiation, the expression of CCN1, an angiogenic protein known to be related to both senescence and osteogenesis, also increased up to passage 10. Furthermore, ectopic expression of small interfering RNA against CCN1 at passage 10 significantly reversed Alizarin Red S staining and osteocalcin expression. Altogether, our study demonstrates the characterization of long-term in vitro cultured T-MSC and that CCN1 may be involved in mediating a passage-dependent increase in osteogenic potential of T-MSC.     

microRNA-25-3p suppresses osteogenic differentiation of BMSCs in patients with osteoporosis by targeting ITGB3

This study was conducted to investigate the impact of the microRNA (miR)-25–3p/ITGB3 axis on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) from patients with osteoporosis (OP). BMSCs isolated from the bone marrow of healthy controls and OP patients were identified by flow cytometry, in which ITGB3 mRNA and miR-25–3p expression was detected by RT-qPCR and ITGB3, Runx2, OPN, ALP, and OSX protein expression by western blot. The binding between ITGB3 and miR-25–3p was assessed by dual-luciferase reporter gene and Ago2-RIP assays. BMSC osteogenic differentiation was observed by alizarin red staining and ALP activity. The differentiation of BMSCs to adipocytes and chondrocytes was measured by oil red O staining and alcian blue staining, respectively. BMSCs were successfully isolated from the bone marrow of healthy controls (normal-BMSCs) and OP patients (OP-BMSCs). ITGB3, Runx2, OPN, ALP, and OSX expression was poorer and miR-25–3p expression was higher in OP-BMSCs than in normal-BMSCs. Mechanistically, ITGB3 was negatively targeted by miR-25–3p. After osteogenic, adipogenic, and chondrogenic differentiation of BMSCs were successfully induced, adipogenic differentiation increased and osteogenic and chondrogenic differentiation decreased in OP-BMSCs compared with normal-BMSCs. Overexpression of ITGB3 facilitated mineralized nodule formation and elevated ALP activity and Runx2, OPN, and ALP expression in OP-BMSCs. miR-25–3p upregulation diminished mineralized nodule formation, ALP activity, and Runx2, OPN, and ALP expression in OP-BMSCs and normal-BMSCs, which was annulled by additional ITGB3 overexpression. miR-25–3p targets ITGB3, thereby suppressing osteogenic differentiation of BMSCs from OP patients.     

Multiple differentiation capacity of STRO-1+/CD146+ PDL mesenchymal progenitor cells

Although mesenchymal progenitor cells can be isolated from periodontal ligament (PDL) tissues using stem cell markers STRO-1 and CD146, the proportion of these cells that have the capacity to differentiate into multiple cell lineages remains to be determined. This study was designed to quantify the proportions of primary human PDL cells that can undergo multilineage differentiation and to compare the magnitude of these capabilities relative to bone marrow-derived mesenchymal stem cells (MSCs) and parental PDL (PPDL) cells. PDL mesenchymal progenitor (PMP) cells were isolated from PPDL cells using the markers STRO-1 and CD146. The colony-forming efficiency and multilineage differentiation potential of PMP, PPDL, and MSCs under chondrogenic, osteogenic, and adipogenic conditions were determined. Flow cytometry revealed that on average 2.6% of PPDL cells were STRO-1(+)/CD146(+), whereas more than 63% were STRO-1(-)/CD146(-). Colony-forming efficiency of STRO-1(+)/CD146(+) PMP cells (19.3%) and MSCs (16.7%) was significantly higher than that of PPDL cells (6.8%). Cartilage-specific genes, early markers of osteoblastic differentiation, and adipogenic markers were significantly upregulated under appropriate conditions in PMP cells and MSCs compared to either their noninduced counterparts or induced PPDL cells. Consistent with these findings, immunohistochemistry revealed substantial accumulation of cartilaginous macromolecules, mineralized calcium nodules, and lipid vacuoles under chondrogenic, osteogenic, or adipogenic conditions in PMP and MSC cultures, respectively, compared to noninduced controls or induced PPDL cells. Thus STRO-1(+)/CD146(+) PMP cells demonstrate multilineage differentiation capacity comparable in magnitude to MSCs and could potentially be utilized for regeneration of the periodontium and other tissues.     

miR-146a调控骨髓间充质干细胞成骨分化的机制研究

目的探讨miR-146a调控骨髓间充质干细胞(BMSC)成骨分化的作用及其分子机制。方法贴壁法分离培养小鼠BMSC,检测成骨分化早期标志物Runx 2的变化,观察BMSC体外成骨分化,利用miRNA特异性的聚合酶链式反应(miRNAspecific qPCR)观察miR-146a的变化情况,并干预miR-146a表达,明确miR-146a对BMSC成骨分化的调控作用。结果成功建立了稳定的BMSC体外培养体系,该细胞能够成功分化为脂肪细胞和成骨细胞;在成骨诱导培养条件下,随着成骨分化,miR-146a水平降低,过表达miR-146a,成骨分化早期标志分子Runx 2表达降低;转染miR-146a拮抗体antago-miR-146a可以补救Runx 2表达的降低。结论 miR-146a负向调控BMSC成骨分化,拮抗miR-146a可以补救BMSC成骨分化的降低。     

Isolation of the multipotent MSC subpopulation from human gingival fibroblasts by culturing on chitosan membranes

Literature has different opinions regarding the percentage of mesenchymal stem cell (MSC)-like population in human gingival tissue. Isolation of these cells is thus important for clinical applications. In this study, two typical but distinct types of gingival fibroblasts (GF), GF-A and GF-B, were grown from human gingival biopsies. They were characterized for surface markers by flow cytometry as well as the expressions of stemness and neural crest marker genes by RT-PCR. The two types of GF were slightly different in their surface markers; however, they had dramatic difference in the expression levels of stemness marker genes and neural crest marker genes. They also demonstrated distinct differentiation capacity. Upon the appropriate induction, GF-A were capable of osteogenic, adipogenic, chondrogenic, and neurogenic differentiation while GF-B only underwent osteogenic differentiation. By culturing either type of GF on chitosan membranes for 24 h, we were able to isolate two distinct subpopulations in each type of GF, i.e. cells with spheroid-forming ability (GF-AS and GF-BS) or those remained flat and attached (GF-AN and GF-BN). We further characterized these cells, and determined the common properties shared by the spheroid-forming subpopulation “S”, as well as those shared by the non-spheroid-forming subpopulation “N”. The subpopulation “S” was capable of the multilineage differentiation, while the subpopulation “N” was only efficient in osteogenic differentiation. GF-A and GF-B had different proportions of subpopulations. Chitosan as the cell culture substratum up-regulated the N-cadherin expression of the “S” but not “N” subpopulation, which may account for the cell sorting effect. This study showed that chitosan membranes could be used for isolation of the spheroid forming subpopulation in human GF that contained multipotent adult stem cells of which the number varied among donors and sites.     

A comprehensive analysis of the dual roles of BMPs in regulating adipogenic and osteogenic differentiation of mesenchymal progenitor cells

Pluripotent mesenchymal stem cells (MSCs) are bone marrow stromal progenitor cells that can differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages. Several signaling pathways have been shown to regulate the lineage commitment and terminal differentiation of MSCs. Here, we conducted a comprehensive analysis of the 14 types of bone morphogenetic protein (BMPs) for their abilities to regulate multilineage specific differentiation of MSCs. We found that most BMPs exhibited distinct abilities to regulate the expression of Runx2, Sox9, MyoD, and PPARgamma2. Further analysis indicated that BMP-2, BMP-4, BMP-6, BMP-7, and BMP-9 effectively induced both adipogenic and osteogenic differentiation in vitro and in vivo. BMP-induced commitment to osteogenic or adipogenic lineage was shown to be mutually exclusive. Overexpression of Runx2 enhanced BMP-induced osteogenic differentiation, whereas knockdown of Runx2 expression diminished BMP-induced bone formation with a decrease in adipocyte accumulation in vivo. Interestingly, overexpression of PPARgamma2 not only promoted adipogenic differentiation, but also enhanced osteogenic differentiation upon BMP-2, BMP-6, and BMP-9 stimulation. Conversely, MSCs with PPARgamma2 knockdown or mouse embryonic fibroblasts derived from PPARgamma2(-/-) mice exhibited a marked decrease in adipogenic differentiation, coupled with reduced osteogenic differentiation and diminished mineralization upon BMP-9 stimulation, suggesting that PPARgamma2 may play a role in BMP-induced osteogenic and adipogenic differentiation. Thus, it is important to understand the molecular mechanism behind BMP-regulated lineage divergence during MSC differentiation, as this knowledge could help us to understand the pathogenesis of skeletal diseases and may lead to the development of strategies for regenerative medicine.     

Replicative aging and gene expression in long-term cultures of human bone marrow stromal cells

Bone marrow stromal cells (BMSCs) can be easily isolated from adult marrow and contain a population of pluripotent progenitors that can give rise to different mesenchymal lineages both in vitro and in vivo. These properties make BMSCs an attractive target for cell-based therapeutic strategies for a variety of disorders. However, because of their low frequency in vivo, to obtain a sufficient number of cells for tissue engineering a step of extensive in vitro expansion is required, which could significantly alter BMSC properties. Therefore, effective therapeutic use of BMSCs requires the design of appropriate approaches for in vitro cell expansion. In this study we have investigated the biological effects of in vitro expansion on BMSC proliferative ability and on their spontaneous differentiation. Telomerase activity and telomere shortening kinetics were evaluated together with variations in osteogenic, chondrogenic, and adipogenic gene expression throughout the BMSC life span. In culture BMSCs never displayed telomerase activity and during in vitro expansion telomere length decreased. Furthermore, gene expression patterns spontaneously varied during expansion, indicating a progressive commitment of the population toward the osteogenic lineage. In conclusion, BMSCs in culture undergo progressive replicative aging and osteogenic differentiation. These observations are relevant to their successful use in clinics and should be considered when designing novel therapeutic strategies.     

成年大鼠骨骼肌干细胞的制备与新型培养方法

 目的：建立高效分离和扩增成年大鼠骨骼肌干细胞的实验方法。方法：通过混合酶消化法从少量成年大鼠骨骼肌样品中分离出骨骼肌干细胞;采用悬浮培养法培养获得骨骼肌干细胞团,批量扩增骨骼肌干细胞;用qRT-PCR和免疫细胞化学染色检测干细胞标志物Nanog、Oct4和骨骼肌干细胞标志物肌源性因子5（myogenic factor 5,Myf5）、配对盒蛋白7 (paired box protein 7,Pax7)的表达;成脂或成骨分化诱导培养基诱导检测骨骼肌干细胞的多功能性。结果：采用混合酶消化法可获得骨骼肌干细胞单细胞,在悬浮的培养条件下能快速克隆成骨骼肌细胞团,贴壁培养后能看到骨骼肌干细胞从细胞团中爬出来,和传统的差速贴壁方法相比悬浮培养法获得的骨骼肌干细胞纯度更高,细胞状态更好,干性因子Nanog、Oct 4和骨骼肌干细胞标志物Myf5、Pax 7的表达更高（P<0.05）;在不同培养基的诱导下能向骨骼肌、成骨和脂肪细胞系分化。结论：悬浮培养法能获得大批量干性好、纯度高、具有多向分化潜能的骨骼肌干细胞,从而为肌肉相关疾病的细胞治疗提供优良的种子细胞。