Simvastatin induces the osteogenic differentiation of human periodontal ligament stem cells

Periodontal ligament stem cells (PDLSCs) are considered as potential mesenchymal stem cell sources for future clinical applications in periodontal regeneration therapy. Simvastation, widely used for lowering serum cholesterol, is known to have a bone stimulatory effect. However, it is not clear whether simvastation affects the differentiation of PDLSCs. This study examined the effects of simvastatin on human PDLSCs in vitro and in vivo. Using the limiting dilution technique, human PDLSCs were isolated and expanded. PDLSCs were cultured with simvastatin (0.01–10 μm ), and the proliferation was measured. The osteogenic differentiation was characterized by alkaline phosphatase (ALP) activity and Alizarin Red‐S staining for calcium deposition. The gene expression levels of osteogenic markers were evaluated by RT‐PCR. In addition, PDLSCs were transplanted into nude mice with ceramic bovine bone powders as carriers to observe the capacity of mineralized tissue formation in vivo. Simvastatin at concentrations <1 μm did not suppress the proliferation of PDLSCs. After the administration of 0.1 μm simvastatin, the expression of ALP, bone sialoprotein, and bone morphogenetic protein‐2 genes were significantly upregulated, and the ALP activity and mineralized nodule formation were significantly higher in the simvastatin‐treated cells than the control cells. In addition, the in vivo transplantation results showed that simvastatin treatment promoted the degree of mineralized tissue formation. Collectively, simvastatin has positive effects on osteogenic differentiation of human PDLSCs in vitro and in vivo. This suggests that simvastatin might be a useful osteogenic induction agent for periodontal bone regeneration.     

Platelet lysate supports the in vitro expansion of human periodontal ligament stem cells for cytotherapeutic use

Human platelet lysate (PL) produced under optimal conditions of standardization and safety has been increasingly suggested as the future ‘gold standard’ supplement to replace fetal bovine serum (FBS) for the ex vivo propagation of mesenchymal stem cells for translational medicine and cell therapy applications. However, the multifaceted effects of PL on tissue‐specific stem cells remain largely unexplored. In the present study, we investigated the stem cell behaviours of human periodontal ligament stem cells (PDLSCs) in media with or without PL. Our data indicate that human PL, either as an adjuvant for culture media or as a substitute for FBS, supports the proliferation and expansion of human PDLSCs derived from either ‘young’ or ‘old’ donors to the same extent as FBS, without interfering with their immunomodulatory capacities. Although PL appears to inhibit the in vitro differentiation of ‘young’ or ‘old’ PDLSCs, their decreased osteogenic potential may be restored to similar or higher levels compared with FBS‐expanded cells. PL‐ and FBS‐expanded PDLSCs exhibited a similar potential to form mineralized nodules and expressed similar levels of osteogenic genes. Our data indicate that large clinically relevant quantities of PDLSCs may be yielded by the use of human PL; however, further analysis of its precise composition and function will pave the way for determining optimized, defined culture conditions. In addition to the potential increase in patient safety, our findings highlight the need for further research to develop the potential of PL‐expanded PDLSCs for clinical use. Copyright © 2016 John Wiley & Sons, Ltd.     

Combination of platelet‐rich plasma within periodontal ligament stem cell sheets enhances cell differentiation and matrix production

The longstanding goal of periodontal therapy is to regenerate periodontal tissues. Although platelet‐rich plasma (PRP) has been gaining increasing popularity for use in the orofacial region, whether PRP is useful for periodontal regeneration is still unknown. The purpose of this study was to determine whether a mixture of periodontal ligament stem cell (PDLSC) sheets and PRP promoted bone regeneration, one of the most important measurement indices of periodontal tissue regenerative capability in vitro and in vivo. In this study, we evaluated the effects of different doses of PRP on the differentiation of human PDLSCs. Then cell sheet formation, extracellular matrix deposition and osteogenic gene expression in response to different doses of PRP treatment during sheet grafting was investigated. Furthermore, we implanted PDLSC sheets treated with 1% PRP subcutaneously into immunocompromised mice to evaluate their bone‐regenerative capability. The results revealed that 1% PRP significantly enhanced the osteogenic differentiation of PDLSCs. Based on the production of extracellular matrix proteins, the results of scanning electron microscopy and the expression of the osteogenic genes ALP, Runx2, Col‐1 and OCN, the provision of 1% PRP for PDLSC sheets was the most effective PRP administration mode for cell sheet formation. The results of in vivo transplantation showed that 1% PRP‐mediated PDLSC sheets exhibited better periodontal tissue regenerative capability than those obtained without PRP intervention. These data suggest that a suitable concentration of PRP stimulation may enhance extracellular matrix production and positively affect cell behaviour in PDLSC sheets. Copyright © 2014 John Wiley & Sons, Ltd.     

不同组织冻存体系对牙周膜干细胞体外扩增的影响

背景：冻存是保证干细胞移植治疗的关键步骤之一。传统的冻存是将细胞直接置于冻存液中进行保存,然而冻存液中二甲基亚砜虽能减少细胞复苏过程中冰晶对细胞膜产生的机械性损伤,但同时又对细胞具有毒性作用,直接影响细胞生存状态,不利于临床移植治疗。目的：寻找适宜牙周膜干细胞体外扩增的牙周周组织冻存的最佳方案。方法：收集健康人牙,刮取牙周组织后将其平均分为3等份,随机分为新鲜组、5%二甲基亚砜组和10%二甲基亚砜组,后2组分别以体积分数5%和10%二甲基亚砜添加冻存1个月后提取牙周膜干细胞。新鲜组直接提取牙周膜干细胞。结果与结论：5%二甲基亚砜组原代细胞游出组织团块所需时间和细胞收获量虽不及新鲜培养组,但却明显优于10%二甲基亚砜组（P〈0.05）。新鲜组、5%二甲基亚砜组和10%二甲基亚砜组第1代牙周膜干细胞克隆形成率、活细胞比率、第3代牙周膜干细胞BrdU细胞增殖能力、MTT细胞生长曲线和牙周膜干细胞表面标志物表达差异没有显著性意义（P〉0.05）。提示5%二甲基亚砜添加冻存体系不仅能比10%二甲基亚砜添加的普通冻存体系明显缩短牙周膜干细胞体外扩增所需时间,增加细胞收获量同时还能保持细胞基本生物学特性,降低二甲基亚砜的总体用量和其在反复冻存复苏细胞过程中对细胞造成的直接损伤,为未来更加安全的实施临床移植治疗提供了保障,是供体组织储存新的选择。     

人牙周膜干细胞与牙周膜细胞生物学特性的比较

背景：牙周膜干细胞生物作用是目前牙周病治疗研究的热点,牙周膜成纤维细胞是其分化的终末功能细胞之一,也是其主要的支持细胞,两者生物学特性的差异研究鲜有报道。目的：比较牙周膜干细胞与牙周膜细胞生物学特性的差异。方法：用组织块法体外对牙周膜细胞以及单细胞克隆分离纯化后的人牙周膜干细胞两种细胞分别进行显微镜下形态观察,CCK8法检测并绘制2种细胞的生长曲线。流式细胞分析比较2种细胞的细胞周期以及细胞表面标记物的表达、实时PCR对2种细胞碱性磷酸酶、增殖细胞核抗原和Scleraxis基因进行检测。结果与结论：牙周膜干细胞与牙周膜成纤维细胞外观差别明显,人牙周膜干细胞的生长曲线培养前5d要低于牙周膜细胞,但在5 d后明显高于牙周膜细胞。人牙周膜干细胞与牙周膜细胞的细胞周期分别为41.1%和23.9%。表面标记物检测结果显示2种细胞虽有相似的表达,但在表达率差异有有显著性意义。实时荧光定量PCR结果显示,人牙周膜干细胞在碱性磷酸酶、增殖细胞核抗原以及Scleraxis基因的表达检测均高于牙周膜细胞。表明牙周膜干细胞在成骨增殖等生物学功能上比牙周膜细胞具有更强的潜能。     

Osteogenic differentiated periodontal ligament stem cells maintain their immunomodulatory capacity

Periodontal ligament stem cells (PDLSCs) have great potential for regenerating periodontal ligament tissue, which is involved in attaching teeth to the underlying alveolar bone. Recently, PDLSCs were characterized as having both low immunogenicity and profound immunomodulation abilities. Further, transplanted PDLSCs differentiate into osteoblasts in vivo. In the present study, we investigated the immunological characteristics of osteogenic differentiated PDLSCs. We found that PDLSCs expressed mesenchymal stem cells markers, including STRO‐1 and CD146, but were negative for CD14, CD34 and CD45. RT–PCR indicated that NCAM1, MSX1 and S100A4 were expressed in PDLSCs. The cells underwent osteogenic and adipogenic differentiation when cultured in defined medium. Osteogenic differentiated PDLSCs failed to stimulate allogeneic T cell proliferation and suppressed phytohaemagglutinin‐triggered T cell proliferation. Indomethacin, an inhibitor of prostaglandin E2 (PGE2) production, restored the T cell proliferation inhibited by osteogenic differentiated PDLSCs. These data confirm that osteogenic differentiated PDLSCs have low immunogenicity and demonstrate that they suppress T cell proliferation in vitro through secretion of PGE2. Copyright © 2012 John Wiley & Sons, Ltd.     

牙周膜干细胞成骨分化中细胞因子的作用

背景：牙周膜干细胞是牙周组织中的成体干细胞,具有高度增殖、自我更新能力和多分化潜能。促进牙周膜干细胞向成骨细胞分化有助于牙周疾病的治疗。目的：观察胰岛素样生长因子1和成纤维细胞生长因子2对牙周膜干细胞向成骨细胞分化的影响。方法：采用胶原酶消化人牙周膜组织,获得牙周膜干细胞,经体外鉴定、扩增后,通过倒置显微镜、苏木精-伊红染色、流式细胞仪对牙周膜干细胞进行生物学检测。分别在成骨细胞诱导培养液中加入成骨诱导液（对照组）及胰岛素样生长因子1和成纤维细胞生长因子2持续诱导7,14d后,进行碱性磷酸酶染色、碱性磷酸酶活性检测,以及茜素红染色,并用实时定量PCR法检测向成骨细胞分化的标志性基因的表达情况。结果与结论：胰岛素样生长因子1刺激组的碱性磷酸酶活性以及钙化结节明显高于对照组,Runx2、Alp、col-1的mRNA呈高表达;成纤维细胞生长因子2刺激组的碱性磷酸酶活性以及钙化结节也高于对照组,Runx2、Alp、col-1的mRNA表达量也高于对照组。提示胰岛素样生长因子1和成纤维细胞生长因子2在不同程度上促进体外培养的牙周膜干细胞向成骨细胞方向分化。     

人骨髓来源细胞外基质对人牙周膜干细胞增殖的影响简

背景：研究发现人来源的骨髓细胞外基质能促进人牙周膜干细胞增殖并保持干细胞的特性。目的：初步观察人骨髓来源的细胞外基质对人牙周膜干细胞增殖能力影响。方法：分离人正常牙周膜细胞及颌骨骨髓细胞,制备骨髓细胞外基质膜片。采用有限稀释法克隆培养纯化人牙周膜干细胞,透射电镜观察细胞超微结构。取P2代人牙周膜干细胞与骨髓细胞来源细胞外基质共同培养,以普通培养基对照,采用CCK8法及细胞周期流式技术检测人骨髓细胞来源细胞外基质对人牙周膜干细胞增殖能力的影响。结果与结论：实验组人牙周膜干细胞较对照组相比细胞增殖能力显著增强（P＜0.05）,且细胞符合其生物学形态及生物学特性,生长状态良好。说明实验建立起的骨髓细胞外基质能够促进牙周膜干细胞的增殖,是一种扩增干细胞的有效方法。     

Human platelet lysate supports the formation of robust human periodontal ligament cell sheets

The use of stem cell‐derived sheets has become increasingly common in a wide variety of biomedical applications. Although substantial evidence has demonstrated that human platelet lysate (PL) can be used for therapeutic cell expansion, either as a substitute for or as a supplement to xenogeneic fetal bovine serum (FBS), its impact on cell sheet production remains largely unexplored. In this study, we manufactured periodontal ligament stem cell (PDLSC) sheets in vitro by incubating PDLSCs in sheet‐induction media supplemented with various ratios of PL and FBS, i.e. 10% PL without FBS, 7.5% PL + 2.5% FBS, 5% PL + 5% FBS, 2.5% PL + 7.5% FBS or 10% FBS without PL. Cultures with the addition of all the designed supplements led to successful cell sheet production. In addition, all the resultant cellular materials exhibited similar expression profiles of matrix‐related genes and proteins, such as collagen I, fibronectin and integrin β1. Interestingly, the cell components within sheets generated by media containing both PL and FBS exhibited improved osteogenic potential. Following in vivo transplantation, all sheets supported significant new bone formation. Our data suggest that robust PDLSC sheets can be produced by applying PL as either an alternative or an adjuvant to FBS. Further examination of the relevant influences of human PL that benefit cell behaviour and matrix production will pave the way towards optimized and standardized conditions for cell sheet production.     

血清预培养促进神经干细胞的增殖

目的 介绍一种时间短、花费少的神经干细胞培养方法。方法 先用含10％胎牛血清的DMEM培养液预培养神经干细胞48h，换含表皮生长因子、碱性成纤维细胞生长因子、B27的DMEM／F12培养液继续培养干细胞球。血清诱导分化后行nestin免疫荧光染色。结果 血清预培养48h后即可见神经干细胞聚球，nestin免疫荧光染色为阳性。结论 血清预培养可使神经干细胞聚球速度加快，促进神经干细胞增殖。     

The effects of fluoxetine on the human adipose‐derived stem cell proliferation and differentiation

Fluoxetine is one of the most commonly used antidepressants. Fluoxetine could prevent the mesenchymal stem cell differentiation in lung fetus of rat. Moreover, the mesenchymal stem cells are also present in adult tissues. Therefore, in the current study, we aimed to investigate the effects of fluoxetine (FLX) on both proliferation and adipogenic/osteogenic differentiation of human adipose‐derived stem cells (ADSCs). After culturing of human ADSCs, these cells were treated with two concentrations of FLX (10 and 20 μm ). Then, cells were differentiated by adding osteogenic and adipogenic media. The effect of FLX on human ADSCs proliferation was evaluated by MTT assay. Fluoxetine role on adipogenic and osteogenic differentiation of human ADSCs was analyzed by oil red and alizarin red staining and RT‐PCR reaction. According to MTT assay, FLX showed a time‐ and concentration‐dependent proliferation response and eventually decreased human ADSCs proliferation. RT‐PCR analysis indicated that FLX significantly diminished the expression of osteogenesis‐related genes such as RUNX2 and alkaline phosphatase (ALP). Data also revealed a significant reduction in the expression of peroxisome proliferator‐activated receptor γ (PPARγ) and fatty acid‐binding protein (FABP) (specific genes of adipogenic lineage). In addition, FLX decreased mineralized matrix and the amount of lipid droplets in human ADSCs by staining methods. Our observation demonstrated that the effects of FLX may be time‐dependent. This drug possesses an increasing phase in proliferation and survival of human ADSCs (first 24 h) following a decreasing phase (after 48 h). Moreover, FLX could attenuate both osteogenic and adipogenic differentiation of human ADSCs.     

利用细胞外基质大规模扩增临床级人脂肪间充质干细胞

背景：如何运用无血清培养体系大规模扩增处于未分化状态的脂肪间充质干细胞,并使其保持“干性”是脂肪间充质干细胞临床应用转化中急待解决的难题。目的：建立含细胞外基质的脂肪间充质干细胞体外培养系统,验证其扩增细胞的高效性、有效性及安全性。方法：在无血清培养条件下,将体外分离得到的脂肪间充质干细胞分别接种在包被细胞外基质的培养板和传统二维塑料培养板上。经过体外扩增后,比较2种条件下细胞数量、细胞表面标记物表达、细胞衰老状况以及体外多向分化能力（诱导成脂、成骨和成软骨）的差异,考察脂肪间充质干细胞在含细胞外基质的培养条件下扩增后的临床安全性。结果与结论：脂肪间充质干细胞扩增到第5代时,包被细胞外基质培养板的细胞产量已经是传统二维塑料培养板的10倍以上,流式细胞检测表明,在含细胞外基质条件下扩增的脂肪间充质干细胞仍保持了干细胞表面特定标记物的表达。细胞衰老检测结果显示,使用包被细胞外基质的培养板扩增脂肪间充质干细胞至第15代时,细胞仍几乎无老化现象,而使用二维塑料培养板扩增的细胞在第5代时已出现明显老化,传代后细胞增殖能力明显下降。体外多向诱导分化实验显示,脂肪间充质干细胞在含细胞外基质条件下扩增至第15代时仍具有成脂、成骨和成软骨的分化功能,并且与第5代时没有明显差异,同时显著优于传统培养条件下的第5代脂肪间充质干细胞。染色体核型分析及小鼠成瘤性试验结果表明,脂肪间充质干细胞在含细胞外基质条件下扩增后仍具备临床应用的安全性。以上结果证实,含细胞外基质的无血清培养系统可以更高效且安全地扩增出具有临床应用潜能的脂肪间充质干细胞。     

MicroRNA‐132 directs human periodontal ligament‐derived neural crest stem cell neural differentiation

Neurogenesis is the basis of stem cell tissue engineering and regenerative medicine for central nervous system (CNS) disorders. We have established differentiation protocols to direct human periodontal ligament‐derived stem cells (PDLSCs) into neuronal lineage, and we recently isolated the neural crest subpopulation from PDLSCs, which are pluripotent in nature. Here, we report the neural differentiation potential of these periodontal ligament‐derived neural crest stem cells (NCSCs) as well as its microRNA (miRNA) regulatory mechanism and function in NCSC neural differentiation. NCSCs, treated with basic fibroblast growth factor and epidermal growth factor‐based differentiation medium for 24 days, expressed neuronal and glial markers (βIII‐tubulin, neurofilament, NeuN, neuron‐specific enolase, GFAP, and S100) and exhibited glutamate‐induced calcium responses. The global miRNA expression profiling identified 60 upregulated and 19 downregulated human miRNAs after neural differentiation, and the gene ontology analysis of the miRNA target genes confirmed the neuronal differentiation‐related biological functions. In addition, overexpression of miR‐132 in NCSCs promoted the expression of neuronal markers and downregulated ZEB2 protein expression. Our results suggested that the pluripotent NCSCs from human periodontal ligament can be directed into neural lineage, which demonstrate its potential in tissue engineering and regenerative medicine for CNS disorders.     

人骨髓间质干细胞分离纯化及基本生物学特性研究

目的　分离纯化人骨髓间质干细胞 (MSC) ,研究其基本生物学特性。方法　用比密 1 .0 73Ficoll淋巴细胞分离液分离成人骨髓MSC ,体外扩增 ,观察细胞生长特性 ,流式细胞仪检测骨髓MSC表面抗原表达及细胞周期 ,染色体技术分析骨髓MSC遗传特性。结果　成人骨髓MSC培养 3d后有散在呈针尖状的贴壁细胞 ,7～ 1 0d后形成集落或融合呈纤维状 ;体外扩增原代可获得 (4～ 6 )× 1 0 5个细胞 ,1 0代可获得 (1～ 5 )× 1 0 1 0 个细胞 ,5～ 6代以后的细胞增殖能力下降 ;流式细胞仪检测结果显示 :CD1 3、CD2 9、CD4 4、CD71表达阳性 ,CD3、CDl4、CD33、CD34、CD38、CD4 5、HLA DR不表达 ;染色体核型正常 ;细胞周期分析显示 ,G0 /G1 期 :79.4 8% ,G2 /M期 :6 .1 0 % ,S期 :1 4 .4 2 %。结论　人骨髓MSC体外具有较好的增殖更新能力 ,3～ 6代的人骨髓MSC作为组织工程细胞具有广阔的应用前景     

Transient serum exposure regimes to support dual differentiation of human mesenchymal stem cells

Human mesenchymal stem cells (MSCs), which can generate both osteoblasts and chondrocytes, represent an ideal resource for orthopaedic repair using tissue‐engineering approaches. One major difficulty for the development of osteochondral constructs using undifferentiated MSCs is that serum is typically used in culture protocols to promote differentiation of the osteogenic component, whereas existing chondrogenic differentiation protocols rely on the use of serum‐free conditions. In order to define conditions which could be compatible with both chondrogenic and osteogenic differentiation in a single bioreactor, we have analysed the efficiency of new biphasic differentiation regimes based on transient serum exposure followed by serum‐free treatment. MSC differentiation was assessed either in serum‐free medium or with a range of transient exposure to serum, and compared to continuous serum‐containing treatment. Although osteogenic differentation was not supported in the complete absence of serum, marker expression and extensive mineralization analyses established that 5 days of transient exposure triggered a level of differentiation comparable to that observed when serum was present throughout. This initial phase of serum exposure was further shown to support the successful chondrogenic differentiation of MSCs, comparable to controls maintained in serum‐free conditions throughout. This study indicates that a culture based on temporal serum exposure followed by serum‐free treatment is compatible with both osteogenic and chondrogenic differentiation of MSCs. These results will allow the development of novel strategies for osteochondral tissue engineering approaches using MSCs for regenerative medicine. Copyright © 2012 John Wiley & Sons, Ltd.     

不同因子影响骨髓间充质干细胞的多向分化

背景：在组织工程领域,关于骨髓间充质干细胞定向诱导分化的研究越来越多,但是细胞培养基中不同成分会对骨髓间充质干细胞的体外增殖分化产生影响。目的：针对培养基中不同因子对骨髓间充质干细胞定向诱导分化的作用加以综述。方法：第一作者应用计算机检索1998年1月至2012年4月Pubmed数据库及万方数据库。检索英文关键词为“bone marrow mesenchymal stromal cells, cell culture medium, differentiation”,中文关键词为“骨髓间充质干细胞,细胞培养,定向诱导分化”,纳入有关不同因子对骨髓间充质干细胞向成骨细胞、软骨细胞和脂肪细胞定向诱导分化作用的文献,排除重复研究。结果与结论：计算机初检共得到184篇文献,根据纳入排除标准,对其中30篇文献进行综述。大体说来,骨髓间充质干细胞向成骨分化的主要因子有地塞米松、转化生长因子、维生素C、维生素D3、β-甘油磷酸钠及乙烯雌酚等;向软骨分化的主要因子有地塞米松、转化生长因子、维生素C、胰岛素样生长因子及成纤维细胞生长因子等;向脂肪细胞转化的主要因子有地塞米松、3-异丁基-1-甲基黄嘌呤、胰岛素和消炎痛等,但是其中一些因子的作用机制及不良反应还不明确,需要进一步的研究与验证。同时,骨髓间充质干细胞在骨髓中的含量较低,不同的分离方法会导致不同的分离率,因此如何选取一种分离率高的分离方法仍有待研究。     

Toll样受体4对牙周膜干细胞免疫学特性的影响

背景：Toll样受体4及其配体脂多糖与牙周疾病的发生、发展密切相关,牙周膜干细胞的免疫学特性在牙周组织修复重建、牙周病的治疗中发挥重要作用,而Toll样受体4及其配体对牙周膜干细胞免疫学特性的影响还不清楚。目的：探讨Toll样受体4对牙周膜干细胞免疫学特性的影响。方法：分离、培养牙周膜干细胞,与10 mg/L的Toll样受体4配体脂多糖共同培养3 d。以未经脂多糖处理的牙周膜干细胞作为对照,观察脂多糖处理的牙周膜干细胞能否引起同种异体淋巴细胞的增殖,以及对混合淋巴细胞反应和植物血凝素引起的淋巴细胞增殖的影响。通过建立Transwell培养系统建立牙周膜干细胞＋植物血凝素＋异体外周血单个核细胞的反应体系,测定细胞上清液中的前列腺素E2浓度。在上述反应体系进行中和实验,观察被牙周膜干细胞抑制了的淋巴细胞重新发生增殖的情况。结果与结论：无论是否与脂多糖共培养,牙周膜干细胞都没有引起等量异体外周血单个核细胞增殖,都能够抑制植物血凝素引起的淋巴细胞增殖和混合淋巴细胞反应,但是脂多糖预处理牙周膜干细胞的免疫抑制作用显著低于无脂多糖组。在牙周膜干细胞＋植物血凝素＋异体外周血单个核细胞的反应体系中,前列腺素E2浓度显著升高。中和实验发现,前列腺素E2的拮抗剂吲哚美辛基本恢复了被牙周膜干细胞抑制的淋巴细胞增殖。提示,脂多糖减弱了牙周膜干细胞的免疫抑制特性,该效应由前列腺素E2减少引起。     

Optimization of the use of a pharmaceutical grade xeno‐free medium for in vitro expansion of human mesenchymal stem/stromal cells

Human bone marrow‐derived mesenchymal stem/stromal cells (hMSCs) are considered promising therapeutic agents in the field of cell therapy and regenerative medicine, mainly due to their relative facility to be isolated, multi‐differentiation potential, and immunomodulatory role. However, their application in clinics requires a crucial step of in vitro expansion. Most of the protocols for hMSCs in vitro culture use foetal bovine serum as medium supplement that, being from animal origin, presents several safety concerns and may initiate xenogeneic immune responses after cells transplantation. This work reports the optimization of a pharmaceutical‐grade xeno‐free strategy for hMSCs in vitro expansion based on the supplementation of basal medium with a pharmaceutical‐grade human plasma‐derived supplement for cell culture (SCC) and 2 human growth factors (bFGF and TGFβ1), plus a coating of human plasma fibronectin (Fn). After 4 weeks in culture, this strategy improves hMSCs expansion yield about 4.3‐fold in comparison with foetal bovine serum supplementation and 4.5‐fold compared with a commercially available xeno‐free medium. hMSCs expanded in SCC‐based formulation maintained their phenotype and differentiation capacity into osteogenic, adipogenic, and chondrogenic lineages, without alterations in cell karyotype. Overall, the SCC‐based medium appears to be an excellent alternative for the xeno‐free expansion of hMSCs as therapeutic agents for clinical applications.