高成脂脂肪干细胞系的分子克隆筛选

目的 探讨并筛选具备高成脂能力的脂肪干细胞系表面标志、可应用于脂肪组织工程的种子细胞,以提高组织工程化脂肪的构建效率.方法 胶原酶消化人脂肪组织,获得脂肪干细胞,培养扩增后成脂诱导,收集诱导成熟的脂肪细胞,天花板贴壁培养得到去分化脂肪细胞.比较去分化脂肪细胞与人脂肪干细胞的增殖能力,成脂分化能力及表面抗原表达的变化.结果 去分化脂肪细胞与脂肪干细胞的形态和增殖能力相似;去分化脂肪细胞的成脂分化能力高于脂肪干细胞;两种细胞表面抗原的表达大致相同,但去分化脂肪细胞的CD54的阳性表达高于脂肪干细胞.结论 CD54的表达可能与去分化脂肪细胞的高成脂分化能力密切相关,可能是高成脂系干细胞的特异性表面抗原标志.     

体外培养的人脂肪间充质干细胞生物学特性的研究

目的:了解体外培养的人脂肪间充质干细胞的生物学特性及其体外成脂和成骨的能力.方法:体外培养人脂肪间充质干细胞并传代计数,行成骨和成脂诱导,流式细胞仪检测其细胞增殖周期与表面分子.结果:人脂肪干细胞经过体外培养均一的阳性表达CD44、CD106,而CD49d、CD34、CD45和HLA-DR表达阴性.细胞周期分析表明:G0/G1、S和G 2/M所占比例分别为79.1%、19.7%和1.3%.分离细胞在诱导体系下可以向成骨和成脂方向分化.结论:脂肪间充质干细胞具有特殊的生物学特点,体外能够向成骨和成脂方向分化.     

兔脂肪干细胞体外成骨诱导培养及成骨活性鉴定的实验研究

目的体外分离培养兔脂肪干细胞（adipose-derived stem cells,ADSCs）,在成骨诱导条件下鉴定其成骨活性。方法取4月龄新西兰大白兔腹股沟区脂肪,Ⅰ型胶原酶消化,分离、培养及传代原细胞,将第3代ADSCs消化后,加入成骨培养液中诱导分化,分别行碱性磷酸酶染色、茜素红染色、VonKoss（a钙结节）染色,以鉴定分化结果。结果体外分离培养的细胞增殖活跃,成骨诱导后碱性磷酸酶染色、茜素红染色及Von Kossa染色均呈阳性表达。结论脂肪干细胞具有成骨分化能力,是一种理想的骨组织工程种子细胞。     

自体富血小板血浆诱导兔脂肪干细胞成软骨分化的实验研究

[目的]体外分离、培养、鉴定兔脂肪干细胞,探讨富血小板血浆体外诱导脂肪干细胞成软骨分化潜能。[方法]取Ⅰ型胶原酶消化兔脂肪后,贴壁法分离培养脂肪干细胞,取第3代细胞分别予以成脂、成骨诱导,证实其多向分化潜能;同时取第3代细胞予以富血小板血浆诱导,2周后倒置显微镜观察细胞形态,行Ⅱ型胶原免疫荧光细胞化学染色、甲苯胺蓝染色和实时荧光定量PCR检测Ⅱ型胶原和聚集蛋白聚糖的表达。[结果]可以从兔脂肪中培养出脂肪干细胞,成脂、成骨诱导证实其多向分化潜能。经自体富血小板血浆诱导的脂肪干细胞,其Ⅱ型胶原免疫荧光细胞化学染色、甲苯胺蓝染色均为阳性。实时荧光定量PCR检测发现经自体富血小板血浆诱导的兔脂肪干细胞Ⅱ型胶原α1链基因和聚集蛋白聚糖基因表达明显高于对照组未经诱导的兔脂肪干细胞(P<0.01)。[结论]自体富血小板血浆可以有效诱导兔脂肪干细胞表达II型胶原和蛋白聚糖,可以诱导兔脂肪干细胞向软骨细胞方向分化。     

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。     

膝关节滑膜间质干细胞分离、培养及其多向分化潜能特性的研究

目的 探讨晚期骨关节炎患者膝关节滑膜间质干细胞(synovium-derived mesenchymalstem cells,SMSCs)体外分离、培养的可行性及其在体外向脂肪细胞、成骨细胞和软骨细胞定向分化的特性.方法 取膝关节滑膜组织,胶原酶消化获得有核细胞.挑选单细胞克隆,筛选获得SMSCs.流式细胞技术检测细胞表面特异性抗原标志.培养至第三代,分别向脂肪细胞、成骨细胞和软骨细胞诱导分化.油红O染色鉴定向脂肪细胞分化;碱性磷酸酶染色、茜素红染色鉴定向成骨细胞分化;甲苯胺蓝染色鉴定向软骨细胞分化.RT-PCR检测脂肪细胞、成骨细胞标志基因.Ⅱ型胶原免疫组化染色检测软骨细胞Ⅱ型胶原的表达.结果 原代SMSCs体外培养呈葵花样细胞集落,传代后可见圆形巨噬样细胞和纺锤形成纤维样细胞,融合后呈成纤维细胞样生长.CD44、CD90呈阳性,CD34、CD71和CD45呈阴性.向脂肪细胞诱导21d,油红O染色阳性;RT-PCR检测有脂蛋白酶、乙二腈及PPARγ2表达;向成骨细胞诱导7、28 d,ALP,茜素红染色阳性,有ALP、Osteopontin及Osteocalcin表达;向软骨细胞诱导21d,甲苯胺蓝染色阳性,Ⅱ型胶原免疫组化染色阳性.结论 晚期骨关节炎患者膝关节滑膜组织可以分离、培养获得SMSCs. SMSCs具有向脂肪细胞、成骨细胞和软骨细胞发生定向分化的潜能.     

吸脂组织中脂肪干细胞分离和定向分化的研究

目的探讨吸脂组织中的干细胞分离和体外诱导分化为表皮样细胞、成骨细胞及脂肪细胞的可能性。方法通过电动负压吸引获取1例行吸脂手术的30岁女性腹部脂肪组织,酶消化法获取脂肪来源干细胞,体外培养扩增,通过流式细胞仪检测表面抗原的表达。取生长良好的第3代人脂肪来源干细胞,分别应用成表皮诱导培养液（70％培养液A＋30％成纤维细胞培养基上清液＋10ng／L表皮生长因子）,成骨诱导培养基（DMEM／10％FBS,0．1μmol／L地塞米松,50μmol／L维生素C,10mmol／Lβ-甘油磷酸钠,100U／ml青霉素,100U／ml链霉素）和成脂肪细胞诱导培养基（DMEM＋10％FBS＋500μmol／L1-甲基-3-异丁基黄嘌呤＋1μmol／L吲哚美辛）诱导20d后,分别对成表皮诱导组进行免疫组化检测CK19表达,成骨诱导组进行碱性磷酸酶检测,成脂诱导组进行油红O检测。结果流式细胞仪鉴定结果示,人脂肪来源干细胞CD44和CD49d为阳性,CD34为阴性。诱导20d后,成表皮诱导组示免疫组织化学鉴定结构显示有CK19的表达;成骨诱导组示细胞碱性磷酸酶染色阳性;成脂肪细胞诱导组示油红O染色,胞质内脂滴均被染成红色,证实为脂性液体。结论从吸出的脂肪组织中分离出脂肪来源干细胞,在体外进行了脂肪干细胞的扩增和传代,所分离的脂肪来源干细胞具备多向分化能力。     

脂肪干细胞原代培养及其向表皮细胞诱导分化的研究

目的 探讨人脂肪干细胞(adipose-derivedstem cells,ADSCs)体外原代培养方法及诱导分化为表皮细胞的可能性.方法 利用酶消化法原代培养ADSCs,免疫细胞化学检测其相关表面抗原CD29、CD34、CD44、CD49d、CD80、CD106的表达;用表皮细胞诱导培养基对ADSCs进行体外诱导,观察细胞的生长情况及形态变化;对诱导前后两种细胞行细胞增殖活性及细胞角蛋白表达检测.结果 利用脂肪抽吸液成功培养出ADSCs,且能稳定传代;免疫组织化学染色证实有特定的干细胞表面标记物表达;对ADSCs成功进行了体外诱导培养,其细胞形态及蛋白表达均有向表皮细胞分化的倾向,并且仍具较高增殖活性.结论 利用酶消化法可在体外成功培养ADSCs,并能持续传代;ADSCs表达特定的干细胞表面抗原;ADSCs可成功进行体外诱导,有向上皮细胞分化的倾向.     

兔骨髓间充质干细胞的分离、扩增及诱导分化

目的探讨兔骨髓间充质干细胞(bone marrow mesenchymal stem cells,BM-MSCs)体外培养、定向诱导分化为成骨细胞和成脂肪细胞的途径,为进一步的实验研究打下基础。方法抽取兔股骨骨髓,以全骨髓贴壁培养法进行体外培养,贴壁细胞传代,倒置显微镜下观察细胞形态。取第3代细胞向成骨细胞和成脂肪细胞诱导,14d后成骨细胞诱导组检测碱性磷酸酶,成脂肪细胞诱导组进行油红O染色。结果全骨髓贴壁培养法可获得BM-MSCs,原代和传代培养的BM-MSCs具有活跃的增殖能力。成骨细胞诱导组碱性磷酸酶检测表达阳性,成脂肪细胞诱导组油红O染色见胞浆内出现大量红染脂滴。结论全骨髓贴壁培养法可有效地分离和扩增BM-MSCs,分离培养的BM-MSCs生长稳定,增殖力强,可向成骨细胞和成脂肪细胞诱导分化。     

大鼠脂肪组织来源干细胞的分离、培养和生物学特性的研究

目的:建立一种分离和培养大鼠脂肪干细胞的方法,并探讨获得的脂肪干细胞的部分生物学特性。方法:取SD大鼠腹股沟处的皮下脂肪,I型胶原酶消化法获取原代脂肪干细胞,接种至含10%胎牛血清的DMEM培养液,培养并适时传代,每日在倒置显微镜下观察记录细胞形态和增殖特征,测定其生长曲线,进行冻存复苏实验。第3代细胞使用成骨诱导液诱导其向成骨细胞分化,进行VonKossa染色;使用成脂诱导液诱导其向成脂细胞分化,进行油红O染色。结果:从大鼠脂肪组织中分离出脂肪干细胞,其在体外生长增殖迅速,呈成纤维样细胞生长。生长曲线表明第3代脂肪干细胞增殖能力最强,可以在地塞米松、维生素C、β-甘油磷酸钠的诱导下,表现出成骨细胞特性,VonKossa染色出现矿化结节;在IBMX(3-异丁基-1-甲基黄嘌呤)、吲哚美辛、胰岛素的诱导下,表现出脂肪细胞特性,油红O染色后发现胞浆内有脂肪滴。脂肪干细胞在液氮中冻存1个月后,其生长增殖活性和多向分化能力没有明显降低。结论:成功建立了一种简单有效的分离和培养大鼠脂肪干细胞的方法,获得的脂肪干细胞生长增殖旺盛,具有多向分化能力,可以方便地保存,有望作为细胞治疗和组织工程的种子细胞。在分离大鼠的脂肪干细胞时,NH4Cl裂解红细胞这一步可以省略,地塞米松在成脂诱导过程中不是必需的。     

In vitro Comparison of Adipogenic Differentiation in Human Adipose-Derived Stem Cells Cultured with Collagen Gel and Platelet-Rich Fibrin

Background: Adipose-derived stem cells (ADSCs) are the most preferred cell type, based on their phenotypic characteristics, plasticity, and favorable immunological properties for applications in soft-tissue augmentation. Hence, the present in vitro study was aimed to evaluate the adipogenic differentiation potential of human ADSCs upon culturing individually with collagen gel and platelet-rich fibrin (PRF). Materials and methods: The collected lipoaspirate was used for establishing ADSCs using enzymatic digestion method. Then, the cells were analyzed for their morphology, viability, proliferation rate, population doubling time (PDT), colony-forming ability, cell surface markers expression, and osteogenic differentiation as biological properties. Further, ADSCs were evaluated for their adipogenicity using induction media alone, and by culturing with collagen gel and PRF individually for prospective tissue augmentation. Results: ADSCs were successfully established in vitro and exhibited a fibroblast-like morphology throughout the culture period. Cells had higher viability, proliferation potential and showed their ability to form colonies. The positive expression of cell surface markers and osteogenic ability confirmed the potency of ADSCs. The ADSCs cultured on collagen gel and PRF, individually, showed higher number of differentiated adipocytes than ADSCs grown with adipogenic induction medium alone. Conclusion: The extent of lipid accumulation by ADSCs was slightly higher when cultured on collagen gel than on PRF. Additional experiments are required to confirm better suitability of scaffold materials for soft-tissue regeneration.     

氯化钴预处理对大鼠脂肪干细胞成脂及增殖的影响

目的观察脂肪来源问充质干细胞（ADSCs）在经氯化钴（CoCl2）预处理的条件下,其增殖、细胞状态变化以及成脂肪化倾向。方法通过有限稀释法分离、培养SD大鼠ADSCs,取第3代ADSCs,分别向骨细胞、脂肪细胞定向诱导分化鉴定,利用流式细胞仪检测大鼠ADSCs细胞表面标志物CD29,CD34,CIM4,CIM5的表达。将ADSCs经不同浓度CoCl2干预,利用Westernblot检测缺氧诱导因子1d（HIF-101）的表达情况。同时,利用MT＇F实验来检测大鼠ADSCs在不同浓度的CoCl2下细胞增殖的改变;并且通过油红O定量检测大鼠ADSCs在经历缺氧后的成脂情况。结果第3代ADSCs在倒置显微镜下观察,大多呈梭形,经流式鉴定,大鼠ADSCs细胞表面标志物CD29、CD44表达率高,而CD34、CD45表达率低,体外诱导培养的大鼠ADSCs能够向成骨细胞和成脂细胞分化,具有较高的自我更新能力。在MTr实验中,400、200ixmol／LCoCl2组,ADSCs增值减弱;而100、50Ixmol／LCoCl2的实验组,缺氧造成的对细胞增殖和细胞毒性与空白对照组相比,没有变化（P〉0．05）。HIF-1d的表达随着培养基中的CoCl2的浓度而增加。在光镜下观察发现,随着CoCl2浓度的升高以及时间的延长,大鼠ADSCs的成脂倾向越发明显（P〈0．001）。结论ADSCs在处于体外适量的CoCl2干预情况时,其增殖并不会受到影响,并且呈显著的增强其成脂倾向。     

人脂肪细胞的可塑性

目的 探索体外培养环境下人成熟脂肪细胞的去分化现象,旨在挖掘其作为种子细胞的潜能,为组织工程研究开辟新思路.方法 自成人吸脂术后抽吸物提取成熟脂肪细胞及脂肪组织来源干细胞(adipose-derived stromal cells,ASCs),天花板贴壁培养法诱导成熟脂肪细胞去分化,观察细胞形态变化,获得去分化脂肪细胞(dedifferentiated adipocytes,DA).相同的条件下,MTT比色法比较DA、ASCs活性并绘制细胞生长曲线;流式细胞仪鉴定DA、ASCs表面分子的表达;油红O染色、茜素红染色、阿尔辛蓝染色分别鉴定DA、ASCs成脂分化、成骨、成软骨分化能力.结果 人成熟脂肪细胞在体外培养环境下能去分化为成纤维细胞状DA;MTT比色法测细胞活性:DA、ASCs均有很强的增殖能力,两者差异无统计学意义;流式细胞仪测定:DA、ASCs中HLA-ABC、CD29、CD44均为阳性,CD45、CD34、CD106均为阴性;成脂分化2周,油红O染色可见DA、ASCs内出现红色脂滴;成骨分化2周,茜素红染色可见DA、ASCs内红色钙盐沉积;成软骨分化2周,阿尔辛蓝染色可见DA、ASCs内软骨基质沉积.结论 成熟的脂肪细胞在体外培养条件下可成为DA,DA具有很强的增殖活性,表达部分干细胞特征性表面蛋白,有成骨、成软骨及强大的成脂分化能力,有望成为组织工程优秀的种子细胞.

Abstract：

Objective To explore the dedifferentiation phenomenon of human mature adipocytes cultured in vitro and to discuss the possibility of using dedifferentiation adipocytes ( DA ) as seed cells.Methods Mature adipocytes and ASCs were harvested from human fat aspirates. Mature adipocytes were cultured and induced to DA by ceiling adherent culture method. Cell morphology were observed during the whole process. Viabilities of DA and ASCs were compared by MTT chromatometry and cell growth curves were drawn based on it. Cell surface markers of DA and ASCs were detected by flow cytometry. The adipogenic,osteogenic and chondrogenic ability of DA and ASCs were assessed by oil red O staining,alizarin bordeaux staining and alcian blue staining, respectively. Regults Human mature adipocytes can dedifferentiate into fibroblast-shaped DA. MTT chromatometry assay demonstrated that DA and ASCs both had strong reproductive activity, with no significant difference between them. Flow cytometry assay demonstrated that both DA and ASCs expressed HLA-ABC, CD29 and CD44, while didn't express CD45,CD34 and CD106. After two weeks of adipogenic differentiation, lipid droplets could be displayed by oil red O staining in both DA and ASCs. After two weeks of osteogenic differentiation, calcium salts mineralization in DA and ASCs could be detected by alizarin bordeaux staining. After two weeks of chondrogenic differentiation, matrix of cartilage cells in DA and ASCs could be detected by alcian blue staining. Conclusions Mature adipocytes can be dedifferentiated into DA in vitro. DA has strong reproductive activity, as well as osteogenic, chondrogenic ability and strong adipogenic ability. It expresses some of the stem cell-related cell surface proteins and is a promising seed cell for adipose tissue engineering.     

Comparison of Osteogenic Potentials of BMP4 Transduced Stem Cells from Autologous Bone Marrow and Fat Tissue in a Rabbit Model of Calvarial Defects

We compared bone marrow stem cells (BMSCs) and adipose-derived stem cells (ADSCs) of adult rabbits under identical conditions in terms of their culture characteristics, proliferation capacity, osteogenic differentiation potentials induced by adenovirus-containing bone morphogenetic protein 4 (Ad-BMP4) in vitro, and capacity to repair calvarial defects in the rabbit model by autologous transplantation ex vivo. According to the results of growth curve, cell cycle, and telomerase activity analysis, ADSCs possess a higher proliferation potential. Both of the Ad-BMP4 transduced MSCs expressed BMP4 mRNA and protein and underwent osteogenic differentiation. Up-regulated mRNA expression of all osteogenic genes was observed in differentiated BMSCs and ADSCs, but with different patterns confirmed by real-time RT-PCR. Deposition of calcified extracellular matrix was significantly greater in differentiated ADSCs compared with differentiated BMSCs. X-ray and histological examination indicated significant bone regeneration in the calvarial defects transplanted with Ad-BMP4 transduced autologous MSCs compared to the control groups. There was no significant difference in new bone formation in Ad-BMP4 transduced MSCs based on quantitative digital analysis of histological sections. The use of ADSCs often resulted in the growth of fat tissue structures in the control groups, and the fat tissue structures were not seen with BMSC cells. Our data demonstrate that BMP4 can be potently osteoinductive in vivo, resulting in bone repair. ADSCs may be an attractive alternative to BMSCs for bone tissue engineering under appropriate stimuli. But the easy adipogenic differentiation needs to be considered when choosing adipose tissue for specific clinical application.     

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.     

脂肪间充质干细胞的基本生物学特性及向成骨细胞诱导分化的实验研究

目的　研究脂肪间充质干细胞的基本生物学特性以及在特定培养条件下向成骨细胞分化 ,探讨其作为骨组织工程的种子细胞的可行性。方法　取 3周龄Lewis大鼠的腹股沟脂肪垫 ,消化法获得脂肪间充质干细胞 ,分别用脂肪诱导培养基和成骨诱导培养基诱导其向脂肪细胞与成骨细胞分化 ,组织化学染色、免疫细胞化学染色和Westernblot检测细胞分化的情况。结果　从成体大鼠脂肪组织中培养出脂肪间充质干细胞 ,原代脂肪间充质干细胞能自发分化为脂肪细胞 ,传代细胞在胰岛素和呋塞米的作用下生成脂滴 ,过氧化物酶体增殖物激活受体 (PPAR)γ表达增强 ,向脂肪细胞分化 ;在呋塞米、抗坏血酸、β 甘油磷酸钠的诱导下 ,脂肪间充质干细胞的碱性磷酸酶(ALP)活性检测显示诱导组与对照组差异有显著性 (P <0 .0 1) ,vonKossa染色出现钙结节 ,骨桥蛋白 (OPN)、骨形态发生蛋白 (BMP) 2免疫细胞化学染色阳性 ,Westernblot检测到诱导后细胞OPN、BMP2的表达。结论　从脂肪组织中可获得具有多分化潜能的间充质干细胞 ,经诱导后可分化为脂肪细胞和成骨细胞 ,有可能成为骨组织工程较理想的种子细胞之一     

Biological alchemy: engineering bone and fat from fat-derived stem cells

Adipose tissue contains a population of pluripotent stem cells capable of differentiating along multiple mesenchymal cell lineages. In this study the authors isolated these fat-derived stem cells successfully from Lewis rats and induced differentiation along adipogenic and osteogenic lineages in vitro and in vivo. Induction was stimulated by exposing stem cells to lineage-specific induction factors. Adipocyte-inducing media contained dexamethasone, insulin, and isobutyl-methylxanthine. Osteoblast inducing media contained dexamethasone, beta-glycerophosphate, and ascorbic acid. Undifferentiated stem cells were maintained in minimal essential media alpha and fetal bovine serum. At 10 days, cells cultured in adipogenic media differentiated into adipocytes in vitro, as evidenced by positive Oil red O staining of lipid vacuoles. At 21 days, cells cultured in osteogenic media differentiated into osteoblasts in vitro as demonstrated by Alizarin red staining of a calcified extracellular matrix and immunohistochemical staining for osteocalcin. Differentiated cells were seeded at a density of 5 x 106 cells onto 15 x 15-mm polyglycolic acid grafts and implanted subcutaneously into three groups of Lewis rats: Group I contained undifferentiated stem cell grafts, group II contained adipocyte grafts, and group III contained osteoblast grafts. At weeks 4 and 8, in vivo fat formation was demonstrated in group II rats, as confirmed by Oil red O staining. At 8 weeks, group III rats demonstrated in vivo bone formation, as confirmed by the presence of osteocalcin on immunohistochemistry and the characteristic morphology of bone on hematoxylin-eosin staining. Group I rats demonstrated no in vivo bone or fat formation at either time interval. These results demonstrate the ability to isolate pluripotent stem cells from adipose tissue, to induce their differentiation into osteoblasts and adipocytes in vitro, and to form bone and fat subsequently in vivo. This is the first published report of in vivo bone formation from fat-derived stem cells. These cells may eventually serve as a readily available source of autologous stem cells for the engineering of bone and fat.