Transdifferentiation of glioblastoma stem-like cells into mural cells drives vasculogenic mimicry in glioblastomas

Recent evidence has shown that glioblastoma stem-like cells (GSCs) can transdifferentiate into endothelial cells and vascular-like tumor cells. The latter pattern of vascularization indicates an alternative microvascular circulation known as vasculogenic mimicry (VM). However, it remains to be clarified how the GSC-driven VM makes a significant contribution to tumor vasculature. Here, we investigated 11 cases of glioblastomas and found that most of them consisted of blood-perfused vascular channels that coexpress mural cell markers smooth muscle α-actin and platelet-derived growth factor receptor β, epidermal growth factor receptor, and vascular endothelial growth factor receptor 2 (Flk-1), but not CD31 or VE-cadherin. This microvasculature coexisted with endothelial cell-associated vessels. GSCs derived from patients with glioblastomas developed vigorous mural cell-associated vascular channels but few endothelial cell vessels in orthotopic animal models. Suppression of Flk-1 activity and gene expression abrogated GSC transdifferentiation and vascularization in vitro, and inhibited VM in animal models. This study establishes mural-like tumor cells differentiated from GSCs as a significant contributor to microvasculature of glioblastoma and points to Flk-1 as a potential target for therapeutic intervention that could complement current anti-angiogenic treatment.     

Type 1 collagen as a potential niche component for CD133‐positive glioblastoma cells

Cancer stem cells are thought to be closely related to tumor progression and recurrence, making them attractive therapeutic targets. Stem cells of various tissues exist within niches maintaining their stemness. Glioblastoma stem cells (GSCs) are located at tumor capillaries and the perivascular niche, which are considered to have an important role in maintaining GSCs. There were some extracellular matrices (ECM) on the perivascular connective tissue, including type 1 collagen. We here evaluated whether type 1 collagen has a potential niche for GSCs. Imunohistochemical staining of type 1 collagen and CD133, one of the GSCs markers, on glioblastoma (GBM) tissues showed CD133‐positive cells were located in immediate proximity to type 1 collagen around tumor vessels. We cultured human GBM cell lines, U87MG and GBM cells obtained from fresh surgical tissues, T472 and T555, with serum‐containing medium (SCM) or serum‐free medium with some growth factors (SFM) and in non‐coated (Non‐coat) or type 1 collagen‐coated plates (Col). The RNA expression levels of CD133 and Nestin as stem cell markers in each condition were examined. The Col condition not only with SFM but SCM made GBM cells more enhanced in RNA expression of CD133, compared to Non‐coat/SCM. Semi‐quantitative measurement of CD133‐positive cells by immunocytochemistry showed a statistically significant increase of CD133‐positive cells in Col/SFM. In addition, T472 cell line cultured in the Col/SFM had capabilities of sphere formation and tumorigenesis. Type 1 collagen was found in the perivascular area and showed a possibility to maintain GSCs. These findings suggest that type 1 collagen could be one important niche component for CD133‐positive GSCs and maintain GSCs in adherent culture.     

内皮抑素和血管生成抑素融合基因修饰的单纯疱疹病毒对胶质瘤干细胞的体外实验研究

目的 探讨内皮抑素和血管生成抑素( Endo - Angio)融合基因修饰的单纯疱疹病毒(VAE)对胶质瘤干细胞(GSCs)的体外溶瘤作用。方法 新鲜高级别（WHOⅢ级、Ⅵ级）人脑胶质瘤标本经原代培养获得GSCs,采用流式细胞术检测其中CD133阳性细胞数,单克隆形成实验检测GSCs的自我更新能力,免疫荧光技术检测未分化GSCs的Nestin及分化后GFAP、NF和MAG的表达;MTS法检测VAE对GSCs增殖活性的影响,RT - PCR和Western blot检测Endo - Angio融合蛋白的表达并检测其对人脑微血管内皮细胞(HBMEC)增殖的影响;最后观察病毒处理后仍存活细胞的自我更新和诱导贴壁情况。结果 (1)从20例高级别胶质瘤标本中培养出4例GSCs,能够表达CD133及Nestin,具有自我更新能力和多向分化能力。(2)VAE能够感染GSCs,4例GSCs增殖活性明显下降(P＜0.05)。(3)VAE感染GSCs 48 h后,基因水平及蛋白水平都有Endo- Angio融合蛋白表达,该融合蛋白使HBMEC增殖活性明显下降(P＜0.05)。(4)VAE感染后仍存活的细胞不再具有形成GSCs球的能力,加入血清诱导后也不再贴壁生长。结论 在体外,VAE能够显著抑制GSCs活性,能够表达具有生物活性的Endo-Angio融合蛋白,为脑胶质瘤溶瘤病毒治疗开辟了新的途径。     

Expression of transcription factor Oct-4 and other embryonic genes in CD133 positive cells from human umbilical cord blood

A significant number of hematopoietic stem/progenitor cells (HSPC) in human umbilical cord blood could serve as a reservoir for the placental vasculature, yet, their morphological and functional features are not completely understood. Here, we describe the characterization of purified CD133(+) progenitor cells from umbilical cord blood, a subset of CD34(+) hematopoietic progenitors that were grown in proliferation medium containing Flt3-ligand, thrombopoietin and stem cell factor. Following isolation and enrichment of the CD133(+) cells by immunomagnetic cell sorting, they remained non-adherent for up to 40 days in culture and expressed different pluripotency markers including Sox-1, Sox-2, FGF-4, Rex-1 and Oct-4.Oct-4 expression was confirmed by laser-assisted single cell picking with subsequent quantitative real-time RT-PCR. The expression of Oct-4 indicates a pluripotent phenotype of CD133(+) cells and appears to be of functional relevance: After three weeks in endothelial differentiation medium, suspended cells became adherent, developed an endothelial cell-like morphology, bound fluoresceine isothiocyanate-labeled Ulex europaeus agglutinin-1, took up acetylated Di-LDL, and expressed other endothelial markers such as PECAM-1 or VEGFR-2. Concomitantly, Oct-4 expression was significantly reduced. Moreover, following treatment with retinoic acid, CD133(+) cells exhibited neural morphology associated with the expression of beta-III-tubulin. CD133(+) cells were found to express the luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor, detected by RT-PCR and immunocytochemistry. The recombinant human chorionic gonadotropin induced proliferation of the CD133(+) cells in a dose-specific manner. Our results indicate that CD133(+) HSPC from umbilical cord blood may have a greater differentiation potential than previously recognized and give rise to proliferative endothelial cells participating in placental vasculogenesis.     

胶质母细胞瘤肿瘤干细胞的分离培养与生物学特性研究

目的从人脑胶质母细胞瘤中分离培养脑肿瘤干细胞（brain tumorstem cells,BTSC）,并研究其生物学特性。方法利用无血清培养基和悬浮培养方法,从6例人胶质母细胞瘤标本中分离培养BTSC,并通过单克隆形成实验观察脑肿瘤干细胞球（brain tumor sphere,BTS）的形成过程。将BTSC接种于含血清培养基,观察其在体外的分化特点。将BTSC子代分化细胞更换培养条件,观察其在无血清培养基中的逆向分化现象。应用细胞免疫荧光染色对BTSC及其分化细胞进行鉴定。结果在人胶质母细胞瘤中成功分离出BTSC,其在无血清培养基中呈悬浮球状生长,具有很强的自我更新和增殖能力;免疫荧光显示其表达CD133。BTSC在含血清培养基中发生贴壁分化。分化后的子代细胞可表达CD133、神经元特异性烯醇化酶（NSE）和胶质纤维酸性蛋白（GFAP）。BTSC子代分化细胞在无血清条件下培养,能够再次增殖形成BTS,呈逆向分化现象。结论人胶质母细胞瘤中存在BTSC,其具有自我更新、无限增殖、多向分化以及逆向分化等生物学特性。     

MiR-29a-Mediated CD133 Expression Contributes to Cisplatin Resistance in CD133<Superscript>+</Superscript> Glioblastoma Stem Cells

CD133 positive (CD133⁺) cells are cancer stem cells in glioblastoma that are associated with poor prognosis and resistance to radiotherapy. However, the role of CD133 in chemoresistance is inconclusive, although recent studies suggest that increased CD133 expression may lead to increased cisplatin resistance under certain circumstances. In this study, we further explored the mechanism underlying CD133-mediated cisplatin resistance in glioblastoma stem cells. We sorted human glioblastoma T98G and U87MG cells into CD133⁺ and CD133^? pools and measured apoptosis and CD133 expression levels in response to cisplatin treatment. We predicted candidate microRNAs that might target CD133 and assessed their levels in cisplatin-treated CD133⁺ cells. Finally, we overexpressed miR-29a in CD133⁺ cells and tested its effects in cisplatin-mediated apoptosis and survival of CD133⁺ tumor bearing mice receiving cisplatin treatment. We found that CD133⁺ glioblastoma stem cells showed more resistance to cisplatin treatment. Cisplatin increased CD133 expression by suppressing miR-29a levels. MiR-29a overexpression improved sensitivity of cisplatin in CD133⁺ cells and significantly suppressed tumor growth in CD133⁺ tumor bearing mice in response to cisplatin treatment. Our data show that miR-29a ameliorates CD133-mediated chemoresistance in glioblastoma stem cells, suggesting it as a potential therapeutic target for treating glioblastoma.     

人胶质瘤干细胞内在自我更新能力

目的 了解胶质瘤干细胞内在的自我更新和增殖能力。方法 观察原代胶质瘤细胞在单纯改良Eagle/F12培养液（DMEM/F12）中胶质瘤干细胞球的形成,并检测其CD133、胶质纤维酸性蛋白（GFAP）、微管相关蛋白（MAP2）、髓磷脂碱性蛋白（MBP）的表达。通过二代球体形成、细胞增殖测定、分化实验分析其自我更新、增殖、多能分化能力。通过裸鼠移植瘤实验观察所分离细胞球细胞与原代培养胶质瘤细胞成瘤能力的差异。结果 在单纯DMEM/F12培养液中形成的胶质瘤细胞球细胞表达神经干细胞标记CD133,不表达分化标志GFAP、MAP2,少数细胞表达MBP。分离出的胶质瘤细胞球细胞可在单纯DMEM/F12培养基中增殖,并能形成CD133阳性的二代细胞球,可分化为GFAP、MAP2、MBP阳性表达的肿瘤细胞。裸鼠成瘤实验显示其成瘤能力显著高于原代胶质瘤细胞。结论 胶质瘤干细胞能在无血清、无外源性细胞因子培养基中形成肿瘤干细胞球,胶质瘤干细胞的自我更新和增殖不依赖于外源性生长因子,它可能拥有自我更新的自身活化机制。     

High expression of RFX4 is associated with tumor progression and poor prognosis in patients with glioblastoma

Abstract

Aim: Glioma stem cells (GSCs) have been shown to contribute to tumor development and recurrence, therapeutic resistance, and cellular heterogeneity of glioblastoma multiforme (GBM). Recently, it has been reported that GSCs lose their self-renewal ability and tumorigenic potential upon differentiation. In this study, we identified Regulatory Factor X4 (RFX4) gene to regulate GSCs’ survival and self-renewal activity in the GBM patients samples.

Materials and methods: We utilized public datasets from the Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), Ivy Glioblastoma Atlas Project, and The Human Protein Atlas to screen candidate genes which are associated with the development of GBM and poor patients survival. Small hairpin RNA (shRNA) lentivirus was applied to knockdown RFX4 gene in GSCs.

Results: We found that RFX4 mRNA expression among the RFX family was particularly reduced during GSC differentiation. RT-qPCR analysis revealed significant downregulation of RFX4 and stem cell markers (CD15 and CD133) mRNA expressions in primary human GBM-derived GSCs cultured under serum condition. Consistently, GSCs showed significantly elevated RFX4 mRNA expression levels compared to normal astrocytes, NHA, whereas glioma cells did not. Furthermore, analysis of the TCGA data set revealed that RFX4 is highly expressed in GBM, and contributes to the lowering of patient survival. Depletion of RFX4 using shRNA lentivirus in patient GBM-derived GSCs decreased neurosphere formation and cell viability.

Conclusion: These results suggest that RFX4 is a potential risk factor for maintaining the stemness of GSCs and making glioma more malignant, and thus, could be a promising target of GBM treatment.     

Functional Differences of miR-125b on the Invasion of Primary Glioblastoma CD133-Negative Cells and CD133-Positive Cells

MicroRNAs (miRNAs) are small noncoding RNAs whose function as modulators of gene expression is crucial for the proper control of cell development, differentiation, and homeostasis. The total number and composition of miRNAs expressed per cell at different stages of development varies widely, and the same miRNA may function differently at different stages of development. In this prospective study, we evaluated the function of miR-125b at different developmental stages of glioblastoma cells, such as primary glioblastoma cells and the corresponding stem cells. CD133 is an important surface marker in glioblastoma stem cells. We found that the upregulation of miR-125b had no effects on the invasion of primary glioblastoma CD133-negative cells but that it could inhibit the invasion of corresponding CD133-positive cells; however, the downregulation of miR-125b also had no effects on the invasion of primary glioblastoma CD133-negative cells but promoted the invasion of CD133-positive cells. Further research into the underlying mechanism demonstrated that the effects of miR-125b on the invasion of glioblastoma CD133-positive cells were associated with the alteration of the expression of MMPs (MMP-2 and MMP-9) and corresponding inhibitors (RECK and TIMP3). Our results demonstrate that miR-125b expression plays an essential role in the invasion of glioblastoma CD133-positive cells but not CD133-negative cells. Therefore, miR-125b may represent a novel target for therapy targeting the invasion of glioblastoma stem cells in the future.     

Engraftment of sorted/expanded human central nervous system stem cells from fetal brain

Direct isolation of human central nervous system stem cells (CNS-SC) based on cell surface markers yields a highly purified stem cell population that can extensively expand in vitro and exhibit multilineage differentiation potential both in vitro and in vivo. The CNS-SC were isolated from fetal brain tissue using the cell surface markers CD133(+), CD34(-), CD45(-), and CD24(-/lo) (CD133(+) cells). Fluorescence-activated cell sorted (FACS) CD133(+) cells continue to expand exponentially as neurospheres while retaining multipotential differentiation capacity for >10 passages. CD133(-), CD34(-), and CD45(-) sorted cells (approximately 95% of total fetal brain tissue) fail to initiate neurospheres. Neurosphere cells transplanted into neonatal immunodeficient NOD-SCID mice proliferated, migrated, and differentiated in a site-specific manner. However, it has been difficult to evaluate human cell engraftment, because many of the available monoclonal antibodies against neural cells (beta-tubulin III and glial fibrillary acidic protein) are not species specific. To trace the progeny of human cells after transplantation, CD133(+)-derived neurosphere cells were transduced with lentiviral vectors containing enhanced green fluorescent protein (eGFP) expressed downstream of the phosphoglycerate kinase promoter. After transduction, GFP(+) cells were enriched by FACS, expanded, and transplanted into the lateral ventricular space of neonatal immunodeficient NOD-SCID brain. The progeny of transplanted cells were detected by either GFP fluorescence or antibody against GFP. GFP(+) cells were present in the subventricular zone-rostral migrating stream, olfactory bulb, and hippocampus as well as nonneurogenic sites, such as cerebellum, cerebral cortex, and striatum. Antibody against GFP revealed that some of the cells displayed differentiating dendrites and processes with neurons or glia cells. Thus, marking human CNS-SC with reporter genes introduced by lentiviral vectors is a useful tool with which to characterize migration and differentiation of human cells in this mouse transplantation model.     

CD105 (Endoglin) exerts prognostic effects via its role in the microvascular niche of paediatric high grade glioma

Paediatric high grade glioma (pHGG) (World Health Organisation astrocytoma grades III and IV) remains poor prognosis tumours, with a median survival of only 15 months following diagnosis. Current investigation of anti-angiogenic strategies has focused on adult glioblastoma multiforme (GBM) with phase III trials targeting vascular endothelial growth factor continuing. In this study we investigated whether the degree of vascularity correlated with prognosis in a large cohort of pHGG (n = 150) and whether different vessel markers carried different prognostic value. We found that CD105 (endoglin) had a strongly significant association with poor prognosis on multivariate analysis (p = <0.001). Supervised hierarchical clustering of genome wide gene expression data identified 13 genes associated with differential degrees of vascularity in the cohort. The novel angiogenesis-associated genes identified in this analysis (including MIPOL-1 and ENPP5) were validated by realtime polymerase chain reaction. We also demonstrate that CD105 positive blood vessels associate with CD133 positive tumour cells and that a proportion of CD105 positive vessel cells demonstrates co-positivity for CD133, suggesting that the recently described phenomenon of vasculogenic mimicry occurs in pHGG. Together, the data suggest that targeting angiogenesis, and in particular CD105, is a valid therapeutic strategy for pHGG.     

Antibody to α4 integrin suppresses natural killer cells infiltration in central nervous system in experimental autoimmune encephalomyelitis

Glioma, the most common tumor of the central nervous system (CNS), currently results in a high rate of morbidity and mortality. The expression of CD133, a stem-like cell marker expressed in the glioma cells, is believed to lead to tumorigenesis in the human brain. Thus, it is necessary to find a proper method to specifically kill the CD133(+) glioma cells. Dendritic cell (DC)/tumor hybrids are proven to be able to induce an effective immune response, leading to killing of glioma cells in vitro. We isolated CD133(+) cells from a population of primary glioma cells, and cultured autologous DCs and T cells at the same time. Next, we electrofused the DCs with the CD133(+) glioma cells and with CD133- ones, in order to explore a new strategy for glioma therapy. We then exposed the T cells to five separate groups of cells: DC/CD133(+) hybrids, DC/CD133(-) hybrids, DCs alone, unsorted glioma cells alone and mixed DCs-glioma cells. A cytotoxicity assay showed that T cells stimulated by either type of hybrid were able to kill cultured autologous glioma cells significantly more effectively than those stimulated by the other three cell types (P<0.05). The amounts of IFN-γ secreted by T cells stimulated by the two types of fused cells were obviously increased compared to those stimulated by the other three cell types (P<0.05). However, no significant differences were noted between the effects of the two hybrids, neither in the cytotoxicity assay nor in the IFN-γ release assay (P>0.05). Therefore, both DC/CD133(+) and DC/CD133(-) hybrids can cause significant T cell immune responses in vitro. There were no significant differences between the immune responses caused by the two types of hybrids.     

胶质母细胞瘤肿瘤干细胞体外分离培养鉴定及生物学特性的研究

目的:探讨人脑胶质母细胞瘤(GBM)肿瘤干细胞的培养方法,观察其体外增殖、分化等生物学特性。方法:应用改良神经干细胞培养基,11例人脑GBM标本中悬浮培养GBM的肿瘤细胞。采用有限稀释法筛选分离具有连续克隆能力的单细胞。通过免疫组化及电镜观察的方法,确定其干细胞特征;通过核型分析探讨该细胞的肿瘤性质;将确定的脑肿瘤干细胞(BTSCs)接种于不含生长因子的干细胞培养基,观察其在体外条件下分化的特点。结果:本组样本中10例(1例污染)获得具有连续克隆和增殖能力及干细胞特征的肿瘤细胞。结论:人脑GBM中存在具有自我更新和增殖潜能的BTSCs,在体外可将其分离、培养和纯化。     

25例恶性胶质瘤中肿瘤干细胞的分离与检测

目的 从不同病理级别胶质瘤组织标本中分离、培养并鉴定胶质瘤干细胞.方法 取25例(23例原发胶质瘤,2例复发胶质瘤)新鲜人脑胶质细胞瘤标本,分离脑肿瘤细胞,接种于含生长因子的无血清达尔伯克(氏)必需基本培养基/F-12(DMEM/F-12)培养基中培养至细胞形成干细胞球,免疫荧光法检测脑肿瘤干细胞CD133、Nestin的表达情况,干细胞球诱导分化后检测细胞表面分化标记物β微管蛋白Ⅲ(β-TubulinⅢ)、神经胶质酸性蛋白(GFAP)表达情况.结果 被检测的23例原发胶质瘤标本中,随着胶质瘤级别的增高,形成神经球的时间缩短,数量增多.2例复发胶质瘤组织分离得到的细胞形成的神经球较原发胶质瘤快而多.免疫荧光方法检测神经球CD133、巢蛋白(Nestin)表达阳性.神经球细胞经诱导分化后β-TubulinⅢ、GFAP表达阳性.结论 胶质瘤组织中存在肿瘤干细胞,不同级别胶质瘤中BTSC数量及增殖能力不同.     

HLA-E contributes to an immune-inhibitory phenotype of glioblastoma stem-like cells

Cancer stem cells are an attractive target for immunotherapeutic approaches to glioblastoma. However, an immune inhibitory phenotype of cells currently classified as "glioma-initiating cells" (GIC) might counteract recognition by immune effector cells. Here, we investigate the contribution of the non-classical MHC molecule HLA-E to the immunosuppressive phenotype of GIC. HLA-E is expressed in GIC lines and its expression is reduced upon differentiation of GIC in serum-containing culture conditions. Constitutive HLA-E inhibits natural killer (NK) cell-mediated lysis of GIC since small-interfering RNA-mediated HLA-E gene silencing enhances the immunogenicity of GIC. Increased GIC lysis was observed both in the CD133+ and in the CD133- compartment. Furthermore, the use of interferon-γ as a possible agent to boost an immune response against glioblastoma cells might be limited by the concurrent upregulation of HLA-E.     

Presence of pluripotent CD133+ cells correlates with malignancy of gliomas

BackgroundPresence of CD133⁺ cancer stem cells has been demonstrated within glioblastoma multiforme (GBM), the most malignant phenotype of gliomas (WHO grade IV). Since GBM frequently develops from low grade gliomas (WHO grade II) we assessed a possible qualitative or quantitative correlation of CD133⁺ cells and glioma grade to get new insights in gliomagenesis.ResultsThe amount of CD133⁺ cells within the bulk tumor mass, analyzed by immunostaining and Western blotting, showed a clear quantitative correlation with glioma grade (WHO° II, III and IV). Most of CD133⁺ cells were arranged in clusters frequently associated to tumor vessels. Protein analysis revealed high cellular coexpression of CD133 with Musashi-I but not CD34 indicating a neural, i.e. local origin of these cells. In vitro, no differences in stem cell properties concerning self-renewal and multi-lineage differentiation have been found for CD133⁺ cells isolated from gliomas of different grades.ConclusionsThese findings indicate a solely quantitative correlation of glioma grade with the presence of neural CD133⁺ cells within tumors supporting the concept of a CD133⁺ stem cell dependent gliomagenesis.     

BMP受体在胶质母细胞瘤起始细胞早期分化中的表达

目的在分离鉴定胶质母细胞瘤U87细胞中的胶质瘤起始细胞(GTICs)的基础上,检测在GTICs细胞中骨形态发生蛋白(BMP)受体1A、B的表达,探讨BMP受体1A、B对GTICs所起的生物学作用。方法利用流式细胞术检测U87细胞中CD133和巢蛋白阳性细胞,细胞免疫荧光和蛋白印迹法检测GTICs和普通细胞中BMP受体1A、B的表达。结果 CD133和巢蛋白阳性表达的细胞在瘤U87细胞中所占的比例分别0.92%和5.72%;GTICs可诱导分化为表达β-微导管蛋白和胶质纤维酸性蛋白的细胞;U87细胞的GTICs中BMP受体1A表达明显增高,而经诱导分化培养后的BMP受体1A的表达又明显降低,BMP受体1B表达变化不明显。结论 BMPs信号通路中BMP受体对调控GTICs的增殖分化可能起着重要作用。     

全反式维甲酸对脑肿瘤干细胞增殖和分化的影响

目的 研究全反式维甲酸(ATRA)对脑肿瘤干细胞(BTSCs)增殖和分化的影响.方法 采用有限稀释法从人脑胶质母细胞瘤新鲜标本中分离、克隆筛选BTSCs;在无血清条件下培养BTSCs.根据培养基中的成分不同分为对照组、ATRA组、ATRA/生长因子组、生长因子组,用MTT法检测BTSCs的增殖效应;在含血清条件下诱导分化BTSCs.根据血清培养基中的成分不同分为ATRA组、对照组.于诱导分化第10天用免疫荧光技术检测BTSCs子代分化细胞CD133和胶质纤维酸性蛋白(GFAP)的表达率;将BTSCs子代分化细胞更换培养条件,观察其在无血清培养基中增殖逆转再次形成脑肿瘤干细胞球(BTS)的比例和形成时间.结果 在无血清条件下,ATRA组BTSCs的增殖速度较对照组明显加快,但低于生长因子组和ATRA/生长因子组,形成的BTS亦小于后两者:在含血清条件下,ATRA组BTSCs子代分化细胞CD133、GFAP的表达率分别为2.29%±0.27%和75.60%±4.03%,对照组为7.05%±0.49%和12.51%±0.77%,前者的分化程度明显高于后者,差异有统计学意义(P<0.05),但前者仍然存在CD133的表达;BTSCs子代分化细胞在回复无血清条件下能够再次增殖形成BTS,ATRA组再形成BTS的比例为4.84%±0.32%,形成时间为(10.07+1.03)d.对照组分别为17.71%±0.78%和(4.08±0.35)d,前者再形成BTS的比例较后者更低、时间更长,差异有统计学意义(P<0.05).结论 ATRA能促进BTSCs增殖并诱导BTSCs子代细胞走向分化,但分化不彻底,细胞不能达到终末分化,可再次形成BTS.