MSCs定向诱导移植修复关节软骨的实验研究

目的 用组织工程方法修复软骨损伤.方法 兔骨髓间充质干细胞体外扩增诱导分化后,移植于兔关节损伤区,术后进行大体及x线观察.结果 实验组移植8周后,软骨损伤区由透明软骨样组织填充,软骨修复良好.结论 自体间充质干细胞移植可修复关节软骨损伤.     

间充质干细胞治疗缺血性脑血管病的研究现状

缺血性脑血管病是一种缺血性疾病,正逐渐成为全世界发病和致死的主要原因之一。在动物模型和临床试验中,间充质干细胞(MSCs)已被用于缺血性脑血管病。大量研究表明,MSCs的治疗是安全的,能改善缺血性疾病患者的症状,但其疗效与多种因素有关,其中包括细胞移植途径、移植时间窗和剂量及MSCs的来源等。本文综述了这些影响因素及采用MSCs治疗缺血性脑血管病的作用机制和临床试验的安全性,为进一步的研究和临床治疗提供帮助。     

脊髓损伤患者骨髓间质干细胞形态学观察

目的骨髓间充质干细胞具有自我复制能力,可以经过不可逆的终末分化过程产生子代细胞,其潜在的多向分化潜力以及可作为细胞治疗这一特点,极有可能成为组织工程较为理想的种子细胞.本实验是对脊髓损伤患者骨髓间充质干细胞形态学观察.方法抽取脊髓损伤患者的骨髓,经密度梯度离心分离、纯化和培养,观察原代和传代细胞的细胞形态.结果原代培养的BM-MSCs最佳的贴壁时间为3 d,生长性状不一,呈散在圆形细胞群、克隆圆形细胞群、散在梭形细胞群、花带状细胞群、旋涡状细胞群,而传代培养的细胞,增殖速度较快,性状一致,排列规则,呈饱满的梭行.结论体外非诱导培养的BM-MSCs方法可能为临床治疗脊髓损伤患者提供种子细胞.     

结肠腺癌中结肠癌干细胞的分离、鉴定

目的从人结肠腺癌组织中分离、鉴定结肠癌干细胞，并初步观察其生物学特性。方法利用新鲜结肠腺癌组织，无血清悬浮成球培养，流式细胞检测ESA、CD44表达情况，体外观察其诱导分化及CK20、Muc表达情况，Balb／C小鼠移植观察其成瘤情况。结果从人原代结肠腺癌中分离、纯化EpCAM^high CD44^＋结肠癌干细胞，结肠癌原代细胞中EpCAM^highCD44^＋细胞比例为1．7％～38％（平均5．4％）。单克隆形成实验证实结肠癌组织中存在肿瘤干细胞。其比例为（2．07±0．11）％，分离获得的EpCAM^highCD44^＋细胞能在无血清培养基中“成球”，在血清诱导下能贴壁分化；将EpCAM^highCD44^＋细胞移植在Balb／C裸鼠体内，表现出很强的致瘤性，移植瘤中EpCAM^highCD44^＋细胞比例为3．6％～43．2％（平均15．2％），所有的移植瘤经组织学测定，均形成腺管样结构，表达结肠特异性分化标志物CK-20、中性上皮粘蛋白（neutral epithelial mucins，Muc）。结论人结肠腺癌组织中存在EpCAM^highCD44^＋细胞群，具有和普通干细胞相类似的无限增殖、自我更新和分化能力。     

人结肠癌CW-2干细胞获取方法研究

目的运用3种不同的方法分离纯化人结肠癌CW-2干细胞，并对其分离纯化效率进行比较，探讨获得癌干细胞的有效方法。方法采用单纯无血清悬浮培养、无血清悬浮培养联合化疗药物、流式细胞分选技术分别富集人结肠癌细胞株CW-2干细胞；然后运用流式细胞术、NOD—SCID小鼠致瘤实验和Transwell侵袭实验分析比较3种方法的富集效率。结果无血清悬浮培养细胞．无血清悬浮培养联合化疗药物处理细胞和流式细胞仪分选技术分选后细胞中具有结肠癌干细胞特性的CD44＋EPCAM_细胞分别为（59．39±4．55）％、（74.36±6．78）％、（86．43±8．43）％；3群细胞的成瘤能力和侵袭能力都存在显著统计学差异（P值〈0．05）：流式细胞分选技术分选后细胞〉无血清悬浮培养联合化疗药物处理细胞〉单纯无血清悬浮培养细胞。结论流式细胞分选技术富集癌干细胞的能力强于单纯无血清悬浮培养和无血清悬浮培养联合化疗药物，无血清悬浮培养联合化疗药物又强于单纯无血清悬浮培养。     

大鼠侧脑室下区神经干细胞具有不易兴奋性

目的建立体外培养大鼠侧脑室下区神经干细胞的方法，观察大鼠侧脑室下区神经干细胞的膜兴奋性。方法无血清培养方法体外分离、纯化孕15～16dwistar胎鼠的侧脑室下区神经干细胞，用免疫荧光鉴定干细胞标记蛋白nestin表达情况、用tuj-1和GFAP免疫染色研究体外NSC的分化情况；取第二代神经干细胞给予DiBACA（3）染色后，经高浓度氯化钾刺激，激光共聚焦显微镜动态扫描，观察侧脑室神经干细胞的兴奋性。结果采用无血清培养基体外分离的神经干细胞具有自我增殖、多向分化潜能等干细胞一般特点，且表达干细胞的标记蛋白nestin；采用DiBAC4（3）染色，高浓度钾刺激后，细胞荧光强度无显著变化，即细胞膜电位无明显改变，神经干细胞具有不易兴奋性。结论采用无血清培养方法成功分离扩增大鼠脑内神经干细胞；由大鼠侧脑室分离而来的神经干细胞具有不易兴奋性。     

神经干细胞对创伤性脑损伤的修复作用

目的建立小鼠创伤性脑损伤动物模型,进行神经干细胞移植,以评价神经干细胞对创伤性脑损伤的修复作用。方法体外培养神经干细胞并进行标记、鉴定,采用改良的Feeney氏自由落体法撞击构建创伤性脑损伤动物模型,脑外伤后1d进行神经干细胞移植,分别在移植当天(移植后0 d)、移植后3 d、7 d、14 d、21 d进行神经功能损伤程度(NSS)评分及疲劳转棒测试,移植后7 d、21 d进行抗Brdu、Nestin、GFAP、β-TubulinⅢ免疫荧光染色。结果移植后7 d、14 d、21 d神经干细胞移植组的NSS评分低于对照组,有统计学差异(P0.05);移植后14 d、21 d神经干细胞移植组的疲劳转棒时间长于对照组,有统计学差异(P0.05);移植后7 d免疫荧光染色发现BrdU/Nestin双阳性细胞,移植后21 d免疫荧光染色发现BrdU/GFAP双阳性细胞及BrdU/β-TubulinⅢ双阳性细胞。结论神经干细胞移植能够改善创伤性脑损伤后的神经功能,移植的神经干细胞能够在宿主脑内存活、迁移并在受损区域分化成星形胶质细胞和神经元。     

RNA干扰CD133基因对结肠癌干细胞生物学行为的影响

目的探讨CD133基因表达、活化被阻断后对结肠癌干细胞生物学行为的影响。方法从EpcAMhighCD44＋结肠癌干细胞中流式分选获得CD133＋细胞，感染LV-CD133shRNA载体慢病毒后观察CD133＋结肠癌干细胞在生长方式、成球能力、克隆形成率、成瘤能力以及ABCC2mRNA的变化；Westernblot分析CD133-细胞中CD133蛋白表达情况。结果EpcAMhighCD44＋结肠癌干细胞中CD133＋细胞比例为89．2％。实验组经过LV-CD133shRNA载体病毒感染后，在干细胞养液中细胞改悬浮生长的方式为贴壁生长，不能形成细胞球。MTT法测定发现细胞增殖减慢，克隆形成率明显下降。将感染细胞移植在Balb／C裸鼠体内，在观察期间，感染LV—CD133shRNA载体病毒的CD133＋细胞无肿瘤形成。ABCG2mRNA表达水平明显降低（P〈0．01）。从EpcAMhighCD44＋结肠癌干细胞中流式分选获得CD133-细胞，其中也有CD133蛋白的表达。结论CD133维持结肠癌干细胞生物学特性。     

组织工程骨膜修复兔大段骨缺损的可行性实验研究

目的探索组织工程骨膜体内成骨修复兔大段骨缺损的可行性。方法培养新西兰大白兔骨髓间充质干细胞(BMSCs),以成骨诱导剂诱导成骨分化后,与猪小肠粘膜下层(SIS)复合构建组织工程骨膜。扫描电镜(SEM)观察细胞与材料复合情况。选4月龄新西兰大白兔24只,制备单侧桡骨干4 cm缺损模型。随机选12只植入组织工程骨膜,作为实验组;另12只骨缺损旷置,作为对照组。术后6周后摄x线片观察,切取整段桡骨作为标本行HE及Masson染色观察。结果BMscs诱导14 d后可成骨分化。SEM显示构建的组织工程骨膜上黏附大量种子细胞。x线片观察:实验组骨缺损处有长柱状新生骨形成,并与截骨端骨性融合,密度与正常骨相近;对照组骨缺损处无成骨征象,密度同周围软组织影。组织学观察:实验组骨缺损处有新骨形成,新生骨组织中可见丰富的血管腔及不规则髓腔样结构;对照组骨缺损处仅为纤维结缔组织,无骨组织形成。结论以SIS和BMSCs构建的组织工程骨膜有修复兔大段骨缺损的可行性。组织工程骨膜有进一步深入研究、开发的价值和前景。     

无血清分离人结直肠癌细胞的体外培养形态及标志物表达特征

目的探讨人结直肠肿瘤干细胞在体外分化过程中细胞形态和干细胞相关标志物CD133的表达变化,为进一步研究结直肠肿瘤干细胞分化走向提供实验依据。方法取来源于人结直肠癌的细胞系HCT116,无血清培养分离出CD133+细胞,加血清诱导分化,相差显微镜下观察其形态变化;在未分化状态下无血清培养第7天和14天与血清诱导分化后收集细胞,利用流式细胞仪检测干细胞标志物CD133的表达量,采用激光共聚焦检测CD133表面标记分子的表达。结果 1)细胞形态:无血清培养分离的CD133+细胞,在生长过程中聚集成规则的细胞球,血清诱导后即贴壁生长,贴壁形态与同来源细胞形态一致,且再次无血清悬浮培养后聚集成球稳定生长。2)标志物变化:结直肠肿瘤干细胞未分化时CD133表面标记分子高表达,流式细胞仪检测未分化细胞CD133第7天表达率为(20.4±0.52)%,第14天表达率为(78.5±2.80)%,分化后表达率为(0.50±0.17)%。结论细胞形态和标志物表达改变均表明高表达CD133+的HCT116结直肠癌肿瘤干细胞可定向分化为同源的结直肠癌细胞,CD133+细胞经血清诱导后表达下调而使细胞失去干细胞特性。     

Response of vascular mesenchymal stem/progenitor cells to hyperlipidemia

Hyperlipidemia is a risk factor for atherosclerosis that is characterized by lipid accumulation, inflammatory cell infiltration, and smooth muscle cell proliferation. It is well known that hyperlipidemia is a stimulator for endothelial dysfunction and smooth muscle cell migration during vascular disease development. Recently, it was found that vessel wall contains a variable number of mesenchymal stem cells (MSCs) that are quiescent in physiological conditions, but can be activated by a variety of stimuli, e.g., increased lipid level or hyperlipidemia. Vascular MSCs displayed characteristics of stem cells which can differentiate into several types of cells, e.g., smooth muscle cells, adipocytic, chondrocytic, and osteocytic lineages. In vitro, lipid loading can induce MSC migration and chemokines secretion. After MSC migration into the intima, they play an essential role in inflammatory response and cell accumulation during the initiation and progression of atherosclerosis. In addition, MSC transplantation has been explored as a therapeutic approach to treat atherosclerosis in animal models. In this review, we aim to summarize current progress in characterizing the identity of vascular MSCs and to discuss the mechanisms involved in the response of vascular stem/progenitor cells to lipid loading, as well as to explore therapeutic strategies for vascular diseases and shed new light on regenerative medicine.     

Natural history of mesenchymal stem cells,from vessel walls to culture vessels

Mesenchymal stem/stromal cells (MSCs) can regenerate tissues by direct differentiation or indirectly by stimulating angiogenesis, limiting inflammation, and recruiting tissue-specific progenitor cells. MSCs emerge and multiply in long-term cultures of total cells from the bone marrow or multiple other organs. Such a derivation in vitro is simple and convenient, hence popular, but has long precluded understanding of the native identity, tissue distribution, frequency, and natural role of MSCs, which have been defined and validated exclusively in terms of surface marker expression and developmental potential in culture into bone, cartilage, and fat. Such simple, widely accepted criteria uniformly typify MSCs, even though some differences in potential exist, depending on tissue sources. Combined immunohistochemistry, flow cytometry, and cell culture have allowed tracking the artifactual cultured mesenchymal stem/stromal cells back to perivascular anatomical regions. Presently, both pericytes enveloping microvessels and adventitial cells surrounding larger arteries and veins have been described as possible MSC forerunners. While such a vascular association would explain why MSCs have been isolated from virtually all tissues tested, the origin of the MSCs grown from umbilical cord blood remains unknown. In fact, most aspects of the biology of perivascular MSCs are still obscure, from the emergence of these cells in the embryo to the molecular control of their activity in adult tissues. Such dark areas have not compromised intents to use these cells in clinical settings though, in which purified perivascular cells already exhibit decisive advantages over conventional MSCs, including purity, thorough characterization and, principally, total independence from in vitro culture. A growing body of experimental data is currently paving the way to the medical usage of autologous sorted perivascular cells for indications in which MSCs have been previously contemplated or actually used, such as bone regeneration and cardiovascular tissue repair.     

Insights into autotransplantation: the unexpected discovery of specific induction systems in bone marrow stromal cells

Many kinds of cells, including embryonic stem cells and tissue stem cells, have been considered candidates for transplantation therapy for neuro- and muscle-degenerative diseases. Bone marrow stromal cells (MSCs) also have great potential as therapeutic agents since they are easily isolated and can be expanded from patients without serious ethical or technical problems. Recently, new methods for the highly efficient and specific induction of functional neurons and skeletal muscle cells have been developed for MSCs. These induced cells were transplanted into animal models of stroke, Parkinson’s disease and muscle degeneration, resulting in the successful integration of transplanted cells and improvement in the behavior of the transplanted animals. Here I describe the discovery of these induction systems and focus on the potential use of MSC-derived cells for ‘auto-cell transplantation therapy’ in neuro- and muscle-degenerative diseases. Received 27 April 2006; received after revision 5 June 2006; accepted 22 August 2006     

Viability of rabbit bone marrow after cryopreservation (in vitro and in vivo)

Summary A technique for preservation of rabbit bone marrow is described, which preserves viability of stem cells in all 22 animals as tested by autologous bone marrow transplantation and in vitro growth. Erythroid precursors survived better than myeloid precursors as observed by in vitro and in vivo recovery.Supported by the Swiss Science Fondation 3.846.0.79.     

Vascular stem/progenitor cells: functions and signaling pathways

Vascular stem/progenitor cells (VSCs) are an important source of all types of vascular cells needed to build, maintain, repair, and remodel blood vessels. VSCs, therefore, play critical roles in the development, normal physiology, and pathophysiology of numerous diseases. There are four major types of VSCs, including endothelial progenitor cells (EPCs), smooth muscle progenitor cells (SMPCs), pericytes, and mesenchymal stem cells (MSCs). VSCs can be found in bone marrow, circulating blood, vessel walls, and other extravascular tissues. During the past two decades, considerable progress has been achieved in the understanding of the derivation, surface markers, and differentiation of VSCs. Yet, the mechanisms regulating their functions and maintenance under normal and pathological conditions, such as in eye diseases, remain to be further elucidated. Owing to the essential roles of blood vessels in human tissues and organs, understanding the functional properties and the underlying molecular basis of VSCs is of critical importance for both basic and translational research.     

The mesenchymoangioblast,mesodermal precursor for mesenchymal and endothelial cells

Mesenchymoangioblast (MB) is the earliest precursor for endothelial and mesenchymal cells originating from APLNR⁺PDGFRα⁺KDR⁺ mesoderm in human pluripotent stem cell cultures. MBs are identified based on their capacity to form FGF2-dependent compact spheroid colonies in a serum-free semisolid medium. MBs colonies are composed of PDGFRβ⁺CD271⁺EMCN⁺DLK1⁺CD73^? primitive mesenchymal cells which are generated through endothelial/angioblastic intermediates (cores) formed during first 3–4 days of clonogenic cultures. MB-derived primitive mesenchymal cells have potential to differentiate into mesenchymal stromal/stem cells (MSCs), pericytes, and smooth muscle cells. In this review, we summarize the specification and developmental potential of MBs, emphasize features that distinguish MBs from other mesenchymal progenitors described in the literature and discuss the value of these findings for identifying molecular pathways leading to MSC and vasculogenic cell specification, and developing cellular therapies using MB-derived progeny.     

Mesenchymal stem cells or cardiac progenitors for cardiac repair? A comparative study

In the past, clinical trials transplanting bone marrow–derived mononuclear cells reported a limited improvement in cardiac function. Therefore, the search for stem cells leading to more successful stem cell therapies continues. Good candidates are the so-called cardiac stem cells (CSCs). To date, there is no clear evidence to show if these cells are intrinsic stem cells from the heart or mobilized cells from bone marrow. In this study we performed a comparative study between human mesenchymal stem cells (hMSCs), purified c-kit⁺ CSCs, and cardiosphere-derived cells (CDCs). Our results showed that hMSCs can be discriminated from CSCs by their differentiation capacity towards adipocytes and osteocytes and the expression of CD140b. On the other hand, cardiac progenitors display a greater cardiomyogenic differentiation capacity. Despite a different isolation protocol, no distinction could be made between c-kit⁺ CSCs and CDCs, indicating that they probably derive from the same precursor or even are the same cells.     

Characterization of murine non-adherent bone marrow cells leading to recovery of endogenous hematopoiesis

Non-adherent bone marrow-derived cells (NA-BMCs) are a mixed cell population that can give rise to multiple mesenchymal phenotypes and that facilitates hematopoietic recovery. We characterized NA-BMCs by flow cytometry, fibroblast colony-forming units (CFU-f), real-time PCR, and in in vivo experiments. In comparison to adherent cells, NA-BMCs expressed high levels of CD11b⁺ and CD90⁺ within the CD45⁺ cell fraction. CFU-f were significantly declining over the cultivation period, but NA-BMCs were still able to form CFU-f after 5 days. Gene expression analysis of allogeneic NA-BMCs compared to bone marrow (BM) indicates that NA-BMCs contain stromal, mesenchymal, endothelial cells and monocytes, but less osteoid, lymphoid, and erythroid cells, and hematopoietic stem cells. Histopathological data and analysis of weight showed an excellent recovery and organ repair of lethally irradiated mice after NA-BMC transplantation with a normal composition of the BM.