Chondrogenic differentiation of amniotic fluid-derived stem cells

For regenerating damaged articular cartilage, it is necessary to identify an appropriate cell source that is easily accessible, can be expanded to large numbers, and has chondrogenic potential. Amniotic fluid-derived stem (AFS) cells have recently been isolated from human and rodent amniotic fluid and shown to be highly proliferative and broadly pluripotent. The purpose of this study was to investigate the chondrogenic potential of human AFS cells in pellet and alginate hydrogel cultures. Human AFS cells were expanded in various media conditions, and cultured for three weeks with growth factor supplementation. There was increased production of sulfated glycosaminoglycan (sGAG) and type II collagen in response to transforming growth factor-β (TGF-β) supplementation, with TGF-β1 producing greater increases than TGF-β3. Modification of expansion media supplements and addition of insulin-like growth factor-1 during pellet culture further increased sGAG/DNA over TGF-β1 supplementation alone. Compared to bone marrow-derived mesenchymal stem cells, the AFS cells produced less cartilaginous matrix after three weeks of TGF-β1 supplementation in pellet culture. Even so, this study demonstrates that AFS cells have the potential to differentiate along the chondrogenic lineage, thus establishing the feasibility of using these cells for cartilage repair applications.     

Congenital anomalies: Treatment options based on amniotic fluid-derived stem cells

Over the past decade, amniotic fluid-derived stem cells have emerged as a novel, experimental approach for the treatment of a wide variety of congenital anomalies diagnosed either in utero or postnatally. There are a number of unique properties of amniotic fluid stem cells that have allowed it to become a major research focus. These include the relative ease of accessing amniotic fluid cells in a minimally invasive fashion by amniocentesis as well as the relatively rich population of progenitor cells obtained from a small aliquot of fluid. Mesenchymal stem cells, c-kit positive stem cells, as well as induced pluripotent stem cells have all been derived from human amniotic fluid in recent years. This article gives a pediatric surgeon’s perspective on amniotic fluid stem cell therapy for the management of congenital anomalies. The current status in the use of amniotic fluid-derived stem cells, particularly as they relate as substrates in tissue engineering-based applications, is described in various animal models. A roadmap for further study and eventual clinical application is also proposed.     

Characterization and hepatogenic differentiation of mesenchymal stem cells from human amniotic fluid and human bone marrow: a comparative study

Since stem cells can differentiate into hepatocyte, stem cell-based therapy becomes a potential alternative treatment for terminal liver diseases. However, an appropriate source of human mesenchymal stem cells (hMSCs) for hepatocytes has not yet been clearly elucidated. The aim of the present study was to investigate the in vitro biological characterization and hepatic differentiation potential of human amniotic fluid-derived mesenchymal stem cells (AF-hMSCs) and human bone marrow-derived mesenchymal stem cells (BM-hMSCs). Our results show that AF-hMSCs possess higher proliferation and self-renewal capacity than BM-hMSCs. Cytogenetic studies indicate that AF-hMSCs are as genetically stabile as BM-hMSCs. Following incubation with specific hepatogenic agents, AF-hMSCs showed a higher hepatic differentiation potential than BM-hMSCs. Expression of several liver-specific markers was significantly greater in AF-hMSCs than in BM-hMSCs, as shown by real time RT-PCR and immunofluorescence (IF). In conclusion, AF-hMSCs possess superior potential for hepatic differentiation, making them more suitable for diverse terminal liver diseases.     

Molecular and phenotypic characterization of human amniotic fluid cells and their differentiation potential

The main goal of the study was to identify a novel source of human multipotent cells, overcoming ethical issues involved in embryonic stem cell research and the limited availability of most adult stem cells. Amniotic fluid cells （AFCs） are routinely obtained for prenatal diagnosis and can be expanded in vitro; nevertheless current knowledge about their origin and properties is limited. Twenty samples of AFCs were exposed in culture to adipogenic, osteogenic, neurogenic and myogenic media. Differentiation was evaluated using immunocytochemistry, RT-PCR and Western blotting. Before treatments, AFCs showed heterogeneous morphologies. They were negative for MyoD, Myf-5, MRF4, Myogenin and Desmin but positive for osteocalcin, PPARgamma2, GAP43, NSE, Nestin, MAP2, GFAP and beta tubulin III by RT-PCR. The cells expressed Oct-4, Rex-1 and Runx-1, which characterize the undifferentiated stem cell state. By immunocytochemistry they expressed neural-glial proteins, mesenchymal and epithelial markers. After culture, AFCs differentiated into adipocytes and osteoblasts when the predominant cellular component was fibroblastic. Early and late neuronal antigens were still present after 2 week culture in neural specific media even if no neuronal morphologies were detectable. Our results provide evidence that human amniotic fluid contains progenitor cells with multi-lineage potential showing stem and tissue-specific gene/protein presence for several lineages.     

人羊膜上皮细胞和胚胎干细胞

人胚胎干细胞有着巨大的医学应用前景,但人胚胎干细胞要求的生长条件很高,体外很难模拟其生长的体内环境,因此控制人胚胎干细胞的生长常不理想,而使用鼠胚胎成纤维细胞(MEF)作为滋养层则存在动物源性污染的问题。该文阐述人羊膜上皮细胞(HAEC)的特点及其作为滋养层培养胚胎干细胞的现状,并探讨基因组DNA甲基化修饰在胚胎干细胞分化过程中的作用,为建立更优化的培养系统提供依据。     

CD117+ amniotic fluid stem cells

Broadly multipotent stem cells can be isolated from amniotic fluid by selection for the expression of the membrane stem cell factor receptor c-Kit, a common marker for multipotential stem cells. They have clonogenic capability and can be directed into a wide range of cell types representing the three primary embryonic lineages. Amniotic fluid stem cells maintained for over 250 population doublings retained long telomeres and a normal karyotype. Clonal human lines verified by retroviral marking were induced to differentiate into cell types representing each embryonic germ layer, including cells of adipogenic, osteogenic, myogenic, endothelial, neuronal and hepatic lineages. AFS cells could be differentiate toward cardiomyogenic lineages, when co-cultured with neonatal cardiomyocytes, and have the potential to generate myogenic and hematopoietic lineages both in vitro and in vivo. Very recently first trimester AFS cells could be reprogrammed without any genetic manipulation opening new possibilities in the field of fetal/neonatal therapy and disease modeling. In this review we are aiming to summarize the knowledge on amniotic fluid stem cells and highlight the most promising results.     

Enhanced neural differentiation of neural stem cells and neurite growth by amniotic epithelial cell co-culture

Amniotic epithelial cells (AECs) were reported to show a neuroprotective effect on neurons, but there was no direct evidence for a functional relationship between neural stem cells (NSCs) and AECs. The aim of this study was to determine whether AECs could stimulate differentiation and expand neurogenesis of NSCs, and whether the roles were due to a diffusible factor or required direct cell-cell contact. AECs were isolated from rat amnion on E14-16 and NSCs were isolated from neocortical tissue. The growth and differentiation of NSCs were compared under different conditions. The results showed that NSCs cultured with FGF-2 proliferated and formed floating neurospheres while those grown in B27 without FGF-2 failed to thrive. Those grown either with AEC conditioned medium or in transwells showed significantly improved survival. Moreover, the neural differentiation and length of neurite were greater in exogenous FGF groups when NSCs were allowed direct contact with AECs. Western blotting, immunocytochemistry and RT-PCR indicated that rat AECs could secrete NT-3 and BDNF. Furthermore, the presence of FGF-2 enhanced the function of AECs. These findings identified that AECs may be regarded as a critical component of NSCs niche and suggested that direct cell-to-cell contact may provide additional and independent support. Such information would circumvent the need for AECs-NSCs co-culture and could potentially facilitate the production of neurons for future clinical applications.     

CD117+ amniotic fluid stem cells: State of the art and future perspectives

Broadly multipotent stem cells can be isolated from amniotic fluid by selection for the expression of the membrane stem cell factor receptor c-Kit, a common marker for multipotential stem cells. They have clonogenic capability and can be directed into a wide range of cell types representing the three primary embryonic lineages. Amniotic fluid stem cells maintained for over 250 population doublings retained long telomeres and a normal karyotype. Clonal human lines verified by retroviral marking were induced to differentiate into cell types representing each embryonic germ layer, including cells of adipogenic, osteogenic, myogenic, endothelial, neuronal and hepatic lineages. AFS cells could be differentiate toward cardiomyogenic lineages, when co-cultured with neonatal cardiomyocytes, and have the potential to generate myogenic and hematopoietic lineages both in vitro and in vivo. Very recently first trimester AFS cells could be reprogrammed without any genetic manipulation opening new possibilities in the field of fetal/neonatal therapy and disease modeling. In this review we are aiming to summarize the knowledge on amniotic fluid stem cells and highlight the most promising results.     

Predicting differentiation potential of human pluripotent stem cells:Possibilities and challenges

The capability of human pluripotent stem cell(hPSC) lines to propagate indefinitely and differentiate into derivatives of three embryonic germ layers makes these cells be powerful tools for basic scientific research and promising agents for translational medicine. However, variations in differentiation tendency and efficiency as well as pluripotency maintenance necessitate the selection of hPSC lines for the intended applications to save time and cost. To screen the qualified cell lines and exclude problematic cell lines, their pluripotency must be confirmed initially by traditional methods such as teratoma formation or by highthroughput gene expression profiling assay. Additionally, their differentiation potential, particularly the lineage-specific differentiation propensities of hPSC lines, should be predicted in an early stage. As a complement to the teratoma assay, RNA sequencing data provide a quantitative estimate of the differentiation ability of hPSCs in vivo. Moreover, multiple scorecards have been developed based on selected gene sets for predicting the differentiation potential into three germ layers or the desired cell type many days before terminal differentiation.For clinical application of hPSCs, the malignant potential of the cells must also be evaluated. A combination of histologic examination of teratoma with quantitation of gene expression data derived from teratoma tissue provides safety-related predictive information by detecting immature teratomas, malignancy marker expression, and other parameters. Although various prediction methods are available, distinct limitations remain such as the discordance of results between different assays and requirement of a long time and high labor and cost,restricting their wide applications in routine studies. Therefore, simpler and more rapid detection assays with high specificity and sensitivity that can be used to monitor the status of hPSCs at any time and fewer targeted markers that are more specific for a given desired cell type are urgently needed.     

人羊膜间充质细胞分离培养及其干细胞特性研究

目的：研究人羊膜间充质细胞（Humanamnioticmesenchymalcells,HAMCs）的分离、培养及其干细胞特性,为羊膜间充质细胞在再生医学的潜在应用奠定实验基础。方法：无菌条件下取正常足月剖腹产胎儿的羊膜剪成碎片,经胰酶胶原酶序贯消化,DMEM/F12培养,倒置显微镜下观察其形态,MTT法检测其生长规律,免疫荧光的方法对细胞进行鉴定,定向诱导方法检测细胞的多向分化潜能。结果：来源于羊膜的间充质细胞,细胞免疫荧光显示SSEA-4,OCT-4阳性,具有很强的增殖能力,并且具有一定的多向分化能力,在特定条件下可分化为脂肪细胞和成骨细胞;结论：羊膜间充质细胞能够在体外分离、培养、扩增,并且具有干细胞特性。羊膜间充质细胞在再生医学和组织工程应用有很好的前景。     

SPC及EGFP共表达载体跟踪人羊水间充质干细胞体外定向分化

目的构建肺泡表面活性蛋白C(SPC)及增强型绿色荧光蛋白(EGFP)共表达载体pcDNA3.1/SPC/EGFP,探讨其在体外跟踪人羊水间充质干细胞(AF-MSCs)定向分化为II型肺泡上皮细胞(AECII)的作用。方法采用PCR和DNA重组技术构建pcDNA3.1/SPC/EGFP表达载体,脂质体转染至AF-MSCs,G418稳定筛选;将AF-MSCs分为阴性对照组、未转染组和转染组,各组体外诱导培养后荧光显微镜观察SPC启动子调控下游EGFP基因表达活性,RT-PCR检测SPA和SPC mRNA表达水平,Western blot检测SPA和SPC蛋白表达以及电镜观察嗜锇性板层小体。结果成功构建pcDNA3.1/SPC/EGFP表达载体,测序结果与SPC启动子及EGFP序列一致;AF-MSCs体外诱导分化后,在阴性对照组中未见绿色荧光细胞,SPA和SPC mRNA及蛋白均为阴性表达,且未发现嗜锇性板层小体;在未转染组中亦未见绿色荧光细胞,而SPA和SPC mRNA(相对表达量为0.072±0.004和0.087±0.012)及蛋白(相对表达量为0.051±0.008和0.063±0.009)均为阳性表达,并发现嗜锇性板层小体;在转染组中可见绿色荧光细胞,SPA和SPC mRNA(相对表达量为0.109±0.011和0.126±0.017)及蛋白(相对表达量为0.075±0.012和0.081±0.006)均为显著表达,与未转染组相比差异均有统计学意义(t值分别为-5.50、-3.16、-2.90和-2.85,均P0.05),亦可见嗜锇性板层小体。结论经pcDNA3.1/SPC/EGFP表达载体转染的AFMSCs在体外适当诱导下能定向分化为AECII,pcDNA3.1/SPC/EGFP表达载体可能成为跟踪AF-MSCs定向分化的工具,为肺组织再生的干细胞治疗提供研究基础。     

肝细胞生长因子对人羊水干细胞迁移和黏附能力的影响

目的分离培养及鉴定羊水干细胞（hAFSC）,并研究肝细胞生长因子（HGF）对羊水干细胞迁移、黏附能力的影响。方法使用细胞贴壁法分离培养羊水干细胞,细胞免疫荧光及westernblot鉴定羊水干细胞,Transwell小室分析HGF对羊水干细胞迁移的作用。明胶贴壁法分析HGF对羊水干细胞黏附能力的作用。两组之间数据的比较采用独立样本t检验。结果分离的羊水干细胞均表达特异性标记物Oct-4、c-kit、SSEA-4、CD105。HGF在体外对hAFSC的迁移有趋化作用,对照组和HGF组每个视野的迁移细胞数分别为38±2.5和80±3.2。对黏附能力有促进作用,对照组和HGF组每个视野的黏附细胞数分别为19±1.5和50±2.7,差异均有统计学意义（P〈0.01）。结论 HGF可趋化羊水干细胞的迁移,增强羊水干细胞的黏附能力。     

人羊膜间充质细胞分离培养及其干细胞特性研究

目的:研究人羊膜间充质细胞(Humanamnioticmesenchymalcells,HAMCs)的分离、培养及其干细胞特性,为羊膜间充质细胞在再生医学的潜在应用奠定实验基础。方法:无菌条件下取正常足月剖腹产胎儿的羊膜剪成碎片,经胰酶胶原酶序贯消化,DMEM/F12培养,倒置显微镜下观察其形态,MTT法检测其生长规律,免疫荧光的方法对细胞进行鉴定,定向诱导方法检测细胞的多向分化潜能。结果:来源于羊膜的间充质细胞,细胞免疫荧光显示SSEA-4,OCT-4阳性,具有很强的增殖能力,并且具有一定的多向分化能力,在特定条件下可分化为脂肪细胞和成骨细胞;结论:羊膜间充质细胞能够在体外分离、培养、扩增,并且具有干细胞特性。羊膜间充质细胞在再生医学和组织工程应用有很好的前景。