成体小鼠骨髓间充质干细胞的培养和纯化

目的建立一种简单易行的小鼠骨髓间充质干细胞(MSC)的分离培养方法,并对其表面标志进行鉴定,为其应用提供实验依据.方法采用差速贴壁法体外分离、扩增MSC,倒置显微镜观察其生长过程,HE染色观察原代培养细胞的生长特性,免疫细胞化学检测MSC表面抗体的表达及细胞的大概纯度.结果用此法进行小鼠骨髓间充质干细胞的培养过程中,血液系细胞在换液过程中被去除,成纤维细胞污染可经差速贴壁法去除.经鉴定获得的骨髓间充质细胞能达到理想的纯度,生长状态良好.结论采用差速贴壁培养法可获得一定纯度的MSC,这种方法简单、便捷、实用,并且用这种方法所分离获得的细胞生长状态好,在体外条件下能大量增殖,原代及传代生长都可形成均一的细胞集落.     

成体小鼠骨髓间充质干细胞的培养和纯化

目的 建立一种简单易行的小鼠骨髓间充质干细胞（MSC）的分离培养方法，并对其表面标志进行鉴定，为其应用提供实验依据。方法 采用差速贴壁法体外分离、扩增MSC，倒置显微镜观察其生长过程，HE染色观察原代培养细胞的生长特性，免疫细胞化学检测MSC表面抗体的表达及细胞的大概纯度。结果 用此法进行小鼠骨髓间充质干细胞的培养过程中，血液系细胞在换液过程中被去除，成纤维细胞污染可经差速贴壁法去除，经鉴定获得的骨髓间充质细胞能达到理想的纯度，生长状态良好。结论 采用差速贴壁培养法可获得一定纯度的MSC。这种方法简单、便捷、实用，并且用这种方法所分离获得的细胞生长状态好，在体外条件下能大量增殖，原代及传代生长都可形成均一的细胞集落。     

小鼠骨髓间充质干细胞的分离培养及形态学观察

目的建立一种简单实用的小鼠骨髓间充质干细胞(MSCs)的分离培养方法,通过了解其细胞生物学特性,为MSCs的应用提供实验依据。方法采用差速贴壁法体外分离、扩增MSCs,倒置显微镜观察原代及传代细胞的形态及生长过程。结果在小鼠骨髓间充质干细胞的培养过程中,血液系细胞在换液过程被去除,成纤维细胞污染经差速贴壁法也可去除。获得的骨髓间充质细胞形态较均一,生长状态良好。结论采用差速贴壁培养法可获得一定纯度的MSCs,此法简单、实用,并且获得的细胞生长状态良好,增殖能力强.     

建立大鼠胚胎神经干细胞克隆的实验研究

为了获得神经干细胞克隆,作者从胚胎大鼠脑前区取出神经组织,经酶消化,机械吹打,有限稀释法培养具有单细胞克隆能力的细胞群,再利用单细胞克隆技术连续传代培养获取亚克隆,采用免疫细胞化学技术检测并鉴定神经干细胞和分化后成熟神经细胞特异抗原的表达.发现从胎脑分离的细胞群具有形成连续克隆能力,并表达特异的神经干细胞蛋白(Nestin);诱导分化后的细胞表达神经元和星形神经胶质细胞的特异抗原(Tubulin和GFAP).因而认为分离细胞克隆具有自我更新和多分化潜能,表达神经干细胞蛋白,有较强的体外增殖和分化成成熟神经细胞的能力,是中枢神经系统的干细胞.     

全骨髓贴壁并差速传代分离纯化大鼠骨髓间充质干细胞:与密度梯度离心法的比较

背景:目前国内外分离纯化骨髓间充质干细胞有两种主要方法:密度梯度离心法及全骨髓贴壁法,前者步骤较复杂,后者简单易操作,但纯化效果不理想。目的:在全骨髓贴壁分离骨髓间充质干细胞基础上,并用差速传代消化法,建立大鼠骨髓间充质干细胞体外分离培养纯化方法。方法:全骨髓贴壁培养法分离并差速消化传代大鼠骨髓间充质干细胞,利用间充质干细胞在消化传代过程中较其他骨髓细胞消化悬浮速度快,以及贴壁快的特点,代替密度梯度离心操作来分离纯化间充质干细胞,对其形态学特征进行观察,并与密度梯度离心法比较两种分离培养法的细胞生长增殖情况;观察碱性磷酸酶及油红染色情况,验证骨髓间充质干细胞的分化能力;检测细胞表面标记物,验证免疫特性及检测其纯度。结果与结论:全骨髓贴壁培养法分离并差速传代大鼠骨髓间充质干细胞,流式细胞鉴定、成骨成脂肪培养结果显示其细胞免疫特性、纯度、分化能力与密度梯度离心法无显著差异,但细胞活力,增殖能力有明显提高。     

提高成年大鼠神经干细胞单克隆形成率的方法

目的　探索提高神经干细胞单克隆形成率的方法并证实所分离培养的神经干细胞仍具有多分化潜能。　方法　对神经干细胞单克隆方法进行改良 ,即在单克隆培养液中加入 1 2原代克隆培养液。将所得到的单细胞克隆球消化、分离、增殖成大量的神经干细胞球 ,用含血清的DMEM分化培养液促其分化。 14d后 ,分别用神经元和胶质细胞的特异性标记物MAP 2标记神经元、GFAP标记星形胶质细胞和CNP标记少突胶质细胞。　结果平均每块含 1 2原代克隆培养液的 96孔培养板中有 2～ 3只孔可形成克隆球 ,而含纯新鲜培养液的培养板中仅 0 5～ 1 0只孔可形成克隆球。这些单细胞克隆球增殖后得到的大量亚细胞系克隆球分化后分别呈MAP 2、GFAP和CNP免疫荧光阳性。　结论　单细胞克隆实验中加入 1 2原代克隆培养液可提高神经干细胞的单克隆形成率 ,单克隆球增殖后得到的大量亚细胞系克隆球亦具有多分化潜能。     

新生大鼠心祖细胞原代分离培养及鉴定

目的原代分离培养及鉴定新生大鼠心祖细胞(CPCs)。方法利用差速贴壁原代分离培养CPCs,形态观察和二乙酸羧基荧光素-琥珀酰亚胺酯(CFDA-SE)示踪法检测细胞增殖。使用Fluo-3/AM钙离子检测法,检测集落增殖细胞中钙离子浓度变化。免疫荧光技术检测集落增殖细胞的干细胞标记物及心肌细胞标记物的表达。结果差速贴壁法获得细胞培养1周左右即观察到集落增殖的细胞,圆/椭圆形或不规则多边形细胞呈铺路石样排列。随培养时间延长,细胞集落增大。培养3周左右,细胞集落中细胞形态发生改变(分化),逐渐出现突起或呈梭形等,集落中出现波动/跳动的心肌细胞。用CFDA SE示踪显示,细胞呈集落增殖特点;Fluo-3/AM钙离子检测显示,集落增殖细胞中分化细胞的钙离子浓度增高。免疫荧光检测显示,不同的集落增殖细胞具异质性,存在c-kit~+/CD34~-、Nanog~+/CD34~-和c-kit~-/Nanog~-细胞集落;增殖的细胞集落存在c-kit和Gata4阳性共表达,随细胞分化出现心肌肌钙蛋白T(c Tn T)细胞集落。结论差速贴壁法原代分离培养的心祖细胞具有集落增殖和分化为心肌细胞的能力,是研究心祖细胞表面标记物、增殖和调控机制的理想材料。     

乳鼠肌源性干细胞向许旺样细胞的诱导分化

背景：有证据显示,肌源性干细胞能够促进肌纤维的再生,增强再生肌组织的功能,并具有分化为神经外膜组织细胞和具有许旺细胞表型细胞的潜能。 目的：分析许旺细胞条件培液诱导小鼠乳鼠源肌源性干细胞向许旺细胞的分化,探讨其作为神经组织工程的种子细胞的可行性。 方法：取C57BL/6乳鼠四肢骨骼肌,采用改良的Preplate技术纯化培养肌源性干细胞,利用抗体Desmin和Sca-1对纯化后的细胞进行免疫细胞化学鉴定。采用许旺细胞条件培液诱导其向许旺细胞分化,并行许旺细胞特异性抗体S100β、P75NTR免疫细胞化学鉴定。 结果与结论：从小鼠骨骼肌中分离得到肌源性干细胞,能在体外稳定增殖传代,Desmin和Sca-1免疫细胞化学染色阳性。肌源性干细胞经体外诱导分化后部分细胞S100β、P75NTR染色阳性。说明应用改良的Preplate方法,可从新生小鼠骨骼肌中分离得到肌源性干细胞;采用许旺细胞条件培液能将其诱导分化为许旺样细胞,有可能成为神经组织工程神经较理想的许旺细胞替代物。     

大鼠肌源性干细胞的分离、纯化和培养

背景:要获得满足临床需要的肌源性干细胞,体外筛选和扩增已成为关键环节。目的:拟建立稳定高效的成年大鼠肌源性干细胞的分离培养、纯化方法。方法:成年SD大鼠麻醉后,无菌条件下取骨骼肌,采用XI型胶原酶、Dispase和胰酶消化法获得肌源性干细胞,以密度梯度离心法和差速贴壁法进行纯化。记录细胞生长曲线,MTT比色法观察不同接种密度对细胞生长的影响,采用免疫细胞化学染色对细胞进行鉴定。结果与结论:原代肌源性干细胞体积较小,贴壁缓慢,折光性较好,多呈球形、梭形或纺锤形,增殖缓慢。传代培养后,加入含体积分数为20%血清浓度的完全培养基,以1×109L-1密度接种时活细胞数量最多,为适宜接种密度,传1~4代细胞生长状态良好。免疫细胞化学染色结果为desmin(+),CD34(+),CD45(-),Sca-1(+),证实通过体外原代培养获得了高纯度的肌源性干细胞,并成功扩增。     

成年大鼠骨骼肌干细胞的制备与新型培养方法

 目的：建立高效分离和扩增成年大鼠骨骼肌干细胞的实验方法。方法：通过混合酶消化法从少量成年大鼠骨骼肌样品中分离出骨骼肌干细胞;采用悬浮培养法培养获得骨骼肌干细胞团,批量扩增骨骼肌干细胞;用qRT-PCR和免疫细胞化学染色检测干细胞标志物Nanog、Oct4和骨骼肌干细胞标志物肌源性因子5（myogenic factor 5,Myf5）、配对盒蛋白7 (paired box protein 7,Pax7)的表达;成脂或成骨分化诱导培养基诱导检测骨骼肌干细胞的多功能性。结果：采用混合酶消化法可获得骨骼肌干细胞单细胞,在悬浮的培养条件下能快速克隆成骨骼肌细胞团,贴壁培养后能看到骨骼肌干细胞从细胞团中爬出来,和传统的差速贴壁方法相比悬浮培养法获得的骨骼肌干细胞纯度更高,细胞状态更好,干性因子Nanog、Oct 4和骨骼肌干细胞标志物Myf5、Pax 7的表达更高（P<0.05）;在不同培养基的诱导下能向骨骼肌、成骨和脂肪细胞系分化。结论：悬浮培养法能获得大批量干性好、纯度高、具有多向分化潜能的骨骼肌干细胞,从而为肌肉相关疾病的细胞治疗提供优良的种子细胞。     

Human muscle-derived cell populations isolated by differential adhesion rates: phenotype and contribution to skeletal muscle regeneration in Mdx/SCID mice

Muscle-derived stem cells (MDSCs) isolated from murine skeletal tissue by the preplate method have displayed the capability to commit to the myogenic lineage and regenerate more efficiently than myoblasts in skeletal and cardiac muscle in murine Duchenne Muscular Dystrophy mice (mdx). However, until now, these studies have not been translated to human muscle cells. Here, we describe the isolation, by a preplate technique, of candidate human MDSCs, which exhibit myogenic and regenerative characteristics similar to their murine counterparts. Using the preplate isolation method, we compared cells that adhere faster to the flasks, preplate 2 (PP2), and cells that adhere slower, preplate 6 (PP6). The human PP6 cells express several markers of mesenchymal stem cells and are distinct from human PP2 (a myoblast-like population) based on their expression of CD146 and myogenic markers desmin and CD56. After transplantation to the gastrocnemius muscle of mdx/SCID mice, we observe significantly higher levels of PP6 cells participating in muscle regeneration as compared with the transplantation of PP2 cells. This study supports some previous findings related to mouse preplate cells, and also identifies some differences between mouse and human muscle preplate cells.     

诱导人眼轮匝肌来源肌源干细胞向许旺细胞样细胞的分化

背景：肌源干细胞的优越性引导学者们尝试从人眼轮匝肌中分离该细胞,同时进行许旺细胞方向的诱导分化,为周围神经的修复提供新的种子细胞来源。 目的：诱导人肌源干细胞向具有许旺细胞特性的细胞分化。 方法：①收集重睑成形术中切除的上睑眼轮匝肌,经酶消化和细胞筛过滤,贴壁培养分离人肌源干细胞,予细胞特异标记物以免疫组织化学染色。②取大鼠坐骨神经分离培养许旺细胞,收集许旺细胞条件培养液。③将人肌源干细胞与许旺细胞条件培养液共培养,观察转化细胞的形态和免疫组织化学染色的变化。 结果与结论：①原代培养4周时可见人肌源干细胞,与传代培养之人肌源干细胞同样呈现明显的小圆形形态,折光性强,少量细胞呈现短梭形。所有人肌源干细胞表现为Desmin阳性,Sca-1染色阳性。②分离培养大鼠坐骨神经来源许旺细胞,S100染色阳性率为(97.4±0.7)%。③经与许旺细胞条件培养液共培养,人肌源干细胞分化后细胞表达许旺细胞特异标记物S100,GFAP和p75。结果提示分离获得人肌源干细胞与许旺细胞条件培养液共培养,可以诱导人肌源干细胞表达许旺细胞特异标记物,初步证实了人肌源干细胞可分化为许旺细胞样细胞。     

兔肌源性干细胞的生物学特性及表型研究

目的:探讨兔肌源性干细胞(MDSCs)的分离及特征和表型研究。方法:取1.5月龄新西兰兔大腿肌肉，采用差速贴壁分离Preplate技术，分别用Ⅺ胶原酶、dispase蛋白酶以及胰蛋白酶分步消化肌肉，并用差速贴壁法分离晚期贴壁的细胞，流式细胞仪(FCM)、免疫细胞化学以及Western blotting等检测初步确定细胞表型。结果:进行连续6 d的差速贴壁分离。第3 d开始得到较多量的圆形或短梭形细胞，簇样生长，体积明显小于骨骼肌细胞及其它杂细胞，10 d左右汇合，有极强的融合生长倾向。细胞在汇合度<30%传代可较好地保持原形态，流式细胞仪检测示MDSCs>80%为desmin+，>70%为Bcl-2+，>95%为CD45-，免疫细胞化学定性示desmin+。Western blotting检测显示随着细胞的纯化，α-SMA表达越来越弱。而细胞高汇合度(>50%)或低血清培养时极易融合成肌管或肌细胞链，skeletal myosin+。结论:MDSCs具有desmin、Bcl-2高水平表达和CD45极低水平表达的特性，具有多向分化能力的MDSCs是组织工程研究的一种新型种子细胞。     

Clonogenic analysis reveals reserve stem cells in postnatal mammals: I. Pluripotent mesenchymal stem cells

Clonal populations of lineage-uncommitted pluripotent mesenchymal stem cells have been identified in prenatal avians and rodents. These cells reside in the connective tissue matrices of many organs and tissues. They demonstrate extended capabilities for self-renewal and the ability to differentiate into multiple separate tissues within the mesodermal germ line. This study was designed to determine whether such cells are present in the connective tissues of postnatal mammals. This report describes a cell clone derived by isolation from postnatal rat connective tissues, cryopreservation, extended propagation, and serial dilution clonogenic analysis. In the undifferentiated state, this clone demonstrates a high nuclear-to-cytoplasmic ratio and extended capacity for self-renewal. Subsequent morphological, histochemical, and immunochemical analysis after the induction of differentiation revealed phenotypic markers characteristic of multiple cell types of mesodermal origin, such as skeletal muscle, smooth muscle, fat cells, cartilage, and bone. These results indicate that this clone consists of pluripotent mesenchymal stem cells. This report demonstrates that clonal populations of reserve stem cells are present in mammals after birth. Potential roles for such cells in the maintenance, repair, and regeneration of mesodermal tissues are discussed.     

The role of muscle-derived stem cells in bone tissue engineering

The formation of bone and repair of bone defect requires a source of pluripotential mesenchymal stem cells. However, the capacity of the human body to generate bone components is limited. In this report, we show that the highly purified myogenic cells by the preplate technique have the capacity to differentiate into osteogenic lineage in vitro. The recombinant human bone morphogenic protein (rh-BMP-2) was immobilized on the molded gelatin composite. Primary muscle cells were isolated from newborn Wistar-rats calf muscle. The cells were then preplated in collagen-coated flasks. After six serial platings, the culture was enriched with small, round cells [pp6]. The effects of immobilized rhBMP-2 on the gelatin scaffold were evaluated by the analysis of alkaline phosphatase (ALP) and osteocalcin in culture medium after seedings of muscle-derived cells [pp6]. The results showed that the cells isolated from pp6 slow adhering cells possessed round mononuclear phenotype, marked ALP stain and matrix mineralization. The synthesis and secretion of ALP from pp6 muscle-derived cells were persistent higher than that of pp1-pp5 groups. The efficacy of rhBMP-2 immobilization on the gelatin scaffolds as manifested as the synthesis and secretion of ALP and osteocalcin from muscle-derived cells was always significantly higher than that of the control samples. In summary, our results suggest that the muscle-derived pp6 cells were capable of inducing and participating in bone formation. These results suggest that muscle tissue is a valuable resource for osteoprogenitor cells to be used in clinical practice to improve bone healing.     

小鼠肌源性干细胞高表达CD34、 Sca-1、Bcl-2和desmin

目的: 探讨小鼠肌源性干细胞(MDSCs)的生物学特性和表型。方法: 取小鼠骨骼肌,用酶分步消化,应用差速贴壁法进行纯化获取MDSCs,观察细胞的形态,对MDSCs分别进行增殖能力、克隆形成率和融合情况等鉴定;通过流式细胞术、免疫荧光法及免疫印迹法(Western blotting)等检测初步确定细胞表型。结果: 经差速贴壁法获取的MDSCs为圆形或短梭形细胞,聚集成簇,呈对数生长;细胞倍增时间(9.69±2.08)h,克隆形成率(13.35±2.54)%;细胞在高汇合度(>50％)或低血清(2％FBS)培养时,极易融合成肌管或肌细胞链;流式细胞术和免疫荧光技术鉴定:CD34、Sca-1、Bcl-2和desmin在原代MDSCs中的表达均>90%;Western blotting检测显示随着细胞的纯化, desmin表达越来越强,α-SMA表达越来越弱。结论: MDSCs通过差速贴壁法获取时纯度较高,它具有高水平表达CD34、Sca-1、Bcl一2和desmin的生物学特性,这4种蛋白可作为鉴定MDSCs的标志物。MDSCs增殖旺盛,可在体外大量扩增,是组织工程研究的一种新型种子细胞。     

尿源性干细胞在体外向尿路上皮细胞和平滑肌细胞的分化

文题释义：干细胞分化：干细胞通过体外诱导的方法,基因组在时间和空间上选择性表达,产生特异性蛋白质,定向成为特定的成熟细胞。 平滑肌：是由平滑肌细胞组成的非横纹肌肌肉组织,分布在动脉和静脉血管管壁、膀胱、子宫、男性和女性生殖道、消化道、呼吸道、眼睛的睫状肌和虹膜。 背景：成体干细胞是存在于已分化组织中的多能干细胞,尿源性干细胞为新发现的成体干细胞,具有取材方便、高度增殖能力、多向分化潜能等优点,在组织工程学领域受到重视。 目的：研究人尿源性干细胞体外分离获取的方法,以及体外向尿路上皮细胞和平滑肌细胞分化的可行性。 方法：收集健康成人尿液标本,通过分离、培养获取尿源性干细胞;应用CCK-8实验检测细胞增殖能力,并绘制细胞生长曲线;应用流式细胞仪检测细胞表型;体外应用专用培养基诱导尿源性干细胞向尿路上皮细胞和平滑肌细胞分化;应用荧光定量PCR、免疫组织化学染色、免疫荧光细胞染色和Western blot鉴定尿源性干细胞的分化能力。 结果与结论：①从健康成人尿液中成功分离提取出尿源性干细胞,细胞具有高度增殖能力,生长曲线呈现S型;②尿源性干细胞高表达间充质干细胞表面标记物,CD29表达率为98.11%,CD90表达率为95.74%;③尿源性干细胞经体外诱导分化14 d后,可以表达尿路上皮细胞特异性基因Cytokeratin 7、Cytokeratin 20、Uroplakin Ⅲ和平滑肌细胞特异性基因α-SMA和SM22;④结果表明,尿源性干细胞经过体外诱导可以向尿路上皮细胞和平滑肌细胞分化,能够为尿路组织修复重建提供理想的种子细胞来源。 ORCID: 0000-0002-6460-3332(赵战魁) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

脂肪干细胞分离、纯化和保存：研究进展与未来方向

BACKGROUND: In 2001, Zuk et al found adipose-derived stem cells (ASCs) from the aspirate of liposuction for the first time, which launched a new era of stem cell research. In recent years, stem cells have been proved to widely exist in many tissues and organs. ASCs are always in the spotlight of plastic and reconstructive surgery, tissue engineering and regenerative medicine because of extensive sources and simple isolation. OBJECTIVE: To review the fat tissue harvesting and ASCs isolation, purification, expansion, and cryopreservation, to discuss the main factors which influence the yield, proliferation capacity and differentiation potential of ASCs, and to predict the future research interests based on current issues. METHODS: On September 10th, 2015, relevant articles were searched in PubMed using the following format: (adipose stem cells[Title]) OR (adipose-derived stem cells[Title]) OR (adipose-derived mesenchymal stem cells[Title]) and in SinoMed using the following format in Chinese: (“adipose-derived stem cells” [Title])or(“adipose-derived mesenchymal stem cells”[Title]). Finally, 81 representative articles were included according to their titles and abstracts. In this review, we also introduced relevant experience about the aforementioned procedures from the Department of Plastic Surgery and Tissue Regeneration and Molecular Cell Engineering Lab of University of Texas, MD Anderson Cancer Center, USA. RESULTS AND CONCLUSION: The widely dispersed fat tissues potentially provide abundant stem cells for tissue engineering and regenerative medicine. Liposuction is a mini-invasive approach for harvesting fat tissues. Collagenase digestion is the major method for ASCs isolation due to its simplicity and high yield in basic research. However, clinical fat transplantation without ASCs isolation or non-collagenase isolation of stromal vascular fraction or ASCs is preferred. The phenotype, proliferation and differentiation capacity of ASCs may be affected by several factors during the fat tissue harvesting and ASCs isolation. Therefore, a standard protocol for ASCs isolation is needed.       

Matrix metalloproteinase inhibition negatively affects muscle stem cell behavior

Skeletal muscle is a large and complex system that is crucial for structural support, movement and function. When injured, the repair of skeletal muscle undergoes three phases: inflammation and degeneration, regeneration and fibrosis formation in severe injuries. During fibrosis formation, muscle healing is impaired because of the accumulation of excess collagen. A group of zinc-dependent endopeptidases that have been found to aid in the repair of skeletal muscle are matrix metalloproteinases (MMPs). MMPs are able to assist in tissue remodeling through the regulation of extracellular matrix (ECM) components, as well as contributing to cell migration, proliferation, differentiation and angiogenesis. In the present study, the effect of GM6001, a broad-spectrum MMP inhibitor, on muscle-derived stem cells (MDSCs) is investigated. We find that MMP inhibition negatively impacts skeletal muscle healing by impairing MDSCs in migratory and multiple differentiation abilities. These results indicate that MMP signaling plays an essential role in the wound healing of muscle tissue because their inhibition is detrimental to stem cells residing in skeletal muscle.