Human amniotic membrane as an alternative source of stem cells for regenerative medicine

The human amniotic membrane (HAM) is a highly abundant and readily available tissue. This amniotic tissue has considerable advantageous characteristics to be considered as an attractive material in the field of regenerative medicine. It has low immunogenicity, anti-inflammatory properties and their cells can be isolated without the sacrifice of human embryos. Since it is discarded post-partum it may be useful for regenerative medicine and cell therapy. Amniotic membranes have already been used extensively as biologic dressings in ophthalmic, abdominal and plastic surgery. HAM contains two cell types, from different embryological origins, which display some characteristic properties of stem cells. Human amnion epithelial cells (hAECs) are derived from the embryonic ectoderm, while human amnion mesenchymal stromal cells (hAMSCs) are derived from the embryonic mesoderm. Both populations have similar immunophenotype and multipotential for in vitro differentiation into the major mesodermal lineages, however they differ in cell yield. Therefore, HAM has been proposed as a good candidate to be used in cell therapy or regenerative medicine to treat damaged or diseased tissues.     

A new technique to expand human mesenchymal stem cells using basement membrane extracellular matrix

Mesenchymal stem cells (MSC) show a very short proliferative life span and readily lose the differentiation potential in culture. However, the growth rate and the proliferative life span of the stem cells markedly increased using tissue culture dishes coated with a basement membrane-like extracellular matrix, which was produced by PYS-2 cells or primary endothelial cells. Furthermore, the stem cells expanded on the extracellular matrix, but not those on plastic tissue culture dishes, retained the osteogenic, chondrogenic, and adipogenic potential throughout many mitotic divisions. The extracellular matrix had greater effects on the proliferation of MSC and the maintenance of the multi-lineage differentiation potential than basic fibroblast growth factor. Mesenchymal stem cells expanded on the extracellular matrix should be useful for regeneration of large tissue defects and repeated cell therapies, which require a large number of stem or progenitor cells.     

The immunobiological development of human bone marrow mesenchymal stem cells in the course of neuronal differentiation

The central nervous system (CNS) has been referred to as the "immunological privileged site". However, it is now clear that the privileged status of the CNS is a result of a balance between immune privilege and effective response. In vitro, human bone marrow mesenchymal stem cells (MSCs) have the ability to differentiate into neurons. Based on this biological attribute we gain the possibility by means of using MSCs as the donors to develop a future cell therapy in clinical application. But using MSCs as donor cells inevitably raises the question as to whether these donor cells would be immunogenic, and if so, would they be rejected after transplantation. To investigate this, human MSCs were cultured in vitro and induced to differentiate along neuronal lineage. The expression of human leukocyte antigen (HLA) class I and class II molecules and the co-stimulatory protein CD80 were increased on the surface of MSCs in the course of neuronal differentiation. But neither of the co-stimulatory proteins, CD40 or CD86, was expressed. After IFN-gamma exposure, the expression of the HLA molecules was further enhanced, but the co-stimulatory proteins were unaffected. MSCs that had been differentiated along neuronal lineage were not capable of inducing the proliferation of peripheral blood lymphocytes (PBLs). Even after IFN-gamma exposure, PBLs remained unresponsive. Furthermore, MSCs differentiated along neuronal lineage suppressed the proliferation of PBLs induced by allogeneic PBLs and mitogens. The mechanisms involved in the immunosuppression may be related to the effect of soluble factors and cell-cell interactions of neuronal differentiated MSCs and PBLs. From the above data we suggested that the low immunogenicity and immunomodulatory function of MSCs in the course of neuronal differentiation in vitro, which will be helpful to further investigation in order to establish the new way for future medical application.     

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。     

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.     

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.     

Functional studies of mesenchymal stem cells derived from adult human adipose tissue

Recent evidence suggests that cells with the properties of human mesenchymal stem cells (hMSCs) can be derived from adult peripheral tissues, including adipose tissue, muscle and dermis. We isolated hMSCs from the stromal-vascular portion of subcutaneous adipose tissue from seven adult subjects. These cells could be readily differentiated into cells of the chondrocyte, osteocyte and adipocyte lineage demonstrating their multipotency. We studied the functional properties of hMSCs-derived adipocytes and compared them with adipocytes differentiated from hMSCs obtained from bone marrow (BM-hMSC). The two cell types displayed similar lipolytic capacity upon stimulation with catecholamines, including a pronounced antilipolytic effect mediated through alpha2A-adrenoceptors, a typical trait in human but not rodent fat cells. Furthermore, both cell types secreted the fat cell-specific factors leptin and adiponectin in comparable amounts per time unit. The fat tissue-derived hMSCs retained their differentiation capacity up to at least fifteen passages. We conclude that hMSCs derived from adult human adipose tissue can be differentiated into fully functional adipocytes with a similar, if not identical, phenotype as that observed in cells derived from BM-hMSCs. Human adipose-tissue-derived MSCs could therefore constitute an efficient and easily obtainable renewable cellular source for studies of adipocyte biology.     

间充质干细胞在肝脏疾病应用的研究现况

间充质干细胞（MSC）属于成体干细胞的一种,是一类具有自我更新和多向分化能力的多能干细胞。其来源丰富,免疫原性低,目前体内外实验均发现MSC可促进损伤肝脏修复,改善症状,提高存活率。通过调节肝脏局部和全身炎症反应和免疫紊乱发挥治疗作用。本文就MSC治疗肝脏疾病的研究现况进行综述。     

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.     

Microenvironment at tissue injury, a key focus for efficient stem cell therapy: A discussion of mesenchymal stem cells

Stem cell therapy is not a new field, as indicated by the success of hematopoietic stem cell reconstitution for various hematological malignancies and immune-mediated disorders. In the case of tissue repair, the major issue is whether stem cells should be implanted, regardless of the type and degree of injury. Mesenchymal stem cells have thus far shown evidence of safety, based on numerous clinical trials, particularly for immune-mediated disorders. The premise behind these trials is to regulate the stimulatory immune responses negatively. To apply stem cells for other disorders, such as acute injuries caused by insults from surgical trauma and myocardial infarction, would require other scientific considerations. This does not imply that such injuries are not accompanied by immune responses. Indeed, acute injuries could accompany infiltration of immune cells to the sites of injuries. The implantation of stem cells within a milieu of inflammation will establish an immediate crosstalk among the stem cells, microenvironmental molecules, and resident and infiltrating immune cells. The responses at the microenvironment of tissue injury could affect distant and nearby organs. This editorial argues that the microenvironment of any tissue injury is a key consideration for effective stem cell therapy.     

Schwann cell mediated trophic effects by differentiated mesenchymal stem cells

Mesenchymal stem cells were isolated from the bone marrow of rats and differentiated to provide a functional substitute for slow growing Schwann cells for peripheral nerve regeneration. To assess the properties of the differentiated mesenchymal stem cell, the cells were co-cultured with dorsal root ganglia and the secretion of the neurotrophic factors and the neurite outgrowth was evaluated. The neurite outgrowth of the dorsal root ganglia neurons was enhanced in co-culture with the differentiated stem cells compared to the undifferentiated stem cells. Differentiated stem cells like Schwann cells were responsible for the stimulation of longer and branched neurites. Using enzyme-linked immunosorbant assays and blocking antibodies, we have shown that this effect is due to the release of brain derived neurotrophic factor and nerve growth factor, which were up-regulated in differentiated mesenchymal stem cells following co-culture. The relevance of the tyrosine kinase receptors was confirmed by the selective tyrosine kinase inhibitor, K252a which abolished the neurite outgrowth of the dorsal root ganglia neurons when co-cultured with the differentiated mesenchymal stem cells similar to Schwann cells. The results of the study further support the notion that mesenchymal stem cells can be differentiated and display trophic influences as those of Schwann cells.     

Osteogenic potential of rat mesenchymal stem cells after several passages

Osteogenic potential of serially passaged rat bone marrow derived mesenchymal stem cells (BMCs) was evaluated for clinical feasibility. Osteogenic differentiation in vitro was evaluated by means of the concentration and mRNA expression of alkaline phosphatase and osteocalcin. For in vivo osteogenesis, BMCs in various degrees of differentiation were implanted into the athymic mice. Although elevated levels of osteogenic markers were prominent in the less passaged BMCs continuously cultured with osteogenic supplements (OS group), they decreased with passaging. Similar to the in vitro experiments, abundant bone and cartilage formations inside the membrane were observed in the P0 through P2 cells of the OS group. In the P3 cells, however, the chambers were filled with fibrous tissues showing the failure of osteogenesis. Establishment of the culture conditions that permit the rapid expansion of BMCs while retaining their potential for differentiation will be required for future clinical applications.     

小鼠骨片间充质干细胞分离培养及鉴定

目的建立小鼠骨片间充质干细胞（MSC）分离培养及扩增的方法。方法取小鼠胫骨和股骨,洗去骨髓后,用胶原酶I消化疏松骨密质,利用MSC具有迁徙和贴壁生长的能力进行分离。并对获取的细胞进行流式鉴定和诱导分化。结果培养2d小鼠骨片边缘爬出成纤维样细胞,呈克隆和鱼群样生长,并可以进行持续传代培养。流式鉴定结果显示这群细胞表达MSC标志Scall（92．7％）,CD29（98．4％）,CD90（91．6％）,不表达造血细胞标志CD34（1．57％）,CD45（3．99％）,CD11b（0．63％）,并可成功诱导分化成骨细胞和脂肪细胞。结论成功建立从小鼠骨片中获得MSC的方法,为实验研究提供可靠的细晌实源．     

Isolation, characterization, and differentiation of stem cells derived from the rat amniotic membrane

Abstract Stem-cell-based therapies may offer treatments for a variety of intractable diseases. A fundamental goal in stem-cell biology concerns the characterization of diverse populations that exhibit different potentials, growth capabilities, and therapeutic utilities. We report the characterization of a stem-cell population isolated from tissue explants of rat amniotic membrane. Similar to mesenchymal stem cells, these amnion-derived stem cells (ADSCs) express the surface markers CD29 and CD90, but were negative for the lymphohematopoietic markers CD45 and CD11b. ADSCs exist in culture in a multidifferentiated state, expressing neuroectodermal (neurofilament-M), mesodermal (fibronectin), and endodermal (α-1-antitrypsin) genes. To assess plasticity, ADSCs were subjected to a number of culture conditions intended to encourage differentiation into neuroectodermal, mesodermal, and endodermal cell types. ADSCs cultured in a defined neural induction media assumed neuronal morphologies and up-regulated neural-specific genes. Under different conditions, ADSCs were capable of differentiating into presumptive bone and fat cells, indicated by the deposition of mineralized matrix and accumulated lipid droplets, respectively. Moreover, ADSCs cultured in media that promotes liver cell differentiation up-regulated liver-specific genes (albumin) and internalized low-density lipoprotein (LDL), consistent with a hepatocyte phenotype. To determine whether this observed plasticity reflects the presence of true stem cells within the population, we have derived individual clones from single cells. Clonal lines recapitulate the expression pattern of parental ADSC cultures and are multipotent. ADSCs have been cultured for 20 passages without losing their plasticity, suggesting long-term self-renewal. In sum, our data suggest that ADSCs and derived clonal lines are capable of long-term self-renewal and multidifferentiation, fulfilling all the criteria of a stem-cell population.     

Isolation and characterisation of mesenchymal stem cells from adult mouse bone marrow

The future use of adult mesenchymal stem cells (MSCs) for human therapies depends on the establishment of preclinical studies with other mammals such as mouse. Surprisingly, purification and characterisation of murine MSCs were only poorly documented. The aim of this study was to purify mouse MSCs from adult bone marrow and to functionally characterise their abilities to differentiate along diverse lineages. Adherent cells from adult C57Bl/6J mouse bone marrow were depleted of granulo-monocytic cells and subsequently allowed to grow on fibronectin-coated dishes in presence of fetal bovine serum and growth factors. The growing fibroblastoid cell population primarily consisted of spindle- and star-shaped cells with significant renewal capacity as they were cultured until 30 passages (about 60 doubling population). We fully demonstrated the MSC phenotype of these cells by inducing them to differentiate along osteoblastic, adipocytic, and chondrocytic pathways. Mouse MSCs (mMSCs) sharing the same morphological and functional characteristics as human MSCs can be successfully isolated from adult bone marrow without previous mouse or bone marrow treatment. Therefore, mMSCs will be an important tool to study the in vivo behaviour and fate of this cell type after grafting in mouse pathology models.     

Overexpressed ATP7B protects mesenchymal stem cells from toxic copper

Wilson’s disease (WD) is characterized by accumulation of high levels of copper in liver due to malfunction of copper transporter ATP7B which is central for copper homeostasis. Here we report for the first time that mesenchymal stem cells (MSC) derived from bone marrow express detectable levels of ATP7B. The role of ATP7B overexpression for MSC survival and selection in high copper was investigated. Hepatoma cell line HepG2 that has a high intrinsic expression of ATP7B served as a control. Using retroviral vector a significant higher expression level of ATP7B could be achieved in MSCs. Whereas copper treatment resulted in cell death in untransduced MSCs, viability assays demonstrated a unique copper resistance of ATP7B overexpressing MSCs that outcompeted HepG2. In long-term cell culture stable transgene expression for up to 9 weeks was shown for ATP7B overexpressing MSCs which rapidly overgrew untransduced cells. Our findings suggest that ATP7B overexpression provides an important selection advantage to MSCs in high copper microenvironments, and may represent novel cell transplants for therapy of WD.     

Effects of human amniotic membrane grafts combined with marrow mesenchymal stem cells on healing of full-thickness skin defects in rabbits

We have investigated the wound-healing effects of mesenchymal stem cells (MSCs) in combination with human amniotic membrane (HAM) when grafted into full-thickness skin defects of rabbits. Five defects in each of four groups were respectively treated with HAM loaded with autologous MSCs (group A), HAM loaded with allologous MSCs (group B), HAM with injected autologous MSCs (group C), and HAM with injected allologous MSCs (group D). The size of the wounds was calculated for each group at 7, 12, and 15 days after grafting. The wounds were subsequently harvested at 25 days after grafting. Sections stained with hematoxylin and eosin were used to determine the quality of wound healing, as based on the characteristics and amount of granulated tissue in the epidermal and dermal layers. Groups A and B showed the most pronounced effect on wound closure, with statistically significant improvement in wound healing being seen on post-operative days 7, 12, and 15. Although a slight trend toward improved wound healing was seen in group A compared with group B, no statistically significant difference was found at any time point between the two groups. Histological examination of healed wounds from groups A and B showed a thin epidermis with mature differentiation and collagen bundle deposition plus recovered skin appendages in the dermal layer. In contrast, groups C and D showed thickened epidermis with immature epithelial cells and increased fibroblast proliferation with only partially recovered skin appendages in the dermal layer. Thus, the graft of HAM loaded with MSCs played an effective role during the healing of skin defects in rabbits, with no significant difference being observed in wound healing between autologous and allologous MSC transplantation. This study was supported by research funds from Dong-A University.     

Characterization of porcine arterial endothelial cells cultured on amniotic membrane, a potential matrix for vascular tissue engineering

The existing of basement membrane improves the development of endothelium while constructing blood vessel equivalent. The amniotic membrane (AM) provides a natural basement membrane and has been used in ocular surface reconstruction. This study evaluated the molecular and cellular characteristics of porcine vascular endothelial cells (ECs) cultured on AM. ECs cultured on AM expressed the endothelial marker vWF and exhibited normal endothelial morphology. Here, we demonstrated that AM enhanced the expression of intercellular molecules, platelet-endothelial cell adhesion molecule-1 (PECAM-1), and adhesion molecule VE-cadherin at the intercellular junctions. The expression level of integrin was markedly higher in ECs cultured on AM than on plastic dish. Furthermore, the AM downregulated the expression of E-selectin and P-selectin in both LPS-activated and non-activated ECs. Consistently, adhesion of leukocytes to both activated and non-activated cells was decreased in ECs cultured on AM. Our results suggest that AM is an ideal matrix to develop a functional endothelium in blood vessel equivalent construction.     

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.