Investigating the expression of pluripotency-related genes in human amniotic fluid cells: A semi-quantitative comparison between different subpopulations,from primary to cultured amniocytes

Various classifications have been proposed for human amniotic subpopulations, including classification of spindle-shaped (SS) and round-shaped (RS) cells, as well as the more referred triple-category of epithelioid (E-type) cells, amniotic fluid-specific (AF-type) cells and fibroblastoid (F-type) cells. The present study aims to investigate these amniotic subpopulations regarding the expression of some stem cell markers, including OCT4, NANOG, SOX2, C-KIT (CD117), C-MYC, KLF4, and THY1 (CD90). Flow cytometry was performed to characterize the isolated clonal subpopulations for a hematopoietic and a mesenchymal marker using PE-CD31 and FITC-CD90, respectively. A semi-quantitative RT-PCR analysis was carried out on the isolates in the second half of their lifespan when the cells were at the stationary phase of the growth curve. Characterization of isolated cells demonstrated that all clones including both epithelioid and fibroblastoid cells, had mesenchymal, not hematopoietic, lineage. RT-PCR analysis also revealed a higher expression of the target genes in epithelioid cells. Furthermore, the expression pattern of the genes and their correlations were remarkably different between primary- and long term-cultured amniocytes. Taken together, our results showed that the primary-cultured cells express the stemness genes equally, whereas the long term-cultured amniocytes exhibited a highly variable manner in the expression pattern of the genes. Diverse derivation site of amniocytes and individual genetic background can potentially explain the observed variation in the expression level of the target genes. These can also explain why amniocytes differ in many respects observed in our study, including survival rate, plastic adhesion, and growth characteristics.     

Osteogenic differentiation of amniotic fluid mesenchymal stromal cells and their bone regeneration potential

In orthopedics, tissue engineering approach using stem cells is a valid line of treatment for patients with bone defects. In this context, mesenchymal stromal cells of various origins have been extensively studied and continue to be a matter of debate. Although mesenchymal stromal cells from bone marrow are already clinically applied, recent evidence suggests that one may use mesenchymal stromal cells from extra-embryonic tissues, such as amniotic fluid, as an innovative and advantageous resource for bone regeneration. The use of cells from amniotic fluid does not raise ethical problems and provides a sufficient number of cells without invasive procedures. Furthermore, they do not develop into teratomas when transplanted, a consequence observed with pluripotent stem cells. In addition, their multipotent differentiation ability, low immunogenicity, and anti-inflammatory properties make them ideal candidates for bone regenerative medicine. We here present an overview of the features of amniotic fluid mesenchymal stromal cells and their potential in the osteogenic differentiation process. We have examined the papers actually available on this regard, with particular interest in the strategies applied to improve in vitro osteogenesis. Importantly, a detailed understanding of the behavior of amniotic fluid mesenchymal stromal cells and their osteogenic ability is desirable considering a feasible application in bone regenerative medicine.     

羊水干细胞治疗维甲酸诱导的大鼠骨质疏松的初步研究

目的:维甲酸(ratinoicacid)诱导雌性大鼠骨质疏松(Ostoporosis,OP)模型,观察并分析羊水干细胞(amniotic fluid-derivedstem cells,AFS)对OP的治疗作用。方法:选取40只S D雌性大鼠,随机分为4组,每组10只。除正常组(A组)外,模型组(B组),一次注入AFS组(C组)和两次注入AFS组(D组)均用维甲酸70 mg/(k g.d)连续灌胃1 4 d,A组用等量灭菌注射用水灌胃。于给药二,三周分别测量各组大鼠血清Ca、Pi、碱性磷酸酶ALP和抗酒石酸酸性磷酸酶TRACP含量,制造模型(简称造模)过程观察动物生活习性体重等变化,实验结束进行股骨骨形态计量学测量,骨薄片病理学观察。结果:造模过程中,大鼠出现少食,竖毛,及体重减轻。B.C组大鼠体重较正常组显著降低(P<0.05)。造模21d后,B组血清Ca2+水平显著升高。模型组骨密度较正常组显著下降(P<0.05),但D组骨密度变化无明显统计学意义。B,C,D组血清ALP,TRACP均明显高于正常组(P<0.05)。骨薄片染色显示模型组骨胶原排列杂乱无序,结构松散,D组有修复迹象。结论:羊水干细胞尾静脉注射可缓解维甲酸诱导的大鼠骨质疏松,且两次次给药的治疗效果好于单次给药。     

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.     

以人皮肤成纤维细胞作为饲养层细胞培养人胚胎干细胞

目的：比较人皮肤成纤维细胞（humandermalfibroblasts,HDFs）与小鼠胚胎成纤维细胞（Mouseembryonicfibroblasts,MEFs）的增殖能力及研究人皮肤成纤维细胞作为饲养层支持人胚胎干细胞（humanembryonicstemcells,hESCs）未分化生长的能力。方法：利用组织贴壁法从人皮肤中分离出HDFs,通过细胞形态的观察和生长曲线的绘制比较HDFs与MEFs的体外增殖能力。将HDFs作为饲养层细胞与hESCs共培养,传代12代后,检测hESCs碱性磷酸酶（AKP）、表面特异性标志及胚胎干细胞特异性转录因子。结果：HDFs可连续传代培养15代以上,10代以下的HDFs增殖迅速,而MEFs自第4代起,增殖能力就明显下降;hESCs在HDFs饲养层上可传代培养12代以上,克隆边界清晰,细胞排列紧密,碱性磷酸酶、表面标志物检测均呈阳性,表达了hESCs特异性转录因子。结论：HDFs比MEFs具有更强的增殖能力;HDFs可作为培养hEscs的饲养层细胞。     

Hypoxic remodelling of Ca2+ signalling in proliferating human arterial smooth muscle

Since Notch signaling plays a critical role in stem cells and oncogenesis, we hypothesized that Notch signaling might play roles in cancer stem cells and cancer cells with a stem cell phenotype. In this study, we accessed potential functions of the Notch pathway in the formation of cancer stem cells using human glioma. Using RT-PCR, we found that most human astrogliomas of different grades expressed moderate to high level of Notch receptors and ligands. mRNA of Hes5 but not Hes1, both of which are major downstream molecules of the Notch pathway, was also detected. In human glioma cell lines BT325, U251, SHG-44, and U87, mRNA encoding different types of Notch receptors were detected, but active form of Notch1 (NIC) was only detected in SHG-44 and U87 by Western blot. Interestingly, proliferation of these two glioma cell lines appeared faster than that of the other two lines in which NIC was not detected. We have over-expressed NIC of Notch1 in SHG-44 cells by constitutive transfection to evaluate the effects of Notch signaling on glioma cells. Our results showed that over-expression of NIC in SHG-44 cells promoted the growth and the colony-forming activity of SHG-44 cells. Interestingly, over-expression of NIC increased the formation neurosphere-like colonies in the presence of growth factors. These colonies expressed nestin, and could be induced to cells expressing neuron-, astrocyte-, or oligodendrocyte-specific markers, consistent with phenotypes of neural stem cells. These data suggest that Notch signaling promote the formation of cancer stem cell-like cells in human glioma. Xue-Ping Zhang, Gang Zheng and Lian Zou are contributed equally to this study.     

人脐带间充质干细胞作为维持人胚胎干细胞生长饲养层细胞的研究

目的寻找可以维持人胚胎干细胞未分化生长的人源性细胞作为饲养层细胞,从而解决使用鼠源性细胞作为饲养层带来的安全问题。方法尝试以人脐带间充质干细胞作为饲养层细胞来培养人胚胎干细胞,检验其是否可以维持人胚胎干细胞的未分化生长状态。用胶原酶消化法分离人脐带间充质干细胞,光镜下观察细胞形态;流式细胞仪检测其表面标志;诱导人脐带间充质干细胞向成骨细胞和脂肪细胞进行分化。将人胚胎干细胞系H1接种于丝裂霉素C灭活后的人脐带间充质干细胞上,每隔5d进行一次传代。培养20代后,对人胚胎干细胞特性进行相关检测,包括细胞形态、碱性磷酸酶染色、相关多能性基因的表达、分化能力。结果从人脐带中分离出的间充质干细胞为梭形,呈平行排列生长或漩涡状生长;细胞高表达CD44、CD29、CD73、CD105、CD90、CD86、CD147、CD117,不表达CD14、CD38、CD133、CD34、CD45、HLA-DR;具有分化成脂肪细胞和成骨细胞的潜能。人胚胎干细胞在人脐带间充质干细胞饲养层上培养20代后,继续保持人胚胎干细胞的典型形态,碱性磷酸酶染色为阳性,免疫荧光染色显示OCT4、Nanog、SSEA4、TRA-1-81、TRA-1-60的表达为阳性,SSEA1表达为阴性,体外悬浮培养可以形成拟胚体。结论人脐带间充质干细胞可以作为人胚胎干细胞的饲养层细胞,支持其生长,并维持其未分化生长状态。     

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.     

Comparison of feto-maternal organ derived stem cells in facets of immunophenotype,proliferation and differentiation

Scientific explorations on feto-maternal organ stem cells revealed its possible applicability in treatment of various diseases. However, establishment of an ideal placental tissue stem cell source in regenerative application is inconclusive and arduous. Hence, this study aims to resolve this tribulation by comparison of mesenchymal stem cells (MSC) from fetal placenta – amniotic membrane (AM-MSC), chorionic plate (CP-MSC) tissue and the maternal placenta-Decidua (D-MSC), thereby facilitating the researchers to determine their pertinent source. The cells were expanded and scrutinized for expression profiling, proliferation and differentiation ability. Remarkable expressions of certain markers in addition to its prospective mesodermal differentiation confirmed their mesenchyme origin. Despite the specified alikeness among these sources, reliable and non-invasive procurement of AM-MSC coupled with its higher growth potency makes it the most constructive stem cell source. However, exhibited similarities demands further investigations on extensive expandability and cytogenetic stability of these sources prior to its therapeutic applicability.     

Human pluripotent stem cells:Towards therapeutic development for the treatment of lifestyle diseases

There are two types of human pluripotent stem cells: Embryonic stem cells(ESCs) and induced pluripotent stem cells(iPSCs),both of which launched themselves on clinical trials after having taken measures to overcome problems: Blocking rejections by immunosuppressants regarding ESCs and minimizing the risk of tumorigenicity by depleting exogenous gene components regarding iP SCs.It is generally assumed that clinical applications of human pluripotent stem cells should be limited to those cases where there are no alternative measures for treatments because of the risk in transplanting those cells to living bodies.Regarding lifestyle diseases,we have already several therapeutic options,and thus,development of human pluripotent stem cell-based therapeutics tends to be avoided.Nevertheless,human pluripotent stem cells can contribute to the development of new therapeutics in this field.As we will show,there is a case where only a short-term presence of human pluripotent stem-derived cells can exert long-term therapeutic effects even after they are rejected.In those cases,immunologically rejections of ESC-or allogenic iP SC-derived cells may produce beneficial outcomes by nullifying the risk of tumorigenesis without deterioration of therapeutic effects.Another utility of human pluripotent stem cells is the provision of an innovative tool for drug discovery that are otherwise unavailable.For example,clinical specimens of human classical brown adipocytes(BAs),which has been attracting a great deal of attention as a new target of drug discovery for the treatment of metabolic disorders,are unobtainable from living individuals due to scarcity,fragility and ethical problems.However,BA can easily be produced from human pluripotent stem cells.In this review,we will contemplate potential contribution of human pluripotent stem cells to therapeutic development for lifestyle diseases.     

Stem cells for reprogramming: could hUMSCs be a better choice?

Human umbilical cord mesenchymal stem cells (hUMSC) are primitive multipotent cells capable of differentiating into cells of different lineages. They can be an alternative source of pluripotent cells since they are ethically and regulatory approved, are easily obtained and have low immunogenicity compared to embryonic stem cells which are dogged with numerous controversies. hUMSC can be a great source for cell and transplantation therapy.     

Mesenchymal cells from human amniotic fluid survive and migrate after transplantation into adult rat brain

Amniotic fluid has been recently suggested as an alternative source of mesenchymal stem cells. However, the fate of amniotic fluid-derived mesenchymal stem cells (AF-MSCs) after in vivo transplantation has yet to be determined. In the present study we explored whether human AF-MSCs could survive and migrate following transplantation into the striatum of normal and ischemic rat. We found that the grafted cells could survive and migrate towards multiple brain regions in the normal animals, while they moved towards the injured region in the ischemic rat. Double-immunostaining analyses showed that the implanted human AF-MSCs express markers for immature neurons (Doublecortin) at 10 days, and for astrocytes (GFAP) at 10, 30 and 90 after transplantation. This study provides the first evidence that human amniotic fluid contains cells having the potential to survive and integrate into adult rat brain tissue and, therefore, to function as effective stem cells for therapeutic strategies.     

Human adult pluripotency: Facts and questions

Cellular reprogramming and induced pluripotent stem cell(IPSC) technology demonstrated the plasticity of adult cell fate, opening a new era of cellular modelling and introducing a versatile therapeutic tool for regenerative medicine.While IPSCs are already involved in clinical trials for various regenerative purposes, critical questions concerning their medium-and long-term genetic and epigenetic stability still need to be answered. Pluripotent stem cells have been described in the last decades in various mammalian and human tissues(such as bone marrow, blood and adipose tissue). We briefly describe the characteristics of human-derived adult stem cells displaying in vitro and/or in vivo pluripotency while highlighting that the common denominators of their isolation or occurrence within tissue are represented by extreme cellular stress. Spontaneous cellular reprogramming as a survival mechanism favoured by senescence and cellular scarcity could represent an adaptative mechanism. Reprogrammed cells could initiate tissue regeneration or tumour formation dependent on the microenvironment characteristics. Systems biology approaches and lineage tracing within living tissues can be used to clarify the origin of adult pluripotent stem cells and their significance for regeneration and disease.     

胰岛类器官研究进展

类器官是将具有多向分化潜能的干细胞或组织细胞在特定环境下培养分化成为能够模拟原生器官结构和功能的三维结构.类器官在各种疾病模型研究及药物筛选中发挥至关重要的作用.近年来,通过体外诱导胰腺组织或多能干细胞分化形成具有胰岛细胞功能的胰岛类器官研究成为热点,为胰岛相关疾病模型、药物研究以及糖尿病的治疗提供了新的手段.本文针对...     

Search for nave human pluripotent stem cells

Normal mouse pluripotent stem cells were originally derived from the inner cell mass(ICM) of blastocysts and shown to be the in vitro equivalent of those pre-implantation embryonic cells, and thus were called embryonic stem cells(ESCs). More than a decade later, pluripotent cells were isolated from the ICM of human blastocysts. Despite being called human ESCs, these cells differ significantly from mouse ESCs, including different morphology and mechanisms of control of pluripotency, suggesting distinct embryonic origins of ESCs from the two species. Subsequently, mouse pluripotent stem cells were established from the ICMderived epiblast of post-implantation embryos. These mouse epiblast stem cells(Epi SCs) are morphological and epigenetically more similar to human ESCs. This raised the question of whether cells from the human ICM are in a more advanced differentiation stage than their murine counterpart, or whether the available culture conditions were not adequate to maintain those human cells in their in vivo state, leading to a transition into Epi SC-like cells in vitro. More recently, novel culture conditions allowed the conversion of human ESCs into mouse ESC-like cells called nave(or ground state) human ESCs, and the derivation of nave human ESCs from blastocysts. Here we will review the characteristics of each type of pluripotent stem cells, how(and whether) these relate to different stages of embryonic development, and discuss the potential implications of nave human ESCs in research and therapy.     

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。