人脐带间充质干细胞分离培养方法的研究

目的探讨人脐带间充质干细胞(MSCs)体外分离培养的最佳方法。方法无菌条件下采集早产儿(不足37周)和足月儿的脐带,分离MSCs,比较胎龄、脐带新鲜程度、分离方法和不同培养基对脐带MSCs原代培养过程的影响,通过免疫荧光法检测脐带MSCs表面标记物的表达情况,观察脐带MSCs的生物学特性。结果足月分娩,新鲜脐带,采用组织块平铺法和MesencultTM培养基,脐带MSCs原代培养成功率较高。相同条件下,早产儿脐带MSCs原代培养成功率低于足月分娩脐带。人脐带MSCs高表达CD44、CD90和CD29。结论筛选出一种人脐带MSCs体外分离培养的最佳方法。     

脐带血来源间充质干细胞体外分离培养条件的优化

脐带血间充质干细胞（umbilical cord blood mesenchymal stem cells，UCB-MSCs）不仅可以作为滋养层细胞支持造血干细胞在体外的大规模扩增，在造血移植过程中还能够降低并发症的发生率以及加速造血重建功能的恢复．但是，目前UCB-MSCs的原代分离培养成功率一般只有30％左右，为进一步提高该成功率，利用正交实验方法对UCB-MSCs体外培养的主要影响因素：细胞的接种密度、细胞因子的组合及用量、是否添加血清和滋养层细胞，进行逐层筛选，并对培养出的间充质干细胞进行了流式细胞分析和向成骨、软骨及脂肪方向的诱导分化检测，以期获得UCB-MSCs培养的最佳方法．实验结果表明，细胞的接种密度是UCB-MSCs培养最显著的影响因素（P〈0．1），接种密度越大，MSCs越容易生长，能够培养出MSCs的几率就越大，其次为细胞因子，添加细胞因子能有效地刺激MSCs的生长．在高接种密度的基础上，添加细胞因子IL-3（15μg／L）和GM-CSF（5μg/L），可大大提高UCB-MSCs体外原代培养的成功率，从30％左右提高到90％以上．流式细胞检测结果显示，所分离培养的细胞表达间充质干细胞的抗原（CD13^＋、CD29^＋、CD44^＋、CD105^＋、CD166^＋），不表达造血细胞的抗原（CD34^-、CD45^-、HLA-DR^-），并能够向成骨、软骨及脂肪方向分化，这与源于骨髓的间充质干细胞相一致．所建立的培养方法能够为UCB-MSCs的临床应用提供大量优质的种子细胞．     

影响骨髓间充质干细胞分离效率的若干因素的研究

骨髓间充质干细胞来源有限,数量稀少,极大地限制了其在组织修复、细胞治疗等方面的应用。为了解决细胞数量限制的问题,从提高分离效率的角度对骨髓间充质干细胞的分离过程进行了研究,考察了培养基种类、初次换液时间、起始接种密度对兔骨髓间充质干细胞分离效率的影响。结果表明,α-MEM培养基较DMEM、DMEM-LG培养基更适于兔MSCs的分离;从骨髓中分离得到的单个核细胞以1×106cells／ml的密度接种,接种24h后换液在短时间内得到的MSCs数目最多。采用以上分离条件,能够获得形态均一、可稳定传代10代以上仍保持旺盛增殖能力的MSCs,并具有向骨、脂肪分化能力。     

人胎儿造血组织来源间充质干细胞基本生物学特性的比较

目的:分离、纯化人胎儿造血组织来源的胎儿骨髓间充质干细胞(MSCs derived from human fetal bone marrow,hfBM-MSCs)和胎儿肝脏间充质干细胞(MSCs derived from human fetal liver,hfL-MSCs),比较其基本生物学特性.方法:取材12-20周流产胎儿,冲洗骨髓腔得到全骨髓细胞悬液;通过二步离心联合羟乙基淀粉沉淀法富集肝非实质细胞,接种、培养、纯化MSCs.绘制MSCs生长曲线.检测MSCs表面标志、细胞周期.诱导MSCs向脂肪细胞分化并鉴定.结果:hfBM-MSCs与hfL-MSCs均为平行排列的纤维状细胞,但前者较细长,后者稍宽大;hfBM-MSCs与hfL-MSCs的原代培养时间、原代培养细胞数有显著性差异:而第二代细胞数无显著性差异;二者生长曲线相似,但hfBM-MSCs的增殖速度较快;细胞周期分析显示G0-G1期细胞在hfBM-MSCs与hfL-MSCs分别占96.1%和91.2%.流式检测结果显示HLA-DR、CD40和CD80在hfBM-MSCs与hfL-MSCs均极低表达,而后者更低;hfBM-MSCs与hfL-MSCs均可成脂分化,但前者更容易向脂肪样细胞分化.结论:在相同时间内,从胎儿骨髓中可以获得更多的MSCs;hfBM-MSCs具有较大的增殖潜能、增殖速度以及分化能力,而hfL-MSCs的免疫原性更弱.     

枸杞多糖诱导人脐血间充质干细胞向神经元样细胞分化的实验研究

目的:探讨枸杞多糖诱导人脐血间充质干细胞(MSCs)向神经元样细胞分化的可行性及其机制。方法:无菌条件下收集正常足月儿的脐带血,经肝素抗凝,用相对密度1.077的淋巴细胞分离液分离脐血的单个核细胞,用低糖DMEM培养基进行培养和纯化扩增。选取第3代细胞进行诱导实验,当传代细胞长满瓶底的80%以上时,先用含15?S和10ng/ml bFGF的DMEM完全培养基预诱导24小时,然后用不含血清含1g/L枸杞多糖的DMEM培养基诱导,光镜下观察细胞形态,用免疫组化技术检测细胞Nestin和NF的表达。结果:预诱导后MSCs没有变化,而经枸杞多糖诱导4h后细胞即出现形态学上的改变,细胞变成不规则形,立体感增强,从胞体伸出突起。免疫组化检测显示,细胞Nestin、NF呈阳性。结论:人脐血间充质干细胞经枸杞多糖诱导可转化为神经元样细胞,其诱导机制可能与枸杞多糖的抗氧化作用有关。     

人脐血间充质干细胞诱导分化为神经细胞的研究

目的探讨体外诱导人脐血间充质干细胞(MSCs)向神经细胞分化的条件,为治疗中枢神经系统损伤提供实用的干细胞来源。方法体外分离、纯化、扩增脐血MSCs,流式细胞仪检测细胞表面标志。采用脑源性神经营养因子BDNF 10ng/ml 维甲酸RA0.5μM 碱性成纤维生长因子bFGF 20ng/ml协同诱导脐血MSCs定向分化。免疫荧光染色检测诱导后细胞的星形胶质细胞特异标志GFAP及神经元特异标志MAP2的表达情况。建立大鼠脊髓横断损伤模型,将BrdU标记的诱导后的细胞移植入损伤的脊髓中,采用BBB运动功能评分标准在术后24h及1、2、3、4、5周各时间点对大鼠进行运动功能评分。用组织学和免疫组化方法检测移植到大鼠脊髓中的BrdU阳性细胞的存活、迁移、分化情况。结果脐血MSCs体外培养三代后,细胞表面CD11b、CD34、CD45和CD44表达阴性。诱导分化7d后,大部分细胞的形态类似神经元,免疫荧光染色检测MAP2阳性细胞占大多数,明显多于GFAP阳性细胞。5周后,细胞移植组大鼠的后肢运动功能恢复情况较对照组好。免疫组织化学结果显示植入的细胞可长时间在宿主脊髓中存活,并向损伤处两端迁移。结论人脐血MSCs于体外在特定的条件下可以诱导分化为神经元样细胞。移植脐血MSCs诱导后的神经细胞可在损伤的脊髓中存活、迁移,并能促进脊髓损伤后行为和功能恢复。     

大鼠骨髓间充质干细胞分离与培养条件的优化研究

目的:建立和优化大鼠骨髓间充质干细胞(mesenchymal stem cells,MSCs)的分离培养条件,以获得群体均一、未分化状态保持良好的MSCs.方法:收集不同周龄大鼠骨髓细胞;以不同浓度Percoll密度梯度离心分离骨髓单个核细胞;以60%低糖DMEM40%MCDB201为基础培养基,培养24h去悬浮细胞;以不同接种密度传代培养;碱性磷酸酶染色和油红O染色考察MSCs向骨和脂肪组织分化的潜能.结果:采用57%Percoll液的分离效果优于70%Percoll液.6周龄(体重约180g)大鼠能在细胞分离的质和量上达到最佳效果.24h进行悬浮细胞去除、5×103/cm2接种密度传代培养,光镜和电镜显示MSCs增殖能力强,功能状态活跃,成脂成骨实验显示多向分化潜能保持良好.结论:优化大鼠周龄、分离液的密度、细胞培养条件及改进培养方法有助于获得多向分化潜能保持良好的均一的MSCs.     

脐血间充质干细胞诱导向神经元样细胞分化的实验研究

目的:研究脐血间充质干细胞生物学特性及向神经元样细胞分化的潜能。方法:采用密度梯度离心结合贴壁培养法自脐血中分离间充质干细胞,观察细胞生长情况,描绘生长曲线,流式细胞仪检测细胞表面标志物,分别向成骨细胞、脂肪细胞、神经元样细胞进行诱导分化,通过茜素红染色、油红O染色检测脐血间充质干细胞成骨、成脂肪细胞诱导分化能力,而以免疫组织化学检测诱导后细胞表面神经标志物的表达。结果:纯化的脐血间充质干细胞贴壁生长,呈均一梭形,生长曲线呈S型,并以P3代增殖能力最强,细胞表面不表达或弱表达CD34、CD35、CD106,高表达CD29、CD44、CD105。成骨诱导2周后,可检测到钙化基质的形成,成脂肪诱导3周后,可检测到脂滴的形成。向神经元样细胞诱导分化后,可观察到典型的神经元样形态改变,且NSE、NF、GFAP阳性表达。结论:分离纯化的脐血间充质干细胞具有较强的增殖能力与分化潜能,并在体外诱导条件下可以向神经元样细胞定向分化。     

人脐带间充质干细胞的分离培养与鉴定

目的:建立并优化人脐带间充质干细胞分离纯化方法,并对其表面标志与多向分化潜能进行鉴定。方法:收集健康足月产胎儿脐带组织,采用组织块贴壁法进行原代培养,流式细胞仪对其表面标志进行检测,通过向成骨成脂分化对其多向分化潜能进行鉴定,RT-PCR对其干细胞特性基因Oct4、Nanog、Sox2、Nestin进行检测。结果:采用组织块贴壁法可在2周左右获得大量间充质干细胞,培养的细胞经流式细胞仪检测,高表达CD29、CD44、CD105、CD106,低表达CD34、CD45;经成骨成脂诱导2周后可分化为成骨细胞和成脂细胞,RT-PCR检测发现原代细胞表达Oct4、Nanog、Sox2、Nestin基因。结论:人脐带间充质干细胞可在体外扩增培养,具有多向分化潜能,可作为组织工程种子细胞来源。     

脐血间充质干细胞诱导向神经元样细胞分化的实验研究

目的：研究脐血间充质干细胞生物学特性及向神经元样细胞分化的潜能。方法：采用密度梯度离心结合贴壁培养法自脐血中分离间充质干细胞,观察细胞生长情况,描绘生长曲线,流式细胞仪检测细胞表面标志物,分别向成骨细胞、脂肪细胞、神经元样细胞进行诱导分化,通过茜素红染色、油红O染色检测脐血间充质干细胞成骨、成脂肪细胞诱导分化能力,而以免疫组织化学检测诱导后细胞表面神经标志物的表达。结果：纯化的脐血间充质干细胞贴壁生长,呈均一梭形,生长曲线呈S型,并以P3代增殖能力最强,细胞表面不表达或弱表达CD34、CD35、CD106,高表达CD29、CD44、CD105。成骨诱导2周后,可检测到钙化基质的形成,成脂肪诱导3周后,可检测到脂滴的形成。向神经元样细胞诱导分化后,可观察到典型的神经元样形态改变,且NSE、NF、GFAP阳性表达。结论：分离纯化的脐血间充质干细胞具有较强的增殖能力与分化潜能,并在体外诱导条件下可以向神经元样细胞定向分化。     

In vitro culture of Keratinocytes from human umbilical cord blood mesenchymal stem cells: the Saigonese culture

There have been many attempts to acquire and culture human keratinocytes for clinical purposes including from keratotome slices in media with fetal calf serum (FCS) or pituitary extract (PE), from skin specimens in media with feeder layers, from suction blister epidermal roofs’ in serum-free culture and from human umbilical cord blood (hUCB) mesenchymal stem cells (MSCs) in media with skin feeder layers. Conversely this study was designed to investigate whether keratinocytes could be obtained directly from hUCB MSCs in vitro. It is widely established that mesenchymal stem cells from human umbilical cord blood have multipotent capacity and the ability to differentiate into disparate cell lineages hUCB MSCs were directly induced to differentiate into keratinocytes by using a specific medium composed of primary culture medium (PCM) and serum free medium (SFM) in a ratio 1:9 for a period of 7 days and tested by immunostain p63 and K1-K10. Cells thus cultured were positive in both tests, confirming the possibility to directly obtain keratinocytes from MSCs hUCB in vitro.     

Different culture method changing CD105 expression in amniotic fluid MSCs without affecting differentiation ability or immune function

MSCs are kind of cultured cells that reside in different tissues as inducers or regulators of physiological and pathological processes. Here, we derived MSCs from amniotic fluid and compared their differentiation ability and immunosuppression effect on PHA-activated PBMC with those of MSCs isolated from umbilical cords. Amniotic fluid MSCs were isolated and cultured on commercial AFC medium and classic MSC medium, and the number and size of colonies were used to evaluate differences in primary and passaged culture. Rate of proliferation, population doubling time, cell morphology, cell surface markers and mRNA expression were measured in subcultured cells. Furthermore, a comparative study was performed with umbilical cord MSCs to assess the ability of differentiation and immunosuppressive effect of PHA-stimulated PBMCs. Amniotic fluid MSCs were isolated and expanded by three methods, and exhibited nearly all the characteristics of umbilical cord MSCs. Compared with umbilical cord MSCs, amniotic fluid MSCs had an enhanced osteogenic and chrondrogenic differentiation capability, and stronger immunosuppression effect of inhibition of PHA-activated PBMC division. Culture with commercial AFCs medium yielded the highest percentage of CD105 expression and showed some advantages in primary cell isolation, cell source-specific marker retention and cell proliferation. We demonstrated that amniotic fluid MSCs exhibited some advantages over umbilical cord MSCs, and different culture media caused cell proliferation, cell surface marker and cell morphology change, but were not associated with varying differentiation capability and immune effects.     

Isolation of three important types of stem cells from the same samples of banked umbilical cord blood

It is known that umbilical cord blood (UCB) is a rich source of stem cells with practical and ethical advantages. Three important types of stem cells which can be harvested from umbilical cord blood and used in disease treatment are hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs). Since these stem cells have shown enormous potential in regenerative medicine, numerous umbilical cord blood banks have been established. In this study, we examined the ability of banked UCB collected to produce three types of stem cells from the same samples with characteristics of HSCs, MSCs and EPCs. We were able to obtain homogeneous plastic rapidly-adherent cells (with characteristics of MSCs), slowly-adherent (with characteristics of EPCs) and non-adherent cells (with characteristics of HSCs) from the mononuclear cell fractions of cryopreserved UCB. Using a protocol of 48?h supernatant transferring, we successfully isolated MSCs which expressed CD13, CD44 and CD90 while CD34, CD45 and CD133 negative, had typical fibroblast-like shape, and was able to differentiate into adipocytes; EPCs which were CD34, and CD90 positive, CD13, CD44, CD45 and CD133 negative, adherent with cobble-like shape; HSCs which formed colonies when cultured in MethoCult medium.     

Hypoxic Preconditioning Increases Survival and Pro-Angiogenic Capacity of Human Cord Blood Mesenchymal Stromal Cells In Vitro

Hypoxic preconditioning was shown to improve the therapeutic efficacy of bone marrow-derived multipotent mesenchymal stromal cells (MSCs) upon transplantation in ischemic tissue. Given the interest in clinical applications of umbilical cord blood-derived MSCs, we developed a specific hypoxic preconditioning protocol and investigated its anti-apoptotic and pro-angiogenic effects on cord blood MSCs undergoing simulated ischemia in vitro by subjecting them to hypoxia and nutrient deprivation with or without preceding hypoxic preconditioning. Cell number, metabolic activity, surface marker expression, chromosomal stability, apoptosis (caspases-3/7 activity) and necrosis were determined, and phosphorylation, mRNA expression and protein secretion of selected apoptosis and angiogenesis-regulating factors were quantified. Then, human umbilical vein endothelial cells (HUVEC) were subjected to simulated ischemia in co-culture with hypoxically preconditioned or naïve cord blood MSCs, and HUVEC proliferation was measured. Migration, proliferation and nitric oxide production of HUVECs were determined in presence of cord blood MSC-conditioned medium. Cord blood MSCs proved least sensitive to simulated ischemia when they were preconditioned for 24 h, while their basic behavior, immunophenotype and karyotype in culture remained unchanged. Here, “post-ischemic” cell number and metabolic activity were enhanced and caspase-3/7 activity and lactate dehydrogenase release were reduced as compared to non-preconditioned cells. Phosphorylation of AKT and BAD, mRNA expression of BCL-XL, BAG1 and VEGF, and VEGF protein secretion were higher in preconditioned cells. Hypoxically preconditioned cord blood MSCs enhanced HUVEC proliferation and migration, while nitric oxide production remained unchanged. We conclude that hypoxic preconditioning protects cord blood MSCs by activation of anti-apoptotic signaling mechanisms and enhances their angiogenic potential. Hence, hypoxic preconditioning might be a translationally relevant strategy to increase the tolerance of cord blood MSCs to ischemia and improve their therapeutic efficacy in clinical applications.     

间充质干细胞促进脐带血干细胞体外扩增的研究进展

非亲缘脐带血移植是治疗造血系统疾病的重要移植方式之一,但脐带血移植面临的最大挑战是造血干细胞(HSCs)数量不足,特别是成人患者受到脐带血干细胞数量的限制,导致造血及免疫恢复延迟,非复发死亡率升高。体外扩增脐带血HSCs(UCB-HSCs)是解决该问题的途径之一。研究发现可以通过模拟骨髓造血龛(niche)这一生态位使HSCs在体外进行自我更新增殖,而间充质干细胞(MSCs)正是造血龛的重要的组成细胞之一。本文将探讨MSCs在UCB-HSCs体外扩增中的应用。重点以MSCs促造血的特点、机制,促进脐带血干细胞增殖的各种策略以及其临床应用和前景做一综述。     

Evaluation of chemokine and cytokine profiles in osteoblast progenitors from umbilical cord blood stem cells by BIO-PLEX technology

We have used cytokine protein array to analyze the secretion of cytokines from an osteoblastic clone derived from human umbilical cord blood mesenchymal stem cells (MSCs) cultured in an osteogenic differentiation medium. The analysis demonstrated the unexpected ability of osteoblast committed cells and their early progenitors to produce significant amounts of a range of soluble immune mediators without in vitro exposure to clinically relevant bacterial pathogens. The cells were expanded and their osteogenic potential analyzed over 45 days of culture was revealed by the expression of osteoblast-specific markers (alkaline phosphatase and Runx2), and by matrix mineralization. Over this culture period, the cells secreted particularly high levels of IL-8, MCP-1 and VEGF, but did not express IL-2, IL-7, IL-17, eotaxin, G-CSF and IFN-gamma. These findings should encourage the use of human umbilical cord blood as a potential stem cells source for bone regeneration.     

Isolation of mesenchymal stem cells from equine umbilical cord blood

Background  

There are no published studies on stem cells from equine cord blood although commercial storage of equine cord blood for future autologous stem cell transplantations is available. Mesenchymal stem cells (MSC) have been isolated from fresh umbilical cord blood of humans collected non-invasively at the time of birth and from sheep cord blood collected invasively by a surgical intrauterine approach. Mesenchymal stem cells isolation percentage from frozen-thawed human cord blood is low and the future isolation percentage of MSCs from cryopreserved equine cord blood is therefore expectedly low. The hypothesis of this study was that equine MSCs could be isolated from fresh whole equine cord blood.     

脐静脉和骨髓来源的间充质干细胞的比较研究

间充质干细胞(MSCs)的来源有限,成人骨髓是MSCs的主要来源,这极大地限制了其在实验和临床中的应用。为拓宽MSCs来源,从细胞形态、生长特性、免疫表型和多向分化能力等四个方面对人脐静脉来源和成人骨髓来源的间充质干细胞进行了比较研究。结果表明,人脐静脉来源和成人骨髓来源的 MSCs具有相似的生物学特征,成纤维细胞样形态生长,并具有强大的体外扩增和多向分化能力。人脐静脉来源的MSCs可替代成人骨髓MSCs,作为满足实验和临床需要的重要来源。