人羊水来源间充质干细胞冻存后生物学特征研究

目的体外分离培养人羊水间充质干细胞(human amniotic fluid-derived mesenchymal stem cells,HAFMSCs),观察低温冻存复苏后HAFMSCs生物学特征,为进一步研究奠定理论基础。方法取12份自愿捐赠的孕16～20周羊水标本,采用改良两步法分离培养HAFMSCs,用含量不同的FBS、DMSO冻存液冻存细胞,液氮冻存12周后42℃水浴复苏,锥虫蓝染色检测细胞存活率,MTT法检测细胞增殖速度并绘制生长曲线,流式细胞仪检测冻存复苏后HAFMSCs表型。对冻存复苏后的HAFMSCs进行成脂、成骨诱导分化培养,并分别采用油红O、von Kossa染色进行鉴定;实时荧光定量PCR分析细胞冻存前后Oct-4、Nanog mRNA表达差异。结果细胞冻存12周后,不同的冻存方案对细胞存活率影响有差异,优化的冻存方案为DMEM/FBS/DMSO=50%/40%/10%。冻存复苏后的HAFMSCs呈漩涡状排列,生长曲线呈S形,与冻存前细胞生长曲线相似。流式细胞仪检测示冻存复苏后细胞的MSCs表型CD29、CD44、CD73、CD90为阳性,造血干细胞表型CD34、CD45为阴性。成脂、成骨诱导21 d,油红O、von Kossa染色均呈阳性。实时荧光定量PCR检测示冻存前后Oct-4、Nanog mRNA表达水平差异无统计学意义(P>0.05)。结论 HAFMSCs具有体外增殖快、分化能力强的优势;并可耐受短期冻存,复苏后细胞存活率高,生物学特征及分化潜能未发生明显变化,冻存液DMEM/FBS/DMSO=50%/40%/10%是较好冻存方案。     

间充质干细胞治疗骨关节炎

骨关节炎是中老年患者最常见的慢性退行性关节疾病,以关节软骨变性破坏、软骨下骨硬化、滑膜增生为主要病理特点。随着我国人口老龄化,发病人群的不断增加,骨关节炎致残率高,所造成的巨大社会经济负担,难以估量。目前现有的治疗手段包括改变生活方式,止痛药物,关节腔注射激素、透明质酸等,均不能阻止早、中期病变进展,而病变晚期,关节置换术虽然能够获得较好的疗效,但术后感染、假体松动等并发症仍不容忽视。随着对间充质干细胞研究的深入,其作为一种具有多向分化潜能的种子细胞,逐步显示出在慢性退行性疾病治疗中的优势。间充质干细胞能够向目的细胞定向分化,同时分泌一系列因子参与组织修复。目前,已有的临床前研究更多的倾向于间充质干细胞通过间接机制发挥治疗作用。间充质干细胞在临床研究中也显示出对骨关节炎的治疗意义。本文对间充质干细胞的特性、作用机制、应用方式和尚未解决的问题进行总结归纳。     

小鼠羊水间充质干细胞的分离、培养及鉴定

目的  探讨小鼠羊水间充质干细胞（AF-MSC）的分离、培养及鉴定。方法  在无菌条件下获取孕鼠子宫,收集羊水后进行过滤和离心,对沉淀细胞团进行培养并传代。观察AF-MSC的形态,分析AF-MSC的增殖特点,采用流式细胞术鉴定AF-MSC的表面标志物,检测AF-MSC的三系分化能力及冷冻复苏后的细胞活力。结果  小鼠AF-MSC呈典型的梭形,融合度 > 80%时会出现典型的漩涡状结构。小鼠AF-MSC传代培养无明显潜伏期,培养2~3 d进入对数增长期,增长速度最快,之后增殖速度减慢,进入平台期。AF-MSC表达干细胞抗原（Sca）-1、CD29、CD44,不表达CD34、CD45。小鼠AF-MSC成骨分化后,矿化结晶被茜素红染成深红色的点状;成软骨分化后,分泌的酸性粘多糖被阿利新蓝染成淡蓝色;成脂分化后,胞质脂滴被油红O染成红色。细胞冷冻复苏后存活率 > 95%,生长状态良好,6 d时增殖能力高于冻存前（P < 0.05）,其他时间增殖能力与冻存前比较,差异无统计学意义（均为P > 0.05）。结论  本实验成功分离了小鼠AF-MSC,过程简便、成本低,且分离的细胞可随传代次数的增加而纯化,冻存不影响其增殖能力。     

间充质干细胞及其作用的研究新进展

间充质干细胞(MSCs)可以成功地从所有哺乳动物组织细胞内分离出来,它的多向分化潜能在再生医学中有很大应用潜力.随着对MSCs的生物学特征、MSCs与其周围内环境之间的相互作用、MSCs归巢的分子调节机制及多向分化能力的深入研究,MSCs将作为细胞修复或外源性基因转染和表达的载体在临床试验中发挥重要作用.     

间充质干细胞及其作用的研究新进展

间充质干细胞(MSCs)可以成功地从所有哺乳动物组织细胞内分离出来,它的多向分化潜能在再生医学中有很大应用潜力.随着对MSCs的生物学特征、MSCs与其周围内环境之间的相互作用、MSCs归巢的分子调节机制及多向分化能力的深入研究,MSCs将作为细胞修复或外源性基因转染和表达的载体在临床试验中发挥重要作用.     

人羊水来源c-kit~+间充质干细胞的生物学特征和心肌诱导分化

目的探讨人羊水来源c-kit~+间充质干细胞(c-kit~+human amniotic fluid-derived mesenchymal stemcells,c-kit~+HAFMSCs)的生物学特征及心肌诱导分化能力。方法通过产前诊断或自愿引产获取50份孕中期羊水标本,经流式细胞仪分选c-kit~+HAFMSCs,MTT法观察细胞增殖情况,并通过流式细胞仪、细胞免疫化学染色观察行细胞表型鉴定。在体外经成骨及成脂诱导分化,于诱导培养后21 d采用yon Kossa染色观察钙沉积颗粒,油红O染色观察脂滴形成情况。实时荧光定量PCR检测细胞经5氮-2’脱氧胞苷心肌诱导前后,NKx2.5、Tbx5、GATA-4、α-MHC 4种心肌特异性基因表达情况。结果经流式细胞仪分选,人孕中期羊水中c-kit~+HAFMSCs含量约为贴壁细胞的3.07%±1.03%;体外增殖迅速,表达MSCs表面标志CD29、CD44、CD73、CD90、CD105,不表达造血干细胞表面标志CD34、CD45;农达人类白细胞抗原(human leukocyte antigen,HLA)-ABC,不表达HLA-DR。细胞免疫化学染色示CD29、CD44、CD73、CD90、CD105、Oct-4染色呈阳性,CD34、CD45染色呈阴性。MTT检测示第5、10、15代c-kit~+HAFMSCs具有相似的生长曲线。成骨和成脂诱导培养后21 d,细胞内有钙盐沉积和脂滴形成。实时荧光定量PCR检测示,心肌诱导后14 d,NKx2.5、Tbx5、GATA-4、α-MHC 4种心肌特异性基因表达均较诱导前明显增加,差异有统计学意义(P<0.05)。结论通过流式细胞仪分选的c-kit~+HAFMSCs在体外可以诱导分化为心肌样细胞,可能成为心肌再生性治疗的种子细胞。     

兔骨髓间充质干细胞分离方法的比较

目的：建立并优化骨髓间充质干细胞分离纯化的方法。方法：从兔的胫骨中抽取骨髓，分别通过直接培养、经Fichu—Hypeque和Perool两种分离介质进行分离后再培养的方法，收获所培养的细胞，然后测量间充质干细胞所占的比例。结果:直接培养、经Fichu—Hypaque、Perool分离后再培养的细胞中，骨髓间充质干细胞比例分别为67．3％、83．6％、93．4％。结论:采用Perool分离方法可以更好的提高MSCs的纯度，提高MSCs的培养效率。     

猪骨髓间充质干细胞的分离培养及分化潜能的鉴定

目的建立猪骨髓间充质干细胞(MSCs)的体外分离培养和鉴定的方法,探讨体外培养的间充质干细胞的一些生物学特点,为利用猪的实验研究提供实验基础.方法从猪的髂嵴穿刺取骨髓,经密度梯度离心得到骨髓单个核细胞,接种后形成单层贴壁的成纤维样的细胞.检测细胞周期,多向诱导分化鉴定分离的细胞.结果体外培养的原代MSCs 12～14d达到融合,传代后仍具有分化成骨的能力,细胞周期显示有80%细胞处于G0/G1期.结论体外培养猪的MSCs具有分化成骨的潜能,生长稳定,传代后仍保持未分化状态.猪骨髓间充质干细胞分离培养体系的建立为基础研究和组织工程提供了一个有价值的动物模型.     

人脐带间充质干细胞移植治疗小脑萎缩

目的 研究人脐带间充质干细胞(Human umbilical-cord mesenchymal stem cells,hUMSCs)治疗小脑萎缩(Cerebellar atrophy,CA)的临床疗效.方法 健康新生儿脐带中分离MSCs,连续体外扩增,采用鞘内注射(2.5×1075 mL)结合静脉滴注(7.5×107100 mL)的方法,治疗57例小脑萎缩患者,每次治疗剂量为1.0× 108个细胞.结果 所有患者随访6个月,治疗3个月后有效率为72.0%,治疗6个月后有效率为79.0%,无明显并发症.结论 应用hUMSCs治疗小脑萎缩安全有效,但远期疗效尚待进一步观察.     

A comparison of clinically relevant sources of mesenchymal stem cell-derived exosomes: Bone marrow and amniotic fluid

Background/Purpose

Exosomes may constitute a more practical alternative to live cells in select stem cell-based therapies. We sought to compare exosomes from two mesenchymal stem cell (MSC) sources relevant to perinatal and pediatric diseases.

Sternal repair with bone grafts engineered from amniotic mesenchymal stem cells

Purpose

We aimed at determining whether osseous grafts engineered from amniotic mesenchymal stem cells (aMSCs) could be used in postnatal sternal repair.

Methods

Leporine aMSCs were isolated, identified, transfected with green fluorescent protein (GFP), expanded, and seeded onto biodegradable electrospun nanofibrous scaffolds (n = 6). Constructs were dynamically maintained in an osteogenic medium and equally divided into 2 groups with respect to time in vitro as follows: 14.6 or 33.9 weeks. They were then used to repair full-thickness sternal defects spanning 2 to 3 intercostal spaces in allogeneic kits (n = 6). Grafts were submitted to multiple analyses 2 months thereafter.

Results

Chest roentgenograms showed defect closure in all animals, confirmed at necropsy. Graft density as assessed by microcomputed tomographic scans increased significantly in vivo, yet there were no differences in mineralization by extracellular calcium measurements preimplantation and postimplantation. There was a borderline increase in alkaline phosphatase activity in vivo, suggesting ongoing graft remodeling. Histologically, implants contained GFP-positive cells and few mononuclear infiltrates. There were no differences between the 2 construct groups in any comparison.

Conclusions

Engineered osseous grafts derived from amniotic mesenchymal stem cells may become a viable alternative for sternal repair. The amniotic fluid can be a practical cell source for engineered chest wall reconstruction.     

Fetal bone marrow homing of donor mesenchymal stem cells after transamniotic stem cell therapy (TRASCET)

Purpose

Donor cell engraftment patterns following transamniotic stem cell therapy (TRASCET) with amniotic fluid mesenchymal stem cells (afMSCs) are incompatible with solely direct amniotic seeding. We sought to determine whether fetal bone marrow is a component of such engraftment and to examine the chronology of afMSC placental trafficking.

Donor mesenchymal stem cell linetics after transamniotic stem cell therapy (TRASCET) for experimental spina bifida

Purpose

We sought to examine donor mesenchymal stem cell (MSC) kinetics after transamniotic stem cell therapy (TRASCET) in experimental spina bifida.

人滑膜间充质干细胞的分离培养与鉴定

背景：软骨一旦损伤将很难自我修复,组织工程学修复损伤软骨是目前的研究热点。组织工程学中找到合适的“种子细胞”至关重要。滑膜间充质干细胞（SMSCs）因较其他来源的间充质干细胞成软骨能力、增殖能力更强,且细胞容易获取、对机体损伤小等优点,越来越受到研究者的青睐。 目的：探讨人SMSCs体外分离、培养的方法与步骤,探索对SMSCs进行鉴定的最新方法。 方法：关节镜下获取11例行关节镜手术患者的滑膜组织,按照Pei等[6]的方法分离培养滑膜干细胞;从细胞形态学观察、细胞生长曲线分析、CCK-8测定增殖活力、流式细胞仪检测细胞周期和细胞表面抗原、对SMSCs进行成脂/成骨/成软骨诱导、免疫细胞荧光染色以及RT-PCR检测成脂/成骨/成软骨相关基因的表达等方面对SMSCs进行鉴定。 结果与结论：按照Pei等的方法可以成功地从人滑膜组织中分离出干细胞,分离出的干细胞具有间充质干细胞特异性表型和多向分化潜能。     

子宫内膜间充质干细胞对小鼠模型薄型子宫内膜的修复作用

目的观察小鼠子宫内膜间充质干细胞对小鼠模型薄型子宫内膜的修复作用。方法选择8周龄动情期规律C57BL/6J小鼠共288只,随机分为空白对照组、模型对照组、干细胞移植组,每组96只。空白对照组不做任何处理;模型对照组宫腔注射95%乙醇损伤后立即原位注射PBS 0.1 ml,3 d后尾静脉注射PBS 1 ml;干细胞移植组宫腔注射95%乙醇损伤后立即原位注射子宫内膜间充质干细胞0.1 ml,3 d后尾静脉注射子宫内膜间充质干细胞1 ml,干细胞浓度为5×105/ml。每组根据取样时间(3 d、7 d、14 d、30 d)不同分为4小组,取样当日,半数小鼠子宫组织取样,4%多聚甲醛固定送检,进行子宫内膜厚度、HE染色、免疫组化等检测;另半数小鼠与确定有生殖能力C57BL/6J雄鼠合笼,进行生育力评估。结果干细胞移植组小鼠子宫内膜厚度在各时间点均显著厚于模型对照组(P<0.05),但与空白对照组仍有一定距离(P<0.05)。HE染色结果显示,干细胞移植后的受损子宫内膜上皮层及基质层均增厚,腺体增加。免疫组化结果显示,干细胞移植组角蛋白、波形蛋白、VEGF、ERα各项指标均有显著上升,与模型对照组有显著性差异(P<0.05)。生育力评估发现,模型对照组生育率接近于0(仅30 d亚组1例),而干细胞移植组生育率显著升高,30 d亚组生育率达50%,接近空白对照组(66.7%)。结论小鼠子宫内膜间充质干细胞对薄型子宫内膜有显著改善。     

Preclinical regulatory validation of an engineered diaphragmatic tendon made with amniotic mesenchymal stem cells

Purpose

Under a Food and Drug Administration directive, we examined definite long-term safety and efficacy aspects of an engineered diaphragmatic tendon graft as a regulatory prerequisite for clinical trials.

Methods

Newborn lambs (N = 27) underwent partial diaphragmatic replacement with a Teflon patch, a composite acellular bioprosthesis, or the same bioprosthesis seeded with autologous amniotic mesenchymal stem cells processed under Good Manufacturing Practice guidelines. Multiple safety and efficacy analyses were performed at different time points up to 14 months of age (ovine adulthood).

Results

There was no mortality. None of the blood tests or full body autopsy specimens showed any abnormality. There was a significantly higher failure rate in animals that received an acellular bioprosthetic graft vs an engineered graft, with no significant differences between Teflon and acellular bioprosthetic implants. Tensile strength and total collagen levels were significantly higher in engineered grafts than in acellular bioprosthetic grafts. On histology, lysozyme and myeloperoxidase stainings were unremarkable in all grafts.

Conclusions

Diaphragmatic repair with a clinically viable autologous tendon engineered with amniotic mesenchymal stem cells leads to improved outcomes when compared with an equivalent acellular bioprosthesis, with no local or systemic adverse effects. Clinical trials of engineered diaphragmatic repair appear practicable within regulatory guidelines.     

Growth stimulatory effects of amniotic fluid and amniotic cell conditioned medium on human cells involved in wound healing

Summary Amniotic membrane has been used successfully as a biological dressing for various kinds of wounds. In order to study exocrine activity in amniotic cells, we assessed the incorporation of 3H-thymidine in human fibroblasts and the increase in the number of human keratinocytes when stimulated with amniotic fluid and conditioned medium from amniotic cells. Both amniotic fluid and conditioned medium were shown to stimulate these cell types, which are crucial to the wound healing process. Amniotic fluid was the most potent of the two media tested. The effect exerted by amniotic fluid and conditioned medium were comparable to that of 10% fetal calf serum and a dose-dependent reponse was seen when the media were diluted. These results indicate that the special healing features seen in fetal wounds bathed in amniotic fluid, together with the wound healing properties of amniotic membrane used as a biological wound dressing, may be due to the exocrine activity of the amniotic cells.Abbreviations ACCM Amniotic cell conditioned medium - AM Amniotic membrane - DMEM Dulbecco's Modified Eagle's medium - FCS Fetal calf serum - HA Hyaluronic acid - PBS Phosphatebuffered saline     

Preclinical regulatory validation of a 3-stage amniotic mesenchymal stem cell manufacturing protocol

Purpose

Because of the 4 to 6-month interval between a diagnostic amniocentesis and birth, clinical application of amniotic mesenchymal stem cell (AMSC)-based therapies demands a 3-stage cell manufacturing process, including isolation/primary expansion, cryopreservation, and thawing/secondary expansion. We sought to determine the feasibility and cell yield of such a staged cell manufacturing process, within regulatory guidelines.

Methods

Human AMSCs isolated from diagnostic amniocentesis samples (n = 11) were processed under Food and Drug Administration-accredited good manufacturing practice. Expanded cells were characterized by flow cytometry and cryopreserved for 3 to 5 months. Cell release criteria included more than 90% CD29+, CD73+, and CD44+; less than 5% CD34+ and CD45+; negative mycoplasma quantitative polymerase chain reaction (QPCR) and endotoxin assay; and at least 70% viability.

Results

Isolation and ample expansion of AMSCs was achieved in 54.5% (6/11) of the samples. Early processing and at least a 2-mL sample were necessary for reliable cell manufacturing. Cell yield before cryopreservation was 223.2 ± 65.4 × 10⁶ cells (44.6-fold expansion), plus a 14.7 × 10⁶-cell backup, after 36.3 ± 7.8 days. Cell viability postthaw was 88%. Expanded cells maintained a multipotent mesenchymal progenitor profile.

Conclusions

Human amniotic mesenchymal stem cells can be manufactured in large numbers from diagnostic amniocentesis, by an accredited staged processing, under definite procurement guidelines. These data further support the viability of clinical trials of amniotic mesenchymal stem cell-based therapies.