肿瘤坏死因子对于骨髓基质干细胞CXCR2表达的影响

目的观察肿瘤坏死因子(TNF-α)对于大鼠骨髓基质干细胞(MSCs)表达CXCR2的影响。方法采用有利于MSCs生长及增殖的全骨髓贴壁法分离及培养干细胞;成骨、成脂诱导检测其分化特性。采用TNF-α干预MSCs细胞培养。细胞分为:对照组、低剂量组(5 ng/m L)和高剂量组(10 ng/m L)。选取P3代细胞,培养10天后提取蛋白,Western blot分析MSCs表达CXCR2变化。结果所培养的MSCs成骨、成脂诱导后染色均阳性,证实所培养的即为MSCs细胞。提高培养液中TNF-α的浓度,则MSCs表达CXCR2的量显著增加,差异具有统计学意义。结论 TNF-α能够刺激MSCs表达CXCR2增加,这可能是急性肺损伤时,MSCs细胞动员和定植的机制之一。     

红芪小分子提取物对MSCs体外增殖与染色体的影响

目的探讨红芪小分子提取物对骨髓间充质干细胞(MSCs)体外增殖及染色体的影响。方法利用水提法结合超滤法提取红芪中小分子物质,并筛选其促进MSCs增殖的最佳浓度。设单独培养的MSCs为对照组,红芪最佳浓度(20 mg/L)培养72 h后MSCs为实验组,于倒置相差显微镜下观察各组细胞形态学的改变;采用MTT法检测各组细胞生长曲线;流式细胞术检测细胞周期;染色体显色与计数分析细胞染色体;Western印迹检测各组细胞基质金属蛋白酶(MMP)-2、MMP-9蛋白表达水平的变化。结果红芪浓度20 mg/L为最佳促进MSCs增殖浓度(P0.05)。倒置相差显微镜下观察对照组细胞呈成纤维细胞样;红芪组细胞呈长梭形,形态类似于对照组。与对照组比较,红芪组细胞生长速度显著增快(P0.05);细胞周期G0/G1期细胞减少,S期和G2/M期细胞增多,但差异无统计学意义(P0.05);染色体及MMP-2、MMP-9蛋白表达水平无异常改变(P0.05)。结论红芪小分子提取物20 mg/L体外培养MSCs 72 h后,对其增殖有明显促进作用,且维持其遗传物质染色体的稳定性,其作用机制可能与调控MSCs的MMP-2、MMP-9等相关蛋白表达有关。     

多发性骨髓瘤成骨细胞活性和Runx2的表达

目的 研究多发性骨髓瘤(MM)患者间充质干细胞(MSCs)向成骨细胞(OB)分化过程中Runx2的表达,探讨MM骨形成障碍的机制.方法 以4例有明显骨质破坏的MM患者为研究对象,2例缺铁性贫血和1例特发性血小板减少性紫癜为对照,体外诱导MSCs向OB分化,采用CCK-8试剂盒检测MSCs向OB分化过程中的增殖能力,以平均光密度值(AOD)表示;碱性磷酸酶(ALP)染色和钙结节(Von-Kossa)染色检测MSCs的OB形成能力,分别以IOD sum/Area sum值和平均钙结节个数表示;逆转录-聚合酶链反应(RT-PCR)技术检测MSCs向OB分化过程中Runx2的表达,以Runx2/GAPDH平均光密度值表示.结果 在MSCs向OB分化过程中,MM患者MSCs的增殖能力、ALP表达、钙结节形成能力、Runx2表达均明显低于对照组(P＜0.01).结论 MM患者骨髓MSCs向OB分化过程中的增殖能力和OB形成能力均下降,可能与Runx2的表达减低有关.     

多发性骨髓瘤患者骨髓间充值干细胞向OB分化中的Runx2表达及体外造血机制

目的探讨多发性骨髓瘤(MM)患者骨髓间充质干细胞(MSCs)向成骨细胞(OB)分化中的Runx2表达及MSCs体外造血机制。方法采用流式细胞术与Von-cosa染色分别检测OB的增殖与形成能力,RT-PCR方法检测MSCs向OB分化中的Runx2表达与MSCs造血因子表达,应用甲基纤维素与MSCs进行常规培养检测MSCs体外造血的能力。结果 OB的增殖能力显著低于对照组,观察组IOD sum/Area sum值与平均钙结节数均显著低于对照组(均P<0.05);MM患者在MSCs诱导前Runx2表达显著低于MSCs诱导后,观察组Runx2平均光密度显著低于对照组(均P<0.05);RT-PCR法可检测白细胞介素(IL)-6、单核细胞集落刺激因子(M-CSF)与肝细胞生长因子(SCF)在MSCs中的表达,而血小板生长因子(TPO)与粒细胞集落刺激因子(GM-CSF)未见表达;MM患者与正常体检者细胞集落产量之间差异无统计学意义(P>0.05)。结论 Runx2的表达减弱可能导致MM患者OB的增殖与形成能力的减弱,MM患者与正常MSCs体外造血的能力无显著差别,联合自体MSCs造血干细胞移植在MM诊治中具有临床应用价值。     

老龄兔骨髓间充质干细胞体外培养影响因素的研究

目的研究体外培养老龄兔骨髓间充质干细胞(MSCs)的影响因素。方法以密度梯度离心技术及贴壁筛选法相结合,在体外分离检测MSCs表面CD29、CD34、CD44、CD45的表达;诱导分化反向证明部分细胞可分化为成骨细胞及脂肪细胞,具有分化潜能,鉴定该细胞群为干细胞。细胞记数法计算MSCs倍增时间;观察供体自然老龄情况下,不同换液时间、不同血清浓度、不同种植密度等因素对MSCs生长的影响。结果MSCs CD29和CD44表达阳性,不表达造血干细胞标志CD34和CD45。高密度接种(3×106~4×106个/cm2),低浓度(8%~10%)血清的低糖DMEM培养,在体外可保持MSCs未分化增殖状态,并获得纯度较高同源性较好的干细胞群,高浓度血清(20%)并不加速细胞生长,而促进分化。结论MSCs具有体外多分化潜能,老龄供体骨髓中MSCs的生长、增殖特性受不同换液时间、不同血清浓度及不同种植密度等因素的影响。     

人脐血间充质干细胞源性心肌细胞的诱导分化研究

目的:探讨体外人脐血间充质干细胞(hMSCs)诱导分化成心肌细胞的可行性和最佳方法。方法:收集健康产妇脐血细胞,分离单个核细胞,从中进一步分离间充质干细胞,传代培养至第三代,应用免疫荧光流式细胞仪标记MSCs特异性抗原CD34,CD44和CD90。5-氮(杂)胞苷(5-aza)诱导分化4周后,免疫组织化学染色和RT-PCR分别检测心肌细胞标志物肌钙蛋白I,GATA4和β-肌球蛋白重链。结果:脐血源性MSCs经5-aza诱导分化后,呈现成纤维细胞样形态和克隆增殖特点。免疫分型与骨髓来源MSCs一致,且免疫组织化学染色和RT-PCR可检测到肌钙蛋白I、GATA4和β-肌球蛋白重链的表达。结论:脐血源性MSCs能够被诱导分化成心肌样细胞,可成为干细胞移植的细胞来源。     

骨髓瘤细胞抑制成骨细胞活性和Runx2表达

目的 建立MM细胞系(U266细胞株)和MSCs共培养体系,研究骨髓瘤细胞对MSCs增殖能力、OB形成能力和Runx2表达的影响.方法 研究对象为2例缺铁性贫血和1例特发性血小板减少性紫癜.采用密度梯度离心结合贴壁培养法分离纯化骨髓MSCs,培养3-5代后,诱导MSCs向OB分化,待细胞融合至60％～70％时,分两组,一组加U266细胞株共同培养(共培养组),另一组不加U266细胞株(对照组).采用CCK-8试剂盒检测MSCs向OB分化过程中的增殖能力,以平均光密度值(AOD)表示;碱性磷酸酶(ALP)染色和钙结节(Von-Kcssa)染色检测MSCs的OB形成能力,分别以IOD sum/Area sum值和平均钙结节个数表示;RT-PCR技术检测MSCs向OB分化过程中Runx2的表达,以Runx2/GAPDH平均光密度值表示.结果 在共培养体系中,U266细胞株和MSCs共培养的前3d,MSCs增殖能力无明显变化,两组AOD值分别为0.731±0.020和0.805±0.152(P ＞0.05),至第6天,共培养组的MSCs增殖能力明显低于对照组,AOD分别为0.560±0.034和2.283±0.145(P ＜0.05).与对照组相比,共培养组MSCs向OB分化过程中ALP的表达显著降低,IOD sum/Area sum值分别为0.138±0.038和0.376±0.044(P ＜0.01),钙结节的形成减少,平均钙结节个数分别为(6.7±2.75)个和(15.3±8.99)个(P＜0.01)和Runx2的表达显著降低,Runx2/GAPDH平均光密度值分别为0.238±0.168和0.653 ±0.343(P ＜0.01).结论 骨髓瘤细胞可抑制骨髓MSCs向OB分化的增殖能力和OB形成能力,也抑制其Runx2表达.     

叶酸诱导骨髓基质干细胞分化为神经细胞的浓度探索

目的 探讨叶酸对骨髓基质干细胞诱导分化为神经细胞的最佳浓度.方法 MSCs由正常成年人骨髓经密度梯度离心加贴壁离心获得,取第5代MSCs以1 mmol/L二巯基乙醇(BME)预诱导24 h后,用羟基茴香醚(BHA)、2%二甲基亚砜(DMSO)和3种不同浓度的叶酸诱导分化,采用免疫细胞化学法检测神经细胞特异性抗原标志神经元特异性烯醇化酶(NSE)、胶原纤维酸性蛋白GFAP的表达,MTT(四唑盐比色)法检测不同时间段对BMSCs增殖的影响.结果 不同剂量叶酸组分化为NSE阳性神经元样细胞的百分率都较对照组高(P<0.01) ,但以40 mg/L中等浓度的叶酸神经细胞最多,且神经元比例高于其他组.结论 叶酸诱导BMSCs向神经元分化以40 mg/L浓度为最佳.     

小鼠胰腺干细胞的分离、培养与鉴定

目的从新生小鼠中分离培养并鉴定胰腺干细胞。方法利用组织块法分离新生小鼠胰腺源性干细胞,经RPMI1640+5%胎牛血清+双抗+1 mmol/L丙酮酸钠+1 mmol/L非必需氨基酸+10 ng/mL表皮生长因子+10 ng/mL成纤维生长因子+2 mmol/L谷氨酰胺的溶液培养,利用免疫组化分析胰腺源性干细胞的兔抗巢蛋白抗体(Nestin)、胰肠同源域因子(PDX-1)和葡葡糖转运子(GLUT-2)抗原表达;经10 mmol/L尼克酰胺诱导产生胰岛样细胞团并利用双硫腙(DTZ)染色分析其胰岛素分泌情况。结果分离培养的胰腺源性干细胞呈Nestin、PDX-1和GLUT-2阳性;诱导的胰岛样细胞团呈DTZ染色阳性。结论分离获得的新生小鼠胰腺源性干细胞在体外可分化为类胰岛,并具有胰岛素分泌功能。     

不同代次大鼠骨髓间充质干细胞Wnt/β-catenin信号通路表达分析

目的观察不同代次大鼠骨髓间充质干细胞(MSCs)体外增殖能力及其纤维分化倾向,为临床治疗及组织工程种子细胞的选择提供依据。方法采用贴壁培养法分离培养大鼠MSCs,流式细胞术鉴定细胞表面标记; MTT法检测不同代次MSCs的增殖情况;蛋白质印迹法(Western Blot)检测MSCs Wnt通路相关蛋白的表达情况;实时聚合酶链式反应(Realtime-PCR)法检测不同代次MSCs中成纤维细胞分子标记表达。结果流式细胞术鉴定结果证实我们培养的大鼠MSCs符合间充质干细胞的特征,且纯度高; MTT法检测显示P3代之后细胞的增殖能力明显高于P1、P2代,Western Blot检测显示细胞内Wnt/β-catenin信号通路蛋白表达随培养代次增强,Realtime-PCR法检测结果证实P9代细胞开始出现成纤维分化倾向。结论 MSCs作为种子细胞不宜超过P9代,P3～P7代具有纯度高、倍增快等优点,是能够满足组织工程细胞植入的种子细胞。     

细胞生长因子体外对大鼠肝干细胞的影响

目的 研究大鼠肝干细胞系WB-F344细胞在体外地生长分化与多种细胞因子的关系。方法 用^3H掺入法检测细胞因子对WB-F344细胞生长增殖作用。用western blot检测WB-F344细胞因子受体的表达。流式细胞仪检测细胞凋亡。结果 发现肝细胞生长因子（HGF）80ng/mlcpm值为982.95，表皮生长因子（EGF）80ng/ml cpm值为906.32，胰岛素80ng/ml cpm值为968.67，成纤维细胞生长因子（FGFα）80ng/ml cpm值为863.98。促进WB-F344细胞的增殖；白细胞介素-6（IL-6）80ng/ml cpm值为368.67，肿瘤坏死因子α80ng/ml cpm值为372.90对WB-F344细胞的生长无明显影响；而转化生长因子（TGFβ）能够诱导WB-F344细胞凋亡（80ng/ml凋亡率为26.89%)并抑制其生长；western blot检测发现WB-F344细胞表面存在HGF，EGF，FGFα，TGFβ等细胞因子受体，提示细胞因子可能通过与其受体结合方式调控WB-F344细胞的生长分化，进一步用HGF，EGF，胰岛素等细胞因子组成并添加地塞米松的分化培养体系，在体外对WB-F344细胞进行定向诱导分化。发现WB-F344细胞在体外能够向肝实质细胞分化，此外HGF还具有刺激肝干细胞离散作用。结论 肝干细胞的生长分化受多种细胞因子的调控。细胞生长因子可能在严重肝损伤后肝干细胞的动员，增殖及分化调控中起重要作用。     

Factors controlling proliferation and progesterone production by bovine granulosa cells in serum-free medium

Bovine granulosa cells seeded in the presence of serum on extracellular matrix-coated dishes proliferate actively when exposed to serum-free medium supplemented with insulin (2 microgram/ml), fibroblast growth factor (FGF, 100 ng/ml), and high density lipoprotein (HDL, 30 microgram protein/ml). The final density of the cultures is 80-120% that of cultures grown in the presence of medium supplemented with optimal concentration (10%) of calf serum. Insulin has the greatest effect on cell proliferation when added alone to serum-free medium, since it induced an increase in cell number that was 35-60% that observed with optimal serum concentration. Somatomedin C can replace insulin when added alone. FGF, epidermal growth factor, or HDL had no significant effect on cell proliferation by themselves. When these factors were added together with insulin, they acted synergistically in stimulating cell proliferation. When cultures were seeded in the total absence of serum, the addition of transferrin (10 microgram/ml) to serum-free medium was required in order for insulin and FGF to be mitogenic. Cultures maintained on extracellular matrix and exposed to serum-free medium alone have a lifespan in culture of 4 generations. Addition of insulin, FGF, and HDL increases the lifespan of the cultures to 12 generations. Bovine granulosa cells, which proliferate in a defined medium, respond to dibutyryl cAMP by releasing progesterone into the medium. Addition of FSH to the defined medium resulted in a 30% decrease in cell proliferation and in a 2.1-fold increase in the amount of progesterone released into the medium in response to dibutyryl cAMP. This release of progesterone reached a level similar to that observed with cultures grown in medium supplemented with optimal concentration of serum and exposed or not to FSH during their growth phase and at confluence. These results demonstrate that bovine granulosa cells can actively proliferate in a serum-free medium and maintain their differentiated function, as indicated by their ability to produce progesterone.     

结肠炎大鼠炎症黏膜提取液对骨髓间质干细胞增殖和表面分化抗原的影响

背景:动物实验和临床试验均表明间质干细胞(MSCs)对受损肠道组织有一定修复作用,然而目前尚不清楚肠道炎症微环境对MSCs移植治疗炎症性肠病(IBD)有何影响。目的:通过体外实验观察结肠炎大鼠模型肠道炎症黏膜提取液对骨髓MSCs增殖和表面分化抗原的影响。方法:以全骨髓贴壁法分离和原代培养大鼠MSCs并行传代扩增。取三硝基苯磺酸(TNBS)结肠炎大鼠模型肠道炎症黏膜提取液(0、1、2、3 ml)与MSCs共培养,倒置相差显微镜观察各组细胞贴壁和生长情况,绝对计数法绘制细胞生长曲线,流式细胞术检测细胞表面分化抗原表达。结果:全骨髓贴壁法可成功分离MSCs,所获细胞CD29、CD44表达阳性,CD105表达低度阳性,CD34、CD45表达阴性。3 ml炎症黏膜提取液处理组MSCs接种6 h后见细胞贴壁,36 h开始克隆性增生,此后细胞增殖速度较空白对照组明显加快,第6 d即达100%融合,但表面分化抗原表达与空白对照组相比无明显变化。结论:结肠炎大鼠炎症黏膜提取液可促进骨髓MSCs增殖,但对细胞表面分化抗原无明显影响。     

内皮细胞特异性分子 1预处理对小鼠骨髓间充质干细胞生物学特性的影响

目的:探讨内皮细胞特异性分子 1（ESM 1）孵育预处理对小鼠骨髓间充质干细胞生物学特性的影响,为干细胞移植提供实验依据。方法: 培养及鉴定骨髓间充质干细胞。提取细胞数及浓度固定的初级培养液中的骨髓间充质干细胞,并分为两组:对照组（不进行预处理）及3个浓度（005 μg/ml、01 μg/ml、015 μg/ml）的ESM 1预处理组。每组设4个复孔,每孔均处理60 min。收集每孔中的条件培养液,检测预处理对骨髓间充质干细胞分泌血管内皮生长因子(VEGF)及碱性磷酸酶(ALP)的影响。结果: ESM 1孵育预处理骨髓间充质干细胞后,其存活率明显增加,而凋亡率显著下降,且骨髓间充质干细胞增殖能力随着ESM 1预处理浓度的增加而逐渐升高,凋亡率却呈相反的下降趋势。ESM 1预处理后,骨髓间充质干细胞分泌VEGF、ALP的水平明显升高,且随着ESM 1浓度的增加,骨髓间充质干细胞分泌VEGF、ALP的水平也相应增加。ESM 1孵育预处理后的骨髓间充质干细胞,细胞形态一致,生长及分化速度加快。结论: ESM 1孵育预处理骨髓间充质干细胞不仅促进骨髓间充质干细胞增殖及降低其凋亡,而且可增加VEGF、ALP的分泌。ESM 1通过活化骨髓间充质干细胞,提高细胞本身的潜能,继而提高其存活、增殖的能力, 为临床急性心肌梗死的治疗提供了广泛的应用前景。     

Adipogenic differentiation alters the immunoregulatory property of mesenchymal stem cells through BAFF secretion

Although it has been widely demonstrated that mesenchymal stem cells (MSCs) exert potent immunosuppressive effect, there is little information as to whether adipogenic-differentiated MSCs (adi-MSCs) share the same property. Here, adi-MSCs enhanced alloantigen or mitogen-stimulated lymphocyte proliferation, whereas undifferentiated MSCs (ud-MSCs) inhibited the proliferation. Transwell experiment showed that the stimulatory effect of adi-MSCs was cell-cell contact-independent, and required soluble factors. Furthermore, the supernatant of cultured adi-MSCs could effectively costimulate T and B-lymphocyte proliferation and activation in the presence of anti-CD3 and anti-mu chain treatment, respectively. Production of cytokines interferon-gamma and tumor necrosis factor-alpha by T cells, and Ig secretion by B cells also were increased by the supernatant of cultured adi-MSCs. Mechanism conducted showed that the mRNA and protein expression of costimulatory molecule B-cell activating factor (BAFF) was upregulated, and soluble BAFF was secreted in MSCs after adipogenic differentiation. By blocking the BAFF molecule with specific monoclonal antibody in the culture, T and B-lymphocyte proliferation and activation was stimulated by adi-MSCs or the supernatants were greatly reduced. In conclusion, adipogenic differentiation may alter the immunoregulatory property of MSCs, leading to stimulation of lymphocytes response. The BAFF molecule secreted by the adi-MSCs was responsible for this event.     

Inhibition of cellular senescence by developmentally regulated FGF receptors in mesenchymal stem cells

Bone-derived mesenchymal stem cells (MSCs) are important cells for use in cell therapy, tissue engineering, and regenerative medicine, but also to study bone development, homeostasis, and repair. However, little is known about their developmental ontology and in vivo identity. Because fibroblast growth factors (FGFs) play key roles in bone development and their receptors are developmentally regulated in bones, we hypothesized that MSCs should express FGF receptors (FGFRs), reflecting their developmental origin and potential. We show here that FGFR1/2 are expressed by rare mesenchymal progenitors in putative MSC niches in vivo, including the perichondrium, periosteum, and trabecular marrow. FGFR1? cells often appeared as pericytes. These cells display a characteristic MSC phenotype in vitro when expanded with FGF-2, which appears to maintain MSC stemness by inhibiting cellular senescence through a PI3K/AKT-MDM2 pathway and by promoting proliferation. FGFRs may therefore be involved in MSC self-renewal. In summary, FGFR1/2 are developmentally regulated markers of MSCs in vivo and in vitro and are important in maintaining MSC stemness.     

Differentiation of rat marrow mesenchymal stem cells into pancreatic islet beta-cells

AIM: To explore the possibility of marrow mesenchymal stem cells (MSC) in vitro differentiating into functional isletlike cells and to test the diabetes therapeutic potency of Islet-like cells. METHODS: Rat MSCs were isolated from Wistar rats and cultured. Passaged MSCs were induced to differentiate into islet-like cells under following conditions: pre-induction with L-DMEM including 10 mmol/L nicotinamide+l mmol/L β-mercaptoethanol+200 mL/L fetal calf serum (FSC) for 24 h, followed by induction with serum free H-DMEM solution including 10 mmol/L nicotinamide+1 mmol/L,β-mercaptoethanol for 10 h. Differentiated cells were observed under inverse microscopy, insulin and nestin expressed in differentiated cells were detected with immunocytochemistry. Insulin excreted from differentiated cells was tested with radioimmunoassay. Rat diabetic models were made to test in vivo function of differentiated MSCs. RESULTS: Typical islet-like clustered cells were observed. Insulin mRNA and protein expressions were positive in differentiated cells, and nestin could be detected in predifferentiated cells. Insulin excreted from differentiated MSCs (446.93&#177;102.28 IU/L) was much higher than that from pre-differentiated MSCs (2.45+0.81 IU/L (P&lt;0.01). Injected differentiated MSCs cells could down-regulate glucose level in diabetic rats. CONCLUSION: Islet-like functional cells can be differentiated from marrow mesenchymal stem cells, which may be a new procedure for clinical diabetes stem -cell therapy, these cells can control blood glucose level in diabetic rats. MSCs may play an important role in diabetes therapy by islet differentiation and transplantation.     

Immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals

Mesenchymal stem cells (MSCs) are largely studied for their potential clinical use. Recently, they have gained further interest after demonstration of an immunosuppressive role. In this study, we investigated whether in vivo injection of MSCs could display side effects related to systemic immunosuppression favoring tumor growth. We first showed in vitro that the murine C3H10T1/2 (C3) MSC line and primary MSCs exhibit immunosuppressive properties in mixed lymphocyte reaction. We demonstrated that this effect is mediated by soluble factors, secreted only on "activation" of MSCs in the presence of splenocytes. Moreover, the immunosuppression is mediated by CD8+ regulatory cells responsible for the inhibition of allogeneic lymphocyte proliferation. We then demonstrated that the C3 MSCs expressing the human bone morphogenetic protein 2 (hBMP-2) differentiation factor were not rejected when implanted in various allogeneic immunocompetent mice and were still able to differentiate into bone. Importantly, using a murine melanoma tumor model, we showed that the subcutaneous injection of B16 melanoma cells led to tumor growth in allogeneic recipients only when MSCs were coinjected. Although the potential side effects of immunosuppression induced by MSCs have to be considered in further clinical studies, the usefulness of MSCs for various therapeutic applications still remains of great interest.     

Osteoarthritic chondrocyte–secreted morphogens induce chondrogenic differentiation of human mesenchymal stem cells

Objective

The potential of stem cells to repair compromised cartilage tissue, such as in osteoarthritis (OA), depends strongly on how transplanted cells respond to factors secreted from the residing OA chondrocytes. This study was undertaken to determine the effect of morphogenetic signals from OA chondrocytes on chondrogenic differentiation of human mesenchymal stem cells (MSCs).

Methods

The effect of OA chondrocyte–secreted morphogens on chondrogenic differentiation of human MSCs was evaluated using a coculture system involving both primary and passaged OA chondrocytes. The findings were compared against findings for human MSCs cultured in OA chondrocyte–conditioned medium. Gene expression analysis, biochemical assays, and immunofluorescence staining were used to characterize the chondrogenic differentiation of human MSCs. Mass spectrometry analysis was used to identify the soluble factors. Numerical analysis was carried out to model the concentration profile of soluble factors within the human MSC–laden hydrogels.

Results

The human MSCs cocultured with primary OA chondrocytes underwent chondrogenic differentiation even in the absence of growth factors; however, the same effect could not be mimicked using OA chondrocyte–conditioned medium or expanded cells. Additionally, the cocultured environment down‐regulated hypertrophic differentiation of human MSCs. Mass spectrometry analysis demonstrated cell–cell communication and chondrocyte phenotype–dependent effects on cell‐secreted morphogens.

Conclusion

The experimental findings, along with the results of the numerical analysis, suggest a crucial role of soluble morphogens and their local concentrations in the differentiation pattern of human MSCs in a 3‐dimensional environment. The concept of using a small number of chondrocytes to promote chondrogenic differentiation of human MSCs while preventing their hypertrophic differentiation could be of great importance in formulating effective stem cell–based cartilage repair.