PDGF-BB和TGF-β1诱导人脂肪基质干细胞向血管平滑肌细胞的分化

目的：探讨人脂肪基质干细胞体外诱导为血管平滑肌细胞的可能性。方法：应用酶消化法从人脂肪组织获得单个核细胞,基础培养液培养,经传代后,第一代细胞用于诱导分化实验。未诱导组培养液为基础培养液,诱导组培养液在基础培养液内添加诱导因子PDGF-BB（50ng/mL）、TGF-β1（5ng/mL）,诱导14d后,光镜下观察诱导细胞形态学的改变;免疫荧光和RT-PCR方法检测平滑肌细胞特异标记的表达情况;流式细胞仪检测诱导阳性率。结果：脂肪基质干细胞在特定细胞因子诱导作用下,细胞生长呈现与平滑肌细胞类似的“峰-谷”生长模式,并表达α-SM-Actin、SM-MHC、Calponin、SM-22α等平滑肌特异性标记;诱导组阳性率与未诱导组差异有统计学意义（P〈0.01）。结论：PDGF-BB、TGF-β1可诱导人脂肪基质干细胞向血管平滑肌细胞表型分化。     

体外诱导毛囊干细胞成血管平滑肌细胞的实验研究

目的探讨体外诱导人毛囊干细胞成血管平滑肌细胞的可行性。方法采用中性蛋白酶(Dispase)分离人毛囊干细胞,用含10 ng/mL PDGF-BB、10%血清的低糖DMEM诱导液对其进行诱导,无PDGF-BB的培养液为对照组,观察每代细胞形态,诱导4代后检测α-平滑肌肌动蛋白(α-SM actin)与肌钙结合蛋白(Calponin)的表达。结果在诱导液的作用下,细胞形态逐步向平滑肌样转变,对照组细胞形态改变不显著。细胞免疫荧光检测显示,至第4代,实验组已明显表达α-SM actin与Calponin,对照组表达不明显;流式细胞仪检测显示,实验组α-SM actin与Calponin阳性表达率近50%,对照组则低于8%;RT-PCR显示,实验组表达Calponin,而对照组未见明显表达。结论使用含10 ng/mLPDGF-BB的低糖DMEM培养液可体外诱导人毛囊干细胞成血管平滑肌细胞。     

周期性张力对骨髓间充质干细胞分化的血管平滑肌细胞的表型调节

目的 探讨周期性张力对大鼠骨髓间充质干细胞(BMSCs)诱导分化的血管平滑肌细胞(VSMCs)的表型的调节机制.方法 原代全骨髓法培养大鼠BMSCs,流式细胞术鉴定细胞.作成脂、成骨、成平滑肌细胞方向诱导,验证BMSCs的多向分化潜能.将细胞分为4组:A组用含10 μg/L转化生长因子-β1(TGF-β1)的完全培养基培养,B组用幅度10％、频率1 Hz周期性张力诱导,C组采用TGF-β1+周期性张力联合诱导,D组用含10％胎牛血清(FBS)的DMEM/F12培养基培养作对照.3d后观察诱导后的细胞形态,并行反转录-聚合酶链反应(RT-PCR)检测肌动蛋白(α-actin)、平滑肌肌球蛋白重链(SM-MHC)、钙调节蛋白1(calponin1)、钙调节蛋白3(calponin3)的mRNA表达水平,Western blot观察细胞内α-actin、SM-MHC、calponin1、calponin3蛋白表达水平.结果 经流式细胞术鉴定,所培养细胞为BMSCs.诱导3d后,A、B、C3组的VSMC标志物均较D组明显升高,以SM-MHC和calponin3增加为主.A组(0.919 2±0.028 1)较B组(0.823 6±0.024 6)的SM-MHC蛋白表达水平高,但低于C组(1.043 1±0.090 7),其差异均有统计学意义(P＜0.05).C组中calponin3 mRNA表达量比蛋白表达量变化更明显,而calponin1蛋白表达量和mRNA表达量同步增高.结论 周期性张力能够诱导BMSCs分化为VSMCs,对分化的VSMCs的表型调节还需要细胞因子的参与.     

体外诱导人骨髓间充质干细胞向血管平滑肌细胞分化的实验研究

目的应用血小板衍生生长因子BB（platelet-derived growthfactor BB,PDGF-BB）,体外诱导人骨髓间充质干细胞（human bone marrowmesenchymal stemcells,hBMSCs）向血管平滑肌细胞表型分化,探讨该方法的可行性及诱导细胞作为组织工程血管平滑肌种子细胞的可行性。方法抽取健康成人志愿者骨髓,经密度梯度离心分离得单个核细胞,PDGF-BB（20ng/ml）诱导hBMSCs向血管平滑肌样细胞（vascular smooth muscle cells,VSMCs）分化,观察细胞形态变化。免疫荧光检测细胞内血管平滑肌肌动蛋白α（vascular smooth muscleα-actin,SMα-actin）,血管平滑肌钙结合蛋白（vascular smoothmuscle calponin,SMcalponin）,血管平滑肌肌球蛋白重链（vascular smooth muscle myosin heavy chain,SMMHC）和细胞血管平滑肌钙结合相关蛋白（smooth muscle22α,SM22α）表达情况;反转录聚合酶链式反应（RT-PCR）检测诱导后血管平滑肌细胞SMα-actin,SMcalponin,SMMHC和SM22α的mRNA表达。Western印记检测SM22α的表达。流式细胞分析技术（fluorescence activated cell sorter,FACS）分析诱导后细胞内SMα-actin,SMcalponin,SMMHC的表达。结果PDGF-BB20ng/ml诱导后可见单层培养的细胞形态呈“成纤维细胞样”。免疫荧光检测示SMα-actin,SMcalponin,SMMHC,SM22α表达阳性;RT-PCR检测SMα-actin,SMcalponin,SMMHC,SM22α的mRNA阳性表达。Western印迹检测SM22α的表达为阳性。FACS分析表明诱导后,SMα-actin,SMcalponin,SMMHC表达均增高,与未诱导组比较差异有统计学意义（P〈0.05,n=3）。结论人骨髓间充质干细胞在PDGF-BB的诱导下可向血管平滑肌细胞表型分化,有望成为血管组织工程血管平滑肌种子细胞的来源。     

诱导骨髓基质细胞成血管平滑肌细胞的研究

目的 探讨体外诱导犬骨髓基质细胞(SMSC)为血管平滑肌细胞(VSMC)及其成血管的能力.方法 用含血小板源性生长因子(PDGF-BB)10ìg/L的内皮细胞培养液-2(EGM-2)诱导,无PDGF-BB的EGM-2培养液为对照,检测a-平滑肌肌动蛋白(a-SM actin)与肌钙结合蛋白(calponin)的表达并接种于聚羟基乙酸(PGA)培养4周后取材.结果 诱导组(n=5)细胞逐步呈平滑肌样转变,表达a-SM actin与calponin,流式细胞仪阳性率分别为(45.16±0.97)%、(37.54 4±1.15)%,可成血管样结构,对照组(n=5)变化不明显,阳性率(7.92±1.04)%、(6.37±0.83)%,成血管样结构能力差.结论 体外可诱导犬BMSC为VSMC并有成血管样结构的能力.     

大鼠脂肪干细胞体外诱导成平滑肌样细胞的研究

目的:研究大鼠脂肪干细胞(ADSCs)体外单层培养条件下诱导分化为平滑肌样细胞的可行性。方法:从SD大鼠的腹股沟脂肪垫分离获取脂肪干细胞,测定其生长曲线。取第4代细胞用成脂诱导液诱导分化,并用油红O染色鉴定。取第4代细胞用成骨诱导液诱导分化,并用Von Kossa染色鉴定。取第4代细胞用含有β-巯基乙醇的成平滑肌诱导液诱导,并用免疫组化的方法检测α平滑肌肌动蛋白(α-SMA)的表达。结果:脂肪干细胞成梭形和多角形,体外生长迅速,生长曲线表明传代2 d后细胞进入对数生长期。第4代细胞成脂诱导后,油红O染色证实细胞内存在脂滴;成骨诱导后,Von Kossa染色证实有矿化结节。脂肪干细胞诱导平滑肌样细胞免疫组化结果,β-巯基乙醇诱导组和未诱导组细胞α-SMA的表达阳性率分别为(29.80±6.89)%、(2.89±1.24)%。诱导组细胞α-SMA的表达阳性率高于未诱导组,差异有统计学意义(P<0.01)。结论:脂肪干细胞经诱导后出现明显的平滑肌细胞特征,可成为平滑肌相关疾病在组织工程研究上新的种子细胞来源。     

人脂肪干细胞向血管平滑肌细胞诱导分化的实验研究

目的：体外诱导培养人脂肪干细胞（adi pose-der i ved st em cel l s,ADSCs）,观察是否可以分化形成血管平滑肌细胞（vascul ar smoot h muscl e cel l s,VSMCs）,为构建组织工程化血管寻找新的种子细胞来源。方法：免疫磁珠法分选收集原代脂肪干细胞,加入PDGF-BB、TGF-β1诱导14天后进行检测。结果：实验组细胞生长呈现血管平滑肌细胞所特有的峰谷样生长,血管平滑肌细胞特有的表面抗原标记物表达阳性。结论：脂肪干细胞定向诱导培养后,具有血管平滑肌细胞的特性,有可能成为构建组织工程化血管新的种子细胞来源。     

应用脂肪干细胞构建组织工程化小口径血管平滑肌层的实验研究

目的探索利用脂肪干细胞在生物反应器内构建组织工程血管平滑肌层的可行性。方法用抽吸的脂肪获取脂肪干细胞,在生长因子TGF-β1和BMP4作用下诱导成平滑肌细胞,然后将诱导的平滑肌细胞接种于PGA上,将细胞-材料复合物置于生物反应器内进行培养,在模拟胚胎发育血流动力学的刺激下(搏动频率:75次/分;扩展量<5%),构建小口径的血管平滑肌组织。培养8周后,取材行组织学和生物力学检测并与正常血管对比。结果脂肪干细胞在TGF-β1和BMP4的诱导下,细胞呈现平滑肌细胞特有的"波峰-波谷"样生长特点,并表达平滑肌细胞的特异性标记物α-SMA、SM22α、calponin和SM-MHC;反应器内培养8周后,构建的管状组织胶原分泌旺盛,具有一定的力学强度和弹性。结论利用脂肪干细胞可在体外生物反应器内构建组织工程化小口径血管平滑肌层,为临床上小血管病变的修复提供了一种新的可能的途径。     

诱导脂肪干细胞向血管内皮细胞分化的实验研究

目的 探讨诱导脂肪干细胞(ADSCs)向血管内皮细胞(ECs)分化的可能性,为ADSCs应用于创伤修复的理论提供实验依据.方法 切取大鼠脂肪组织,用酶消化法分离、培养ADSCs,流式细胞仪检测第3代ADSCs的CD49d和CD106的表达以鉴定ADSCs.实验组用含30%大鼠血管匀浆液的条件培养液诱导大鼠ADSCs,空白对照组用含10%胎牛血清DMEM培养液培养大鼠ADSCs,各组诱导3 d后经免疫组化和流式细胞仪检测ADSCs中CD34和血管性假血友病因子(vWF)相关抗原的表达变化.结果 流式细胞仪检测ADSCs的CD49d和CD106阳性率分别为(98.32±0.37)%和(1.67±0.61)%;血管条件培养液诱导组细胞CD34和vWF阳性率分别为(77.14±0.76)%和(75.46±0.37)%,较空白对照组(1.38±0.31)%和(1.70±0.23)%均升高,差异有统计学意义(P<0.01).结论 ADSCs可以被诱导向ECs表型分化,提示ADSCs具有参与创伤后血管形成的潜能,可以应用于创伤修复的治疗.     

复方冬红合剂对血管平滑肌细胞作用的实验研究

目的:探讨复方冬红合剂对血管平滑肌细胞(vascular smooth muscle cells,VSMCs)的作用。方法:分别用稀释度为1∶800～1∶200的复方冬红合剂培养液孵育兔主动脉的VSMCs;用MTT法检测VSMCs的增殖,以划痕实验检测其移行状态;用PI-AnnexinⅤ双标记及caspase-3检测细胞的凋亡状态,并与正常对照组作比较。结果:①复方冬红合剂可抑制VSMCs增殖,其抑制作用随稀释度降低而增强,并在稀释度为1∶200时最为显著(P0.05);②复方冬红合剂可抑制VSMCs移行,其抑制作用也随稀释度降低而增强,并在稀释度为1∶200时最显著(P0.05);③复方冬红合剂并不导致VSMCs凋亡。结论:复方冬红合剂可能通过抑制VSMCs的增殖和移行,而并非经调节其细胞凋亡,来发挥抑制内膜增生的作用。     

Growth potential of orbital cavernous hemangioma suggested by vascular endothelial growth factor and its receptor flk-1

Orbital cavernous hemangiomas (CHs) manifest as slowly developing symptoms indicative of slow growth. The present study investigated the involvement of angiogenic factors and their receptors in the growth of orbital CHs. Surgical specimens of orbital CHs were obtained from nine patients. Formalin-fixed, paraffin-embedded specimens were stained immunohistochemically using antibodies against Ki-67, CD31, alpha-smooth muscle actin (alpha-SMA), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and VEGF receptors (flt-1 and flk-1). CD31 was expressed in the single layer of endothelial cells lining the vascular cavity. The thick vascular walls were positive for alpha-SMA, indicating that the vascular walls were smooth muscle cells. Ki-67 antigen immunostaining was mostly positive in the vascular walls and the staining index ranged from 0% to 6.8% (mean +/- standard deviation, 2.7 +/- 1.9%). VEGF and bFGF immunostaining were positive in all specimens. Flt-1 immunostaining was negative in all specimens, but flk-1 immunostaining was positive in both endothelial cells and smooth muscle cells. These results suggest that both VEGF and its receptor flk-1 are important in the growth of orbital CH.     

脂肪组织来源的基质细胞研究进展

目的介绍近期国内外脂肪组织来源的基质细胞研究进展. 方法广泛查阅近期有关文献,归纳分析此种细胞的培养和分化的研究状况. 结果脂肪组织中存在一种多能的基质细胞,体外培养条件下这种细胞生长状态类似成纤维细胞,可长期增殖,细胞绝大多数为中胚层来源,混有少量的血管周细胞、内皮细胞和平滑肌细胞,在一定的诱导条件下能分化为脂肪细胞、软骨细胞、成骨细胞、成肌细胞和神经细胞. 结论脂肪组织来源的基质细胞可替代间充质干细胞作为组织工程的另一种干细胞.     

The role of alpha-smooth muscle actin and platelet-derived growth factor-beta receptor in the progression of renal damage in human IgA nephropathy

BACKGROUND: The degree of tubulointerstitial damage can be considered a better indicator of renal function outcome in IgA nephropathy (N) than the extent of glomerular sclerosis. MATERIALS AND METHODS: To investigate the pathogenetic mechanisms of interstitial injury in IgAN, we used immunohistochemistry and in situ hybridization to evaluate the glomerular and tubolointerstitial expression of PDGF-beta receptor (R) and alpha-smooth muscle actin (SMA), two markers of mesenchymal cell activation, and correlated these findings with the histopathologic and clinical features of the disease. We studied 155 IgAN patients, divided into three groups based on the histological findings (mild, moderate and severe histological lesions). RESULTS: In normal kidneys and in patients with mild histological lesions, the interstitial areas showed scattered peritubular cells positive for PDGF-betaR and alpha-SMA, with a distribution resembling the capillary network. In the glomeruli several cells (mainly in the mesangial area) stained for PDGF-betaR, but only very few cells were positive for alpha-SMA. Alpha-SMA and PDGF-betaR staining, as expected, was also observed in vascular smooth muscle cells. Compared to patients with mild histological lesions, alpha-SMA expression was strikingly increased in patients with moderate to severe lesions, particularly in areas of tubulointerstitial fibrosis. In these patients, PDGF-betaR gene and protein expression, at the tubulointerstitial level, paralleled that in alpha-SMA. Both signals were significantly correlated with the interstitial damage (interstitial infiltrate and fibrosis). Interestingly, these patients showed a different pattern of distribution of alpha-SMA and PDGF-betaR in the glomeruli: PDGF-betaR expression was upregulated, whereas no changes were seen in alpha-SMA staining. In addition, glomerular PDGF-betaR staining was significantly correlated with mesangial cell proliferation, while alpha-SMA was not. Image analysis showed that 40.2+/-10.3/1,000 microm2 of interstitial cells were positive to both PDGF-betaR and alpha-SMA, but only 2.8+/-1.8/1,000 microm of glomerular cells expressed both signals. CONCLUSIONS: Our study supports the hypothesis that interstitial PDGF-betaR and alpha-SMA positive cells may play a key role in the pathogenesis of tubulointerstitial damage.     

人脂肪组织来源的基质细胞分化为神经元样细胞的体外研究

目的 探讨人脂肪组织来源的基质细胞（adipose tissue-derived stromal cells，ADSCs）向神经元样细胞分化的可能性，为神经移植探索新的细胞来源。方法 采用胶原酶消化法分离培养成人的ADSCs，含血清的培养基进行培养，胰蛋白酶消化传代，采用第3～9代的ADSCs进行诱导。应用异丁基甲基黄嘌呤、消炎痛、胰岛素和地塞米松，诱导其向神经元样细胞和脂肪细胞分化，采用苏丹黑B和免疫细胞化学方法对ADSCs进行鉴定。结果 成功培养出ADSCs来源的基质细胞，细胞在体外生长形态类似成纤维细胞，可维持在未分化状态并稳定增殖，体外扩增可达20代。细胞表达波形蛋白和巢蛋白，大部分细胞还表达平滑肌肌动蛋白和βⅢ管蛋白；应用异丁基甲基黄嘌呤、消炎痛、胰岛素和地塞米松，可诱导ADSCs向神经元样细胞和脂肪细胞分化，其中0．1％～0．2％的细胞分化为神经元样细胞，40％～50％分化为脂肪细胞。分化的神经元样细胞具有典型的神经元形态，并能表达神经元标志物；部分分化的神经元样细胞仍然表达平滑肌肌动蛋白。结论 脂肪组织中存在能分化为神经元样细胞的基质细胞，并能克服间充质细胞的限制，分化为神经元样细胞，但这种细胞是否为有功能的神经元，还需深入研究。     

Cyclic strain induces vascular smooth muscle cell differentiation from murine embryonic mesenchymal progenitor cells

BACKGROUND: Hemodynamic forces play a crucial role in regulating vascular cell phenotype. However, the underlying molecular mechanisms are largely unknown. The objective of this study was to test our hypothesis that cyclic strain could affect smooth muscle cell (SMC) differentiation. METHODS: A murine embryonic mesenchymal progenitor cell line (C3H/10T1/2) was cultured with or without cyclic strain for 6 days. Changes in cell morphology were studied with fluorescence dye Calcein-AM staining. Expression of specific SMC markers, smooth muscle specific alpha-actin (alpha-SMA), and smooth muscle myosin heavy chain (SMMHC), was determined by real-time polymerase chain reaction (PCR) and Western blot. Transforming growth factor- beta (TGF-beta) was used as a positive control. RESULTS: With cyclic strain, CH3/10T1/2 cells demonstrated spindle-shaped morphology and parallel alignment. Cells exposed to cyclic strain illustrated significantly increased mRNA levels of alpha-SMA and SMMHC by 3- and 2-fold, respectively, compared with static cells (P<.05). In addition, cells cultured under cyclic strain with TGF-beta (2 ng/ml) supplementation demonstrated increased mRNA levels of alpha-SMA and SMMHC by 10- and 2-fold, respectively, compared with static cells (P<.05). Furthermore, protein levels of alpha-SMA and SMMHC were also significantly increased by more than 3-fold in cyclic strain-treated cells compared with static cultures (P<.05). TGF-beta synergistically enhanced the effect of cyclic strain on alpha-SMA mRNA expression in CH3/10T1/2 cells. CONCLUSIONS: This is the first study to demonstrate that cyclic strain significantly induces expression of two of the most important SMC markers in a murine embryonic mesenchymal progenitor cell line. Cyclic strain and TGF-beta have a synergistic effect on alpha-SMA mRNA expression.     

BrdU 标记家兔脂肪组织来源干细胞的体外研究

目的 通过采用 BrdU 标记连续培养的家兔脂肪组织来源干细胞(adipose-derived stromal stem cells,ADSCs),检测其最佳标记时间、标记剂量及毒性作用,探讨其作为干细胞标记示踪方法的可行性. 方法 8～12 周龄健康新西兰大白兔 6 只,雌雄不限,体重 1.5～2.0 kg.切取腹股沟皮下 1～2 mL 脂肪组织,采用贴壁法体外分离培养ADSCs,并进行鉴定.取第 3 代细胞以终浓度分别为5、10、15 和 20 μg/mL 的 BrdU 进行标记,分别记为A、B、C及 D 组;另 1 孔不含 BrdU,作为空白对照(E 组).标记12、24、48 和 72h后,采用免疫组织化学检测各组细胞阳性率,苔盼蓝排染法行细胞计数观察标记后细胞活性. 结果 原代培养的ADSCs形态主要为宽大、扁平、短梭形细胞;传代后细胞形态主要为长梭形;第3代 ADSCs经诱导均能向成骨细胞和脂肪细胞分化.免疫组织化学观察:第3代 ADSCs 经 BrdU 标记后,在荧光显微镜下胞核呈绿色荧光.孵育 12h,A、B、C、D 组细胞标记阳性率逐渐增高,分别为 30.6%±2.3%、32.4%±1.9%、45.8%±1.8%、50.8%±3.1%,C、D组与 A 组比较,差异有统计学意义(P<0.01);24 h,A、B、C及D组标记率分别为45.9%±2.0%、87.9%±3.3%、90.6%±2.9% 及 91.7%±3.2%;48 h 和 72 h,A、B、C、D 组标记情况与24 h 相似;E 组各时间点细胞标记阳性率为 0.孵育 24、48及72 h,B、C及D组细胞阳性率与 A 组比较,差异均有统计学意义(P<0.01).孵育12、24、48 及 72 h细胞计数,各组细胞活性均在90%以上,A、B、C、D组与E组比较,差异无统计学意义(P>0.05). 结论 BrdU 标记 ADSCs 的最佳时间为 48 h,最佳浓度为 10μg/mL;BrdU 对细胞标记率及安全性高.     

胚胎面突外胚间充质细胞的培养鉴定及向平滑肌细胞的自分化研究

目的 探讨胚胎面突外胚间充质细胞在体外的生长特性、表型特征及在无分化抑制剂-白血病抑制因子(LIF)的DMEM／F12培养基中向平滑肌细胞自分化的过程。方法 取孕50天人胚面突，用消化法培养获得外胚间充质细胞，倒置显微镜下观察细胞形态和生长情况，免疫组织化学鉴定细胞分化表型。用无LIF的DMEM／F12培养基培养，2天后进行抗α-平滑肌肌动蛋白(α—SMA)单克隆抗体免疫组织化学检测，通过RT—PCR在mRNA水平检测α-SMA的表达，透射电镜观察细胞超微结构。结果 培养的细胞呈单层生长，成纤维状，长梭形，有2～4个突起，抗人自然杀伤细胞标志、波形丝蛋白、S-100蛋白、神经元特异性烯醇化酶、肌红蛋白及Ⅶ因子染色阳性，抗神经胶质纤维酸性蛋白、神经纤维细丝蛋白、α-SMA、角蛋白染色阴性。去除LIF2天后，抗α-SMA免疫组织化学检测呈阳性，RT-PCR显示细胞在RNA水平表达平滑肌细胞特异的α-SMA，透射电镜可见超微结构与平滑肌细胞相类似。结论 所获得的外胚间充质细胞具有干细胞特性。在无分化抑制剂存在时，面突外胚间充质细胞呈明显向平滑肌细胞的自然分化。     

Use of an alpha-smooth muscle actin GFP reporter to identify an osteoprogenitor population

Identification of a reliable marker of skeletal precursor cells within calcified and soft tissues remains a major challenge for the field. To address this, we used a transgenic model in which osteoblasts can be eliminated by pharmacological treatment. Following osteoblast ablation a dramatic increase in a population of alpha-smooth muscle actin (alpha-SMA) positive cells was observed. During early recovery phase from ablation we have detected cells with the simultaneous expression of alpha-SMA and a preosteoblastic 3.6GFP marker, indicating the potential for transition of alpha-SMA(+) cells towards osteoprogenitor lineage. Utilizing alpha-SMAGFP transgene, alpha-SMAGFP(+) positive cells were detected in the microvasculature and in the osteoprogenitor population within bone marrow stromal cells. Osteogenic and adipogenic induction stimulated expression of bone and fat markers in the alpha-SMAGFP(+) population derived from bone marrow or adipose tissue. In adipose tissue, alpha-SMA(+) cells were localized within the smooth muscle cell layer and in pericytes. After in vitro expansion, alpha-SMA(+)/CD45(-)/Sca1(+) progenitors were highly enriched. Following cell sorting and transplantation of expanded pericyte/myofibroblast populations, donor-derived differentiated osteoblasts and new bone formation was detected. Our results show that cells with a pericyte/myofibroblast phenotype have the potential to differentiate into functional osteoblasts.