米非司酮抑制人子宫内膜基质细胞增殖

目的在改良子宫内膜细胞原代分离培养方法的基础之上,探讨米非司酮对原代分离培养的人子宫内膜基质细胞增殖的抑制作用。方法利用改良后的"二次消化法"分离培养增殖期人子宫内膜基质和上皮细胞,进行细胞形态学观察,并通过流式细胞术检测人子宫内膜基质和上皮细胞的波形蛋白和角蛋白表达阳性的细胞比率,计算分离培养的基质和上皮细胞的纯度;采用四甲基偶氮唑盐(MTT)法检测米非司酮对原代分离培养人子宫内膜基质细胞的半数抑制浓度(IC50)。结果原代分离培养的基质细胞较上皮细胞生长迅速。流式细胞检测结果显示,分离培养的基质细胞波形蛋白阳性率达97%,上皮细胞角蛋白阳性率达90%。米非司酮处理人子宫内膜基质细胞2d或3d后,结果均显示,随着米非司酮浓度增加,基质细胞的细胞抑制率(IR)逐渐升高。米非司酮作用基质细胞2d后,50和100μmol/L组的IR值均高于对照组,差异均具有统计学意义(P0.05);米非司酮作用3d后,25、50和100μmol/L组的IR值均高于对照组,差异均有统计学意义(P0.05)。通过对IR值进行直线回归分析,结果表明:米非司酮处理2d和3d对原代人子宫内膜基质细胞的IC50分别为52.85μmol/L和86.46μmol/L。结论改良后的"二次消化法"可获得高纯度人子宫内膜基质细胞和上皮细胞。米非司酮对原代人子宫内膜基质细胞有抑制作用,并呈时间和剂量依赖效应。     

骨髓基质细胞共培养体系促进人脐血CD133+细胞的体外扩增

目的探讨来自白血病患者骨髓细胞的基质细胞层建立的扩增体系体外扩增人脐带血造血干/祖细胞的可行性。方法应用白血病患者缓解期骨髓基质细胞,结合造血细胞生长因子体外扩增人脐带血CD133 细胞(实验组),对扩增细胞的免疫表型、集落形成能力等生物学特性进行动态观察,并与单独应用造血细胞生长因子的常规扩增(对照组)进行比较。结果基质细胞层上接种的细胞易于收集;实验组在扩增各时间点的有核细胞数(NC)明显高于对照组,而且有核细胞中CD34 细胞、CD133 细胞、CD34 CD38-细胞以及CD34 CD133 细胞的比例也明显高于对照组;实验组扩增各时间点有核细胞各种集落的种植率明显高于对照组,有核细胞、CD34 细胞、CD133 细胞、CD34 CD38-细胞以及细胞集落的扩增倍数明显高于对照组。结论含有基质细胞的扩增培养体系,不仅可以加速人脐带血造血干/祖细胞的体外扩增,而且可以延缓造血干/祖细胞的分化。     

脂肪基质血管片段分离的研究进展

脂肪基质血管片段(SVF)是脂肪组织经分解并去掉上清后得到的底层细胞团,SVF含有多种细胞群,包括脂肪来源干细胞(ADSC),内皮祖细胞,免疫细胞,平滑肌细胞,周细胞和其他基质成分。SVF在临床中有诸多应用研究,如脂肪移植,糖尿病,促进组织再生血管化等。脂肪组织的分离方法决定了SVF的数量和质量,本文对目前SVF分离技...     

表皮生长因子对子宫内膜细胞体外增殖的影响

本研究观察了表皮生长因子（ＥＧＦ）在体外对子宫内膜上皮细胞及基质细胞增殖的影响。采用酶消化加物理方法分离人子宫内膜上皮细胞及基质细胞,分别在体外培养,细胞汇合后消化,一部分用盖片法培养,用特异性抗体鉴定细胞纯度,另一部分做细胞增殖实验。在细胞中加入不同浓度的ＥＧＦ,培养２４、４８、７２小时,用ＭＴＴ法及流式细胞仪测定ＥＧＦ对子宫内膜上皮细胞及基质细胞增殖的作用。结果：ＥＧＦ浓度为５．０及１０．０ｎｇ／ｍｌ时,明显刺激子宫内膜上皮细胞及基质细胞的增殖,ＭＴＴ与流式细胞仪两法一致。ＥＧＦ不刺激细胞凋亡。结论：采用酶消化结合物理方法分离的人子宫内膜基质细胞及上皮细胞,方法简便、快速,细胞纯度高,在体外生长良好,可用于体外研究着床及异位子宫内膜生长的机制。当ＥＧＦ浓度为５．０及１０．０ｎｇ／ｍｌ时,确实刺激子宫内膜细胞的增殖。     

人胆囊癌细胞系GBC-SD侧群细胞致瘤特性的实验研究

目的 研究人胆囊癌细胞系GBC-SD侧群细胞(side population cells,SP)的致瘤特性。方法 利用流式细胞术从GBC-SD中分选SP、非SP细胞(non-SP),分别进行软琼脂克隆形成实验和非肥胖性糖尿病联合免疫缺陷(NOD/SCID)小鼠的移植瘤形成实验。通过流式细胞术检测5例人胆囊癌组织中SP细胞的比例。结果人胆囊癌细胞系GBC-SD中存在SP细胞,其比例约为0.87％;SP细胞的克隆形成率高于non-SP细胞（14.74％±3.53％比5.17％±1.05％,t=2.75,P＜0.05）。NOD/SCID小鼠移植瘤实验表明,5×103个SP细胞可成瘤(4/7),而non-SP细胞成瘤则需要1×105个细胞(1/7),且来源于SP细胞亚群的NOD/SCID小鼠移植瘤中存在SP、non-SP细胞。SP细胞存在于人胆囊癌组织中,其范围为0.27％～2.3％。结论来源于GBC-SD的SP细胞亚群具有类似肿瘤十细胞的高致瘤性。     

人子宫内膜体外构建的实验研究

目的探讨体外构建人子宫内膜的新方法。方法将采用筛网分离法获得的原代人子宫内膜基质细胞和腺上皮细胞依次接种到自制的液态胶原材料上共培养,形成子宫内膜组织,并与二维条件下正常培养的子宫内膜基质细胞和腺上皮细胞进行比较。采用组织学和免疫组织化学方法观察所构建的子宫内膜的组织学特征,以及两种细胞在不同培养条件下的生长和分布情况。结果分离获得的子宫内膜基质细胞和腺上皮细胞的纯度分别达到95%和90%。在Ⅰ型液态胶原形成的凝胶内,子宫内膜基质细胞排列紧密,腺上皮细胞呈腺体样结构。结论两种细胞与Ⅰ型液态胶原凝胶复合后构建的子宫内膜具有正常子宫内膜组织的结构。     

生殖周期中小鼠子宫生长抑素细胞的研究

为了解生殖周期中小鼠子宫生长抑素细胞分布及生长抑素含量的变化。采用免疫细胞化学技术观察小鼠子宫生长抑素细胞分布情况 ;以放射免疫方法检测生殖周期中小鼠子宫生长抑素的水平。结果显示 ,生殖周期中各期小鼠子宫内膜固有层均有生长抑素阳性细胞反应 ,多出现于子宫腺体周围或近肌层处固有层内 ,肌层间结缔组织偶见。妊娠第 17天 ,壁蜕膜内结缔组织及胎盘绒毛膜上皮下方胚性结缔组织 ,富含生长抑素阳性细胞。妊娠期小鼠子宫生长抑素水平明显下降 ,与间情期比较 ,有显著性差异 (P<0 .0 5)。结论 :生殖周期中各期小鼠子宫壁上有生长抑素阳性细胞分布 ;妊娠晚期壁蜕膜和胎盘绒毛膜上皮下方胚性结缔组织 ,含有相当数量生长抑素阳性细胞。妊娠期小鼠子宫生长抑素水平减少。生长抑素的细胞来源及其在生殖周期中对小鼠子宫的调节有待进一步研究     

ATF6在转基因敲除鼠人工诱导蜕膜化模型中的研究

目的 利用小鼠人工诱导蜕膜化模型，探讨转录活化因子6(ATF6)在小鼠子宫内膜中的作用机制。方法 构建繁育野生型(Atf6^+/+)和ATF6敲除型(Atf6^-/-)小鼠，并构建小鼠人工诱导蜕膜化模型。雌鼠交配后见阴栓第1天记为Day 1。Day 4进行诱导蜕膜化，蜕膜化后48 h和72 h(即Day 6和Day 7)收集小鼠子宫进行相应指标检测，实时荧光定量PCR方法和蛋白质免疫印迹法检测Atf6 mRNA与蛋白质在蜕膜化子宫中的表达情况；免疫组织化学方法检测野生型(Atf6^+/+)和ATF6敲除型(Atf6^-/-)小鼠人工诱导蜕膜化模型中ATF6和催乳素(PRL)在子宫中的阳性信号表达情况。收集人子宫内膜生理周期中不同时相子宫内膜组织，进行石蜡包埋切片，免疫组织化学方法检测ATF6在子宫内膜中的阳性信号表达情况。结果 与小鼠未蜕膜子宫相比，Day 6和Day 7蜕膜化子宫中的Atf6 mRNA表达水平显著升高(P<0.05),细胞核内的蛋白水平也显著增高(P<0.05);Day 7时子...     

着床期小鼠子宫内膜促血管生成素-2的表达

目的　检查促血管生成素- 2(Ang- 2)蛋白在小鼠着床期子宫内膜的分布及mRNA的表达,以探究Ang- 2基因表达在着床过程中的作用和生物学意义(OD)。　方法　取妊娠2、4、6和8 d的小鼠子宫内膜(蜕膜),运用免疫组织化学S P法和原位杂交技术,检测着床期子宫内膜中Ang- 2蛋白的表达及mRNA的转录水平。结合图像分析技术检测不同时期子宫内膜中Ang -2表达的平均光密度(OD)。　结果　结果显示,Ang 2特异性免疫反应产物在基质细胞及腔上皮细胞胞浆中表达,随着妊娠天数的增加,表达逐渐增强(P<0.01)。原位杂交显示,Ang- 2 mRNA自妊娠第2天起即表达于基质细胞和腺上皮;第4天血管壁胞浆中也出现阳性表达,且其表达强度在妊娠第 6、8 天时逐渐增加。平均 OD值具有显著性差异(P<0.01)。　结论　在小鼠妊娠过程中Ang- 2和Ang 2 mRNA在着床前小鼠子宫内膜中即开始表达,并随妊娠进程而表达增强,提示Ang 2基因在“胎 母”对话的信号传导过程中起调节作用。     

IL-18及IL-18结合蛋白在人正常月经周期子宫内膜的表达

目的探讨白细胞介素(IL)18及IL18结合蛋白(IL18BP)在人正常月经周期子宫内膜上的表达。方法取因子宫肌瘤或子宫脱垂行全子宫切除术的患者子宫内膜,按月经周期和子宫内膜形态学检查结果分为增生期和分泌期2组,每组10例。免疫组织化学法检测子宫内膜的IL-18及IL-18BP表达;实时定量聚合酶连锁反应(qRT-PCR)检测子宫内膜IL-18及IL-18BP mRNA的表达。结果 IL-18、IL-18BP在人增生期和分泌期子宫内膜中均有表达,且随着月经周期其表达呈现时空性变化。IL18在增生期表达于腔上皮、腺上皮细胞膜及基质细胞,与增生期相比,分泌期基质细胞IL18的阳性表达显著降低(MOD值0.33±0.07 vs.0.52±0.12,P0.05),而IL18BP的表达则显著增加(MOD值0.33±0.20 vs.0.10±0.07,P0.05)。qRT-PCR结果显示,在增生期和分泌期子宫内膜标本均有IL18和IL18BP mRNA表达,与增生期相比,分泌期IL18表达显著下降(P0.05),IL18BP表达显著增强(P0.05),IL18BP/IL18比值显著增高(P0.05)。结论 IL18和IL18BP在整个月经周期人子宫内膜上均有表达,且其表达呈现时空性的变化,可能与月经周期过程中子宫内膜的崩解修复有关。     

Induction of chondrogenesis and superficial zone protein accumulation in synovial side population cells by BMP‐7 and TGF‐β1

Synovial cells are known to contain a sub‐population of cells with multipotent differentiating capacity including chondrocytes. However, the stem/progenitor cells in synovial cells have not been well characterized. Stem/progenitor cells can exclude Hoechst 33342 dye, and the cell fraction with this property is called “side population (SP).” SP cells are present in many adult tissues. The aim of this investigation was to identify, isolate, and characterize SP cells from bovine synovium. Hoechst dye efflux and fluorescence activated cell sorting showed that synovial cells contained 0.60% SP cells. In the presence of verapamil, an inhibitor of ABC transporters critical for the dye efflux property, the SP cell fraction was not observed, indicating the critical role of ABC transporters. Isoforms of ABC transporters (ABCG2 and ABCB1 mRNA) were highly expressed in SP cells derived from the synovial cells by real‐time RT‐PCR analysis. Bone morphogenetic protein‐7 (BMP‐7) induced type II collagen mRNA expression characteristic of chondrogenesis in articular cartilage with both SP and non‐SP cells. In addition, expression of SZP mRNA, a marker of the surface layer of articular cartilage, was significantly up‐regulated by BMP‐7, and the protein accumulation of SZP was stimulated by both BMP‐7 and TGF‐β1. Thus, synovial cells contain ABC transporter‐dependent SP cells. These findings demonstrate that side population cells of synovium differentiate toward an articular chondrocyte phenotype of the surface layer and have direct implications for tissue engineering and regeneration of articular cartilage. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:485–492, 2008     

Long-term transgene expression and survival of transgene-expressing grafts following lentivirus transduction of bone marrow side population cells

BACKGROUND: Successful transduction of hematopoietic stem cells is essential if gene therapy is to be used clinically to induce immunologic tolerance. METHODS: Hoechst 33342 staining was used to isolate a population of bone marrow cells enriched for stem cells, termed side population (SP) cells. Murine bone marrow SP cells were transduced with HLA-A2.1-expressing VSV-G-pseudotyped lentivirus or retrovirus vectors under identical conditions. RESULTS: After transduction without prestimulating cytokines, which minimizes cell cycling and helps maintain stem cell pluripotency, the HLA-A2.1 gene was found in the DNA of 56% of CFU-GM colonies derived from lentivirus-transduced SP cells, but in only 4% of colonies derived from retrovirus-transduced SP cells. Lentivirus and retrovirus transduction including cytokine prestimulation produced the same degree of integration as that following lentivirus-transduction of non-prestimulated cells. Transplantation of 5,000 lentivirus-transduced SP cells into lethally irradiated mice resulted in long-term expression of the HLA-A2.1 transgene in peripheral blood progeny of bone marrow SP cells and prolonged skin graft survival across this class I MHC barrier until the time of animal sacrifice. CONCLUSIONS: Recombinant lentivirus, but not retrovirus vectors, effectively transduced SP cells that were not prestimulated with cytokines and lentivirus-transduced SP cells successfully repopulated lethally irradiated C57BL/6 mice, animals where there is no selective advantage to repopulation with transduced cells. Transplantation of a relatively small number of transduced SP cells led to prolonged transgene mRNA expression and antigen-specific survival of grafts expressing the foreign MHC transgene.     

Characterization of benign and malignant prostate epithelial Hoechst 33342 side populations

BACKGROUND: The prostate epithelial stem cell has been proposed as the primary origin of neoplastic change in prostate cancer. However, the isolation and characterization of unexpanded prostate epithelial stem cells have proven problematic. METHODS: A prostate epithelial side population (SP) has been isolated utilizing a modified Hoechst 33342 dye efflux assay from both benign and malignant prostate tissue. CD45(-ve), integrin alpha2(+ve) Hoechst 33342 SP and NSP cells were isolated by FACS, immunophenotyped and functionally characterized in 3D culture. RESULTS: FACS analysis revealed a verapamil sensitive SP accounting for 0.93 +/- 0.12% and 0.57 +/- 0.11% of the total epithelial population from both benign and malignant prostates. The benign SP phenotype revealed a heterogeneous cell population consisting predominantly of small basal cells containing minimal cytoplasm. Conversely, the malignant SP was of undetermined acinar origin and with a complete loss of expression of the CDK2 inhibitor p21(WAF1/Cip1). In vitro androgen-enhanced 3D culture of the benign and malignant SP cells led to the production of spheroids which had acinus like morphology and expressed primitive and basal cell markers. Incorporation of the CD133 marker isolated a further SP sub-fraction accounting for 0.037 +/- 0.01% of epithelial cells. CONCLUSIONS: Our observations are consistent with the Hoechst 33342 dye efflux assay isolating a stem cell enriched population which can be further sub-fractionated by CD133 selection. Moreover, the loss of the CDK inhibitor in malignancy is consistent with the hypothesis that neoplastic change originates in the stem cell compartment.     

Side population (SP) cells isolated from fetal rat calvaria are enriched for bone, cartilage, adipose tissue and neural progenitors

In some tissues, stem cells are enriched within the side population (SP) cells characterized by the efficient efflux of Hoechst 33342, but few data are yet available to address whether such is the case in bone tissue. When we Hoechst-stained and FACS-analyzed freshly isolated 20- or 21-day fetal rat calvaria (RC) cells, a small fraction of cells (0.15 +/- 0.05%) comprised of a distinct SP. When SP, non-SP and total/unfractionated (Total) RC cells were plated at a density of 30 cells per microtiter well, the percentage of wells containing bone-forming progenitors (CFU-O) was significantly higher in the SP compared to the non-SP or Total populations (13 +/- 4% vs. 1.8 +/- 0.4% and 0.7 +/- 0.4% respectively). The SP was also highly enriched for CFU-alkaline phosphatase (CFU-ALP) and CFU-fibroblast (CFU-F). While Dex increased the recruitment of CFU-O and CFU-F in the SP, it did not increase the frequency of CFU-ALP. Limiting dilution analysis showed a non-linear relationship between cell densities (1, 5, 10, 20 and 30 cells/microtiter well) and the frequency of readout CFU-O, CFU-ALP and CFU-F in all populations, suggesting a cell non-autonomous component to proliferation-differentiation of these progenitor types. When the developmental potential of SP cells for chondrocyte, adipocyte and neural lineages was assessed, SP cells were also found to be enriched for progenitors of all three lineages. These data demonstrate that Hoechst staining and SP sorting by flow cytometry are a useful strategy for the enrichment of CFU-O and possibly other precursors present in RC cell populations.     

胰腺癌肿瘤干细胞样侧群细胞的分离鉴定

目的 分离鉴定胰腺癌中的侧群(SP)细胞亚群.方法 应用Hoechst33342染色,流式细胞仪检测5个胰腺癌细胞系及3个原代培养的临床胰腺癌标本中sP细胞的含量.以PANC-1为例,通过平板克隆形成试验和NOD-SCID小鼠异种移植成瘤实验比较SP细胞与non-SP细胞的克隆形成能力及成瘤能力,通过对体外培养的SP细胞和SP细胞衍生肿瘤的HoechsG3342复染SP再分析判断其是否具有分化潜能.结果 除了BXPC-3,其他胰腺癌细胞系及原代培养标本都存在Verapamil敏感的SP细胞.SP细胞与non-SP细胞比较具有较高的克隆形成能力[(43.67±3.10)%比(8.33±1.63)%,P＜0.01],并且能够分化产生non-SP细胞并维持自身SP细胞的比例在一个较稳定的水平.SP细胞的成瘤能力是non-SP细胞的100倍以上.而且SP细胞在体内亦可发生不对称分裂生成SP细胞和non-SP细胞.结论 SP细胞可能是胰腺癌干细胞的候选细胞之一.     

细胞株源人前列腺肿瘤干细胞的分选与鉴定

目的:论证人前列腺癌(prostate cancer,PCa)细胞株中是否存在干细胞亚群。方法:分别用免疫表型法和侧群(side population,SP)细胞法从5种人PCa细胞株(Du145、IA8、LNCaP、TSU-PrL和PC-3)中富集类干细胞,再应用软琼脂克隆形成试验初步验证类干细胞亚群的体外生长方式及成瘤能力。选择LNCaP源SP细胞(LNCaP/SP),依次采用免疫细胞化学技术、Transwell、MTT以及裸鼠致瘤试验,分别检测其干细胞标记物的表达情况、鉴定其体外增殖和侵袭能力以及动物体内的致瘤和转移潜能。结果:5种细胞株中均难以分选出免疫表型为CD133+CD44+的细胞亚群。除PC-3外,其余4株细胞可分选出呈现典型克隆性生长特点的SP细胞。体外克隆形成率在IA8、LNCaP和TSU-PrL源SP细胞与非侧群(non-side population,NSP)细胞间有显著性差异(P<0.05)。与LNCaP/NSP相比,LNCaP/SP的体外增殖和侵袭能力显著增强,同时阳性表达整合素α2、Nanog、CD44、OCT4以及ABCG2等5种干细胞标记物。而且,LNCaP/SP的皮下成瘤率、骨转移率及瘤体体积亦显著高于LNCaP/NSP(P<0.01)。结论:SP分选法更适合富集人PCa细胞株中类干细胞,LNCaP/SP细胞是PCa细胞株LNCaP中的肿瘤干细胞(cancer stem cell,CSC)。     

Identification of side population cells (stem-like cell population) in pediatric solid tumor cell lines

Purpose

Recent evidence has supported the cancer stem cell theory that cancer contains a small number of cancer stem cells (CSC) as a reservoir of cancer cells. Only the CSC, but not most of the remaining constituent cancer cells, are thought to be responsible for tumorigenesis, progression, and metastasis as well as cancer relapse, suggesting that the CSC should be targeted to eradicate the cancer. Side population (SP) cells isolated by fluorescence-activated cell sorting (FACS) using Hoechst dye are known to be enriched in stem cells in various normal tissues as well as cancers. The authors investigated whether such stem-like SP cells may exist in pediatric solid tumors (PSTs).

Materials and Methods

Sixteen pediatric tumor cell lines including 7 neuroblastomas, 4 rhabdomyosarcomas, and 5 Ewing's sarcomas were used for FACS analysis. Analysis of SP cells based on the exclusion of the DNA binding dye, Hoechst 33342, with and without verapamil using FACS was performed.

Results

One Ewing's sarcoma cell line did not show an SP fraction, and only a small fraction of SP cells (0.12%-14.6%) was detected in the other 15 cell lines. These SP cells were all sensitive to verapamil.

Conclusions

This study suggested that most PSTs would contain a small fraction of SP cells (possible stem-like population). Targeting the CSC will provide a novel treatment strategy to eradicate refractory PSTs.     

Presence of nonhematopoietic side population cells in the adult human and nonhuman primate pancreas

Side population (SP) cells defined by their ability to efflux Hoechst dye 33342 (Hst), demonstrate functional stem cell capabilities in adult murine tissues and may represent organ-specific stem cells. We examined adult human (Hu) and rhesus macaque (Rh) pancreatic tissue for the presence of SP cells. METHODS: Hu cadaver (n = 4) and Rh donor (n = 5) pancreata were dispersed with collagenase and separated by density gradient centrifugation to relatively enrich fractions for islet, ductal, and acinar tissue in human and islet and nonislet tissue in Rh. Single cell suspensions were incubated with varying Hst concentrations to determine optimal conditions for SP cell analysis. Cellular heterogeneity was assessed using a panel of monoclonal antibodies positive for hematopoietic and/or endothelial cells. RESULTS: Hu SP cells comprised approximately 0.12%, 0.08%, and 0.45% of the gated populations for Hu islet, ductal, and acinar fractions respectively. In Rh, 5.5% and 3.7% of the islet and nonislet fractions were identified as SP cells. FACS analysis of Hu pancreas-derived SP cells indicated that greater than 95% were CD45(-), and only 6% were CD34(+)CD45(-). A similar phenotype was detected in Rh pancreas-derived SP cell populations: greater than 70% were CD45(-) and less than 2% were endothelial lineage positive. CONCLUSIONS: SP cells are found in both islet- and nonislet-enriched fractions of the adult Hu and Rh pancreas. The majority of pancreatic SPs are CD45(-) and CD34(-), suggesting nonhematopoietic lineage. Further preclinical study is needed to establish the phenotype and functional role of adult tissue-specific versus tissue-resident stem cells.     

Failure of differentiation into mature myotubes by muscle precursor cells with the side-population phenotype after injection into irreversibly damaged striated urethral sphincter

We previously showed that the injection of a heterogeneous population of muscle precursor cells (MPCs) into striated urethral sphincter irreversibly damaged by electrocoagulation results in the formation of functional myotubes. To gain further insights into the role played by the different types of MPCs, we elected to sort the MPCs by Hoechst 33342 staining/fluorescence-activated cell-sorter analysis before injection. We found that the side population (SP) cells (muscle stem cells) injected in isolation survived, whereas the main population (MP) cells did not. However, the SP cells failed to differentiate into mature myotubes, as observed previously with unfractionated MPCs containing both SP and MP cells. This result suggests that interactions between SP and MP cells are required for the formation of myotubes in a nonregenerating muscle. In the setting of sphincter insufficiency, injection of MPCs at different stages of maturation may be a better option than purified muscle stem cells.