低氧对MSCs增殖和分化的影响

间充质干细胞（mesenchymal stemcells, MSCs）是具有自我更新、多向分化和强可塑性的细胞,具有分化为血液、骨、软骨、脂肪、肌肉、表皮、上皮、神经等组织的潜能,受到再生医学研究的关注。目前已有研究表明将MSCs 移植到多种损伤组织中都能改善损伤组织的功能。文章在简要回顾了低氧环境对MSCs增殖和分化的研究内容和有关理论争论基础上重点介绍了缺氧诱导因子（HIF）通路对MSCs 增殖和分化的影响。文章阐述了低氧环境对MSCs向成骨,成软骨,成脂及成神经元方向分化的影响。由于人体组织内生理条件下的氧张力远远小于大气中的氧张力（21%）,采用低氧培养MSCs 的研究方法得出的结论将更加贴近实际MSCs在人体内的增殖、分化情况。因此研究MSCs 在低氧张力环境中增殖、分化的能力将为MSCs 能成功移植到体内并发挥作用提供保障。     

去卵巢对SD大鼠MSCs多向分化能力的影响

通过比较有、无诱导条件下正常与骨质疏松症（OP）大鼠骨髓间充质干细胞（MSCs）骨向、脂向及软骨向的分化情况,观察去卵巢对SD大鼠MSCs多向分化能力的影响。实验分为四组：正常组、正常诱导组、OP组、OP诱导组;分别在有或无成骨、成脂、成软骨诱导条件下,评价各组的分化情况。检测发现,无诱导条件下,正常大鼠MSCs骨向及软骨向分化的能力强于OP大鼠,而OP大鼠MSCs的脂向分化能力强于正常大鼠;诱导条件下,正常大鼠MSCs对骨向及软骨向诱导剂的反应能力强于OP大鼠,而OP大鼠MSCs对成脂诱导剂的反应能力强于正常大鼠。这些结果表明,去卵巢后大鼠MSCs骨向分化能力及对成骨诱导剂的反应力下降,脂向分化能力及对成脂诱导剂的反应力增强,对软骨诱导剂的反应力下降。     

骨髓间质干细胞向心肌细胞分化的可塑性及应用研究进展

减少心肌缺血后损伤,促进心肌细胞和血管再生是治疗心肌缺血损伤、心力衰竭的重要思路,而干细胞移植为该思路带来了新的曙光。骨髓间质干细胞（-mesenchymal stem cells,MSCs）,也称为骨髓基质细胞,能分化为骨、软骨和脂肪细胞表型。研究表明,MSCs还能分化为内皮细胞、神经细胞、平滑肌细胞、骨骼肌细胞和心肌细胞表型。MSCs具有多向分化的潜能,且自体移植可以避免免疫排斥反应,同时也易于在体外大量扩增。研究显示,MSCs移植能抑制损伤心肌的重塑和改善心肌功能。因此,骨髓间质干细胞移植给人们展示了一个诱入的前景。本文综述了近年来有关MSCs特性的新认识,尤其是MSCs向心肌细胞方向分化的可塑性、影响因素和信号转导机制,以及MSCs治疗心肌梗死的动物实验和临床研究进展。     

人体皮肤成纤维细胞的快速分离及多向分化能力鉴定

目的建立快速分离人皮肤成纤维细胞的方法,并探讨成纤维细胞在成脂、成骨、成软骨和成神经的多向分化潜能。 方法利用组织块培养法分离人体皮肤成纤维细胞,通过形态学观察、流式分析、Vimentin蛋白染色鉴定成纤维细胞;再利用生长曲线、核型分析、线粒体染色分析不同传代细胞的增殖速度,线粒体及染色体形态的改变;最后进行成纤维细胞成脂、成骨、成软骨和成神经的诱导分化实验,鉴定其多向分化潜能。两代细胞增殖速度及线料体相对量的比较采用t检验统计分析。 结果分离的皮肤成纤维细胞呈典型梭状及多角形;高表达细胞表面标记物CD90 （NCF1,NCF2占比分别为99.9%,98.7%）和CD73 （NCF1,NCF2占比分别为98.2%,85.6%）,但极少表达造血干细胞标记物CD34 （NCF1,NCF2占比分别为1.8%,2.6%）;细胞Vimentin蛋白表达呈阳性,阳性率为100%;对细胞生长曲线进行分析,表明分离后不同代次细胞增殖差异无统计学意义（t?= 1.586,P?= 0.1567）;线粒体相对含量统计分析,同一株细胞系第5代（相对荧光强度值：6876±577.8）与第10代（相对荧光强度值：7371±471.9）之间的差异无统计学意义（t?= 0.664,P?= 0.543）;核型分析分别显示传代后保持染色体数目为正常46条且形态无明显异常;经诱导后成纤维细胞可向成脂、成骨、成软骨和类神经分化。 结论利用组织块培养法分离出的人体皮肤成纤维细胞状态稳定,增殖能力强,具有成脂、成骨、成软骨和成神经多向分化诱导潜能,为细胞移植修复骨损伤、软骨损伤和神经损伤性疾病的临床研究提供细胞来源及实验依据。     

去卵巢骨质疏松山羊骨髓基质细胞成骨分化能力的研究

目的：研究在构建的去卵巢骨质疏松山羊动物模型中,骨髓基质细胞（MSCs）的生物学特性以及其成骨能力。方法：建立去卵巢骨质疏松山羊动物模型,使用全骨髓法获取去卵巢骨质疏松山羊（实验组）和正常山羊（对照组）MSCs,流式细胞仪检测实验组和对照组细胞周期及增殖指数（PI）;地塞米松诱导21d时油红O染色,观察成脂分化比例;成骨诱导液诱导14d,碱性磷酸酶（ALP）染色、检测ALP表达量。结果：对照组PI高于实验组;地塞米松诱导后实验组脂肪细胞比例明显高于对照组;成骨诱导第7d,对照组ALP的表达量明显高于实验组。结论：去卵巢骨质疏松山羊的MSCs增殖和成骨分化能力都降低,可能与骨质疏松症的发病机理有关。     

黄芪多糖对低氧环境中BMSCs成脂分化的影响

探讨黄芪多糖(APS)对低氧环境中骨髓间充质干细胞(BMSCs)成脂诱导分化的影响。采用四甲基偶氮唑盐(Methyl thiazolyl tetrazolium,MTT)法筛选促进BMSCs增殖的最佳APS浓度,干预不同氧浓度下(3%、6%、10%和20%)成脂诱导培养的BMSCs,通过油红O染色观察细胞内脂滴形成,Real-time PCR和Western blotting检测成脂相关基因过氧化物酶体增殖物激活受体γ_2(PPAR-γ_2)和脂蛋白脂肪酶(LPL)的mRNA和蛋白水平。结果表明,与对照组相比,40μg/mL APS能显著促进不同氧浓度下BMSCs的增殖(P0.05);含有40μg/mL APS的成脂诱导剂能提升低氧环境中BMSCs内脂滴含量及PPAR-γ_2和LPL的蛋白和mRNA水平,在氧浓度为10%时其促进作用较显著(P0.05),差异有统计学意义。40μg/mL APS具有促进低氧环境中BMSCs增殖和成脂诱导分化的作用,其促分化作用与细胞培养的氧环境相关。     

骨髓来源的间充质干细胞分化能力的鉴定

本文研究了人骨髓来源的间充质干细胞(MSCs)的成骨及成脂分化的潜能.通过加入诱导成骨的诱导剂,人的MSCs出现成骨分化的机箱,通过碱性磷酸酶活性测定,茜素红染色及主要调控基因BMP2和Runx2的表达,确定了MSCs具有成骨分化的潜能.对于成脂分化,通过油红O染色,及主要标志基因PPARγ的表达确定其具有成脂分化的潜能.所以,从骨髓分离的到的MSCs纯度达到标准,并且具有成骨成脂分化的多向潜能,是一种理想的实验模型细胞.     

间充质干细胞长期传代培养条件的研究

间充质干细胞(mesenchymal stem cells,MSCs)是存在于成体组织间质部分的多能前体细胞,在体外具有自我更新增殖及向成脂、成骨、成软骨分化的潜能,在组织工程和细胞治疗方面具有广阔的应用前景。MSCs在体外长期培养获得足够数量的细胞是MSCs应用的一个重要因素。然而,目前还没有建立MSCs长期传代培养的最适培养体系。该文分别从培养体系中的基础培养基、血清和生长因子对于MSCs细胞长期传代培养的影响进行了论述,旨在为建立MSCs体外长期传代生长的最适培养体系提供理论依据。     

骨髓间充质干细胞对移植肾缺血再灌注损伤保护作用的实验研究

目的 观察骨髓间充质干细胞（MSCs）对移植肾缺血再灌注损伤（IRI）模型修复的保护作用,及其作用机制的思路。方法 （1）采用密度梯度离心法结合贴壁分离法分离培养纯化SD大鼠骨髓MSCs,观察其形态,流式细胞仪检测细胞表面标记,检测骨髓MSCs向成骨和成脂细胞分化的潜能;（2）成年雌性SD大鼠28只,随机分组：正常对照组（control group,n=6）,假手术对照组（sham-operated group,n=6）,移植肾IRI组（vehicle-treated I/R group,n=8）,经尾静脉输注间充质干细胞（MSCs）移植肾IRI组（MSCs-treated via tail vein I/R group,n=8）。检测肾功能指标血尿素氮（BUN）和肌酐（Cr）水平变化,评定肾小管的凋亡指数和增殖指数,测定肾组织起氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）活性及微量丙二醛（MDA）水平,以及对肾脏病理学变化进行观察。结果 （1）分离培养的骨髓MSCs纯度高、生物学特征稳定;（2）移植肾IRI组肾功能指标（BUN36.9±4.8,Scr279.9±22.6）、氧化应激指标明显升高,组织形态学出现肾间质水肿明显,肾小管上皮细胞空泡样变性,近曲小管管壁肿胀,管腔变小。而经尾静脉输注MSCs移植肾IRI组大鼠肾功能指标（BUN22.6±7.8,Scr223.6±26.7）和氧化应激指标得到明显改善（P〈0.05）,组织形态学肾小管上皮细胞细胞核固缩、碎裂和溶解等细胞坏死和变性征象明显减轻,肾小管上皮细胞增殖指数（PI）高于IRI组,肾小管上皮细胞凋亡指数（AI）低于IRI组,两组间差异有统计学意义（P〈0.05）。结论 骨髓MSCs输注能促进肾脏IRI损伤后肾脏细胞增殖,抑制肾脏细胞凋亡,降低血清Creatinine和BUN,在一定程度上促进IRI后肾功能的恢复,通过抑制氧自由基的生成减轻肾组织的损伤程度,改善肾功能。     

多能成体祖细胞培养条件促进人脂肪干细胞的视网膜神经保护功能

目的：检测多能成体祖细胞（MAPC）的培养条件对猴骨髓间充质细胞（BMMSCs）和人脂肪干细胞（hADSCs）生长的影响,旨在获得更适合治疗视网膜变性疾病的供体细胞。方法通过细胞形态观察、MTT实验、克隆形成率、PCR检测、以及成脂、成骨、成软骨分化潜能检测等,研究MAPC培养条件下猴BMMSCs和hADSCs的特征,并用DMEM/LG和MAPC培养条件培养的hADSCs进行RCS大鼠视网膜下腔移植,通过视网膜电图（ERG）和TUNEL检测,判断细胞移植治疗对视功能及视网膜细胞凋亡的影响。结果与常规培养基相比,MAPC培养条件能促进猴BMMSCs增殖,细胞变小,但传2代后,细胞变得宽大扁平,出现衰老征象;然而,MAPC培养条件下的hADSCs细胞增殖能力及克隆形成率均增强,形成的克隆较大可稳定传10代以上,且具有成脂、成骨、成软骨的多向分化潜能,细胞表面标记物及细胞因子出现差异表达：CD140b、CD90、CD47、HGF和PEDF显著上调,CD73、CD105和IL-6显著下调。与对照组相比,移植DMEM/LG和MAPC培养条件培养的hADSCs（P4）3周后,RCS大鼠的B波波幅明显升高,外核层细胞凋亡明显减少。结论 MAPC培养条件培养的hADSCs显示出更好的视网膜神经保护作用,适合用于治疗视网膜退行性疾病。     

Oxygen tension regulates the osteogenic, chondrogenic and endochondral phenotype of bone marrow derived mesenchymal stem cells

The local oxygen tension is a key regulator of the fate of mesenchymal stem cells (MSCs). The objective of this study was to investigate the effect of a low oxygen tension during expansion and differentiation on the proliferation kinetics as well as the subsequent osteogenic and chondrogenic potential of MSCs. We first hypothesised that expansion in a low oxygen tension (5% pO(2)) would improve both the subsequent osteogenic and chondrogenic potential of MSCs compared to expansion in a normoxic environment (20% pO(2)). Furthermore, we hypothesised that chondrogenic differentiation in a low oxygen environment would suppress hypertrophy of MSCs cultured in both pellets and hydrogels used in tissue engineering strategies. MSCs expanded at 5% pO(2) proliferated faster forming larger colonies, resulting in higher cell yields. Expansion at 5% pO(2) also enhanced subsequent osteogenesis of MSCs, whereas differentiation at 5% pO(2) was found to be a more potent promoter of chondrogenesis than expansion at 5% pO(2). Greater collagen accumulation, and more intense staining for collagen types I and X, was observed in pellets maintained at 20% pO(2) compared to 5% pO(2). Both pellets and hydrogels stained more intensely for type II collagen when undergoing chondrogenesis in a low oxygen environment. Differentiation at 5% pO(2) also appeared to inhibit hypertrophy in both pellets and hydrogels, as demonstrated by reduced collagen type X and Alizarin Red staining and alkaline phosphatase activity. This study demonstrates that the local oxygen environment can be manipulated in vitro to either stabilise a chondrogenic phenotype for use in cartilage repair therapies or to promote hypertrophy of cartilaginous grafts for endochondral bone repair strategies.     

The involvement of histone methylation in osteoblastic differentiation of human periosteum‐derived cells cultured in vitro under hypoxic conditions

Although oxygen concentrations affect the growth and function of mesenchymal stem cells (MSCs), the impact of hypoxia on osteoblastic differentiation is not understood. Likewise, the effect of hypoxia‐induced epigenetic changes on osteoblastic differentiation of MSCs is unknown. The aim of this study was to examine the in vitro hypoxic response of human periosteum‐derived cells (hPDCs). Hypoxia resulted in greater proliferation of hPDCs as compared with those cultured in normoxia. Further, hypoxic conditions yielded decreased expression of apoptosis‐ and senescence‐associated genes by hPDCs. Osteoblast phenotypes of hPDCS were suppressed by hypoxia, as suggested by alkaline phosphatase activity, alizarin red‐S‐positive mineralization, and mRNA expression of osteoblast‐related genes. Chromatin immunoprecipitation assays showed an increased presence of H3K27me3, trimethylation of lysine 27 on histone H3, on the promoter region of bone morphogenetic protein‐2. In addition, mRNA expression of histone lysine demethylase 6B (KDM6B) by hPDCs was significantly decreased in hypoxic conditions. Our results suggest that an increased level of H3K27me3 on the promoter region of bone morphogenetic protein‐2, in combination with downregulation of KDM6B activity, is involved in the suppression of osteogenic phenotypes of hPDCs cultured in hypoxic conditions. Although oxygen tension plays an important role in the viability and maintenance of MSCs in an undifferentiated state, the effect of hypoxia on osteoblastic differentiation of MSCs remains controversial. In addition, evidence regarding the importance of epigenetics in regulating MSCs has been limited. This study was to examine the role hypoxia on osteoblastic differentiation of hPDCs, and we examined whether histone methylation is involved in the observed effect of hypoxia on osteogenic differentiation of hPDCs.     

Forced expression of Sox2 or Nanog in human bone marrow derived mesenchymal stem cells maintains their expansion and differentiation capabilities

Mesenchymal stem cells (MSCs) derived from human bone marrow have capability to differentiate into cells of mesenchymal lineage. The cells have already been applied in various clinical situations because of their expansion and differentiation capabilities. The cells lose their capabilities after several passages, however. With the aim of conferring higher capability on human bone marrow MSCs, we introduced the Sox2 or Nanog gene into the cells. Sox2 and Nanog are not only essential for pluripotency and self-renewal of embryonic stem cells, but also expressed in somatic stem cells that have superior expansion and differentiation potentials. We found that Sox2-expressing MSCs showed consistent proliferation and osteogenic capability in culture media containing basic fibroblast growth factor (bFGF) compared to control cells. Significantly, in the presence of bFGF in culture media, most of the Sox2-expressing cells were small, whereas the control cells were elongated in shape. We also found that Nanog-expressing cells even in the absence of bFGF had much higher capabilities for expansion and osteogenesis than control cells. These results demonstrate not only an effective way to maintain proliferation and differentiation potentials of MSCs but also an important implication about the function of bFGF for self-renewal of stem cells including MSCs.     

Isolation of mesenchymal stem cells from human placenta: comparison with human bone marrow mesenchymal stem cells

The presence within bone marrow of a population of mesenchymal stem cells (MSCs) able to differentiate into a number of different mesenchymal tissues, including bone and cartilage, was first suggested by Friedenstein nearly 40 years ago. Since then MSCs have been demonstrated in a variety of fetal and adult tissues, including bone marrow, fetal blood and liver, cord blood, amniotic fluid and, in some circumstances, in adult peripheral blood. MSCs from all of these sources can be extensively expanded in vitro and when cultured under specific permissive conditions retain their ability to differentiate into multiple lineages including bone, cartilage, fat, muscle, nerve, glial and stromal cells. There has been great interest in these cells both because of their value as a model for studying the molecular basis of differentiation and because of their therapeutic potential for tissue repair and immune modulation. However, MSCs are a rare population in these tissues. Here we tried to identify cells with MSC-like potency in human placenta. We isolated adherent cells from trypsin-digested term placentas and examined these cells for morphology, surface markers, and differentiation potential and found that they expressed several stem cell markers. They also showed endothelial and neurogenic differentiation potentials under appropriate conditions. We suggest that placenta-derived cells have multilineage differentiation potential similar to MSCs in terms of morphology and cell-surface antigen expression. The placenta may prove to be a useful source of MSCs.     

The monoclonal antibody SH-2, raised against human mesenchymal stem cells, recognizes an epitope on endoglin (CD105).

Mesenchymal stem cells are multipotent cells resident in the bone marrow throughout adulthood which have the capacity to differentiate into cartilage, bone, fat, muscle, and tendon. A number of monoclonal antibodies raised against human MSCs have been shown to react with surface antigens on these cells in vitro. A protein of molecular mass 92 kDa was immunoprecipitated using the SH-2 monoclonal antibody. This was purified and identified by peptide sequencing analysis and mass spectrometry as endoglin (CD105), the TGF-beta receptor III present on endothelial cells, syncytiotrophoblasts, macrophages, and connective tissue stromal cells. Endoglin on MSCs potentially plays a role in TGF-beta signalling in the control of chondrogenic differentiation of MSCs and also in mediating interactions between MSCs and haematopoietic cells in the bone marrow microenvironment.     

Short-term exposure of multipotent stromal cells to low oxygen increases their expression of CX3CR1 and CXCR4 and their engraftment in vivo

The ability of stem/progenitor cells to migrate and engraft into host tissues is key to their potential use in gene and cell therapy. Among the cells of interest are the adherent cells from bone marrow, referred to as mesenchymal stem cells or multipotent stromal cells (MSC). Since the bone marrow environment is hypoxic, with oxygen tensions ranging from 1% to 7%, we decided to test whether hypoxia can upregulate chemokine receptors and enhance the ability of human MSCs to engraft in vivo. Short-term exposure of MSCs to 1% oxygen increased expression of the chemokine receptors CX3CR1and CXCR4, both as mRNA and as protein. After 1-day exposure to low oxygen, MSCs increased in vitro migration in response to the fractalkine and SDF-1alpha in a dose dependent manner. Blocking antibodies for the chemokine receptors significantly decreased the migration. Xenotypic grafting into early chick embryos demonstrated cells from hypoxic cultures engrafted more efficiently than cells from normoxic cultures and generated a variety of cell types in host tissues. The results suggest that short-term culture of MSCs under hypoxic conditions may provide a general method of enhancing their engraftment in vivo into a variety of tissues.     

Regulation of differentiation of mesenchymal stem cells into musculoskeletal cells

Mesenchymal stem cells (MSCs) are multipotent cells that have the capability of differentiating into several different cells such as osteoblasts (bone), chondrocytes (cartilage), adipocytes (fat), myocytes (muscle) and tenocytes (tendon). In this review we highlight the different regulators which determine the lineage a particular MSC will differentiate into. Mesenchymal stem cells are increasingly being used in tissue regeneration and repair. Strict regulation of differentiation of MSCs is essential for a positive outcome of the particular tissue treated with MSCs, especially due to the fact that capacity to differentiate decreases with increasing age of the donor.     

Increased Proliferation and Analysis of Differential Gene Expression in Human Wharton's Jelly-derived Mesenchymal Stromal Cells under Hypoxia

Multipotent mesenchymal stromal cells (MSCs) from Wharton''s jelly (WJ) of umbilical cord bear higher proliferation rate and self-renewal capacity than adult tissue-derived MSCs and are a primitive stromal cell population. Stem cell niche or physiological microenvironment plays a crucial role in maintenance of stem cell properties and oxygen concentration is an important component of the stem cell niche. Low oxygen tension or hypoxia is prevalent in the microenvironment of embryonic stem cells and many adult stem cells at early stages of development. Again, in vivo, MSCs are known to home specifically to hypoxic events following tissue injuries. Here we examined the effect of hypoxia on proliferation and in vitro differentiation potential of WJ-MSCs. Under hypoxia, WJ-MSCs exhibited improved proliferative potential while maintaining multi-lineage differentiation potential and surface marker expression. Hypoxic WJ-MSCs expressed higher mRNA levels of hypoxia inducible factors, notch receptors and notch downstream gene HES1. Gene expression profile of WJ-MSCs exposed to hypoxia and normoxia was compared and we identified a differential gene expression pattern where several stem cells markers and early mesodermal/endothelial genes such as DESMIN, CD34, ACTC were upregulated under hypoxia, suggesting that in vitro culturing of WJ-MSCs under hypoxic conditions leads to adoption of a mesodermal/endothelial fate. Thus, we demonstrate for the first time the effect of hypoxia on gene expression and growth kinetics of WJ-MSCs. Finally, although WJ-MSCs do not induce teratomas, under stressful and long-term culture conditions, MSCs can occasionally undergo transformation. Though there were no chromosomal abnormalities, certain transformation markers were upregulated in a few of the samples of WJ-MSCs under hypoxia.