The potential of mesenchymal stem cells derived from amniotic membrane and amniotic fluid for neuronal regenerative therapy

The mesenchymal stem cells (MSCs), which are derived from the mesoderm, are considered as a readily available source for tissue engineering. They have multipotent differentiation capacity and can be differentiated into various cell types. Many studies have demonstrated that the MSCs identified from amniotic membrane (AM-MSCs) and amniotic fluid (AF-MSCs) are shows advantages for many reasons, including the possibility of noninvasive isolation, multipotency, self-renewal, low immunogenicity, anti-inflammatory and nontumorigenicity properties, and minimal ethical problem. The AF-MSCs and AM-MSCs may be appropriate sources of mesenchymal stem cells for regenerative medicine, as an alternative to embryonic stem cells (ESCs). Recently, regenerative treatments such as tissue engineering and cell transplantation have shown potential in clinical applications for degenerative diseases. Therefore, amnion and MSCs derived from amnion can be applied to cell therapy in neuro-degeneration diseases. In this review, we will describe the potential of AM-MSCs and AF-MSCs, with particular focus on cures for neuronal degenerative diseases. [BMB Reports 2014; 47(3): 135-140]     

人羊膜来源成体干细胞的多向分化潜能

干细胞治疗被认为是一种非常有潜力的治疗手段,其中成体干细胞由于不存在伦理问题,更为广大学者所青睐。本研究成功从人羊膜间质细胞中分离纯化出具有自我更新能力和多向分化潜能的成体干细胞。首先从羊膜间质细胞中通过极限稀释法进一步分离得到羊膜来源成体干细胞(Amnion-derived stemcells,ADSC),分析其形态、生长方式及主要的免疫表型,并在体外分别将其向脂肪、成骨、内皮、肝细胞及神经细胞诱导分化。结果发现,ADSC在适宜条件下能够向3个胚层的细胞分化,经连续传代30次,其形态及表型稳定,并仍保持多向分化潜能。证实了ADSC的干细胞特性,可能为细胞治疗及干细胞工程提供种子细胞的新来源。     

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。     

Fetal adnexa derived stem cells from domestic animal: progress and perspectives

The fetal adnexa such as umbilical cord, amnion and amniotic fluid have been proposed as ideal sources of different stem cell lineages. Use of adnexal tissue has many potential advantages, including the noninvasive nature of the isolation procedure, the large tissue mass from which cells can be harvested with high efficiency and the potential of these cells to differentiate. Moreover, particularly in human medicine, the harvesting of these tissues is more ethically acceptable making these sources of stem cells very attractive for regenerative therapies and biotechnological applications. The adnexal tissue cells preserve some of the characteristics of the primitive embryonic layers from which they originate. Indeed, many studies indicate that these stem cells exhibit some features of embryonic stem cells as expression of embryonic markers and proliferation capability, without showing immunogenicity. However, the differentiation potential of these cells, either in vivo or in vitro, is intermediate between the pluripotent embryonic stem cells and the multipotent adult stem cells. Non-embryonic extra-fetal derived stem cells have opened new perspectives for developmental biology and for regenerative medicine, not only in humans but also in animals. In this update, we report the state of the art of fetal adnexa-derived stem cells from domestic animals and analyze their applications and potential uses in veterinary medicine.     

Induced in vitro differentiation of pancreatic-like cells from human amnion-derived fibroblast-like cells

There is growing evidence that the human amnion contains various types of stem cells. As amniotic tissue is readily available, it has the potential to be an important source of material for regenerative medicine. In the present study, we evaluated the potential of human amnion-derived fibroblast-like (HADFIL) cells to differentiate into pancreatic islet cells. Two HADFIL cell populations, derived from two different neonates, were analyzed. The expression of pancreatic cell-specific genes was examined before and after in vitro induction of cellular differentiation. We found that Pdx-1 , Isl-1 , Pax-4 , and Pax-6 showed significantly increased expression following the induction of differentiation. In addition, immunostaining demonstrated that insulin, glucagon, and somatostatin were present in HADFIL cells following the induction of differentiation. These results indicate that HADFIL cell populations have the potential to differentiate into pancreatic islet cells. Although further studies are necessary to determine whether such in vitro -differentiated cells can function in vivo as pancreatic islet cells, these amniotic cell populations might be of value in therapeutic applications that require human pancreatic islet cells.     

Induced in vitro differentiation of neural-like cells from human amnion-derived fibroblast-like cells

There is growing evidence that the human amnion contains various types of stem cell. As amniotic tissue is readily available, it has the potential to be an important source of material for regenerative medicine. In this study, we evaluated the potential of human amnion-derived fibroblast-like (HADFIL) cells to differentiate into neural cells. Two HADFIL cell populations, derived from two different neonates, were analyzed. The expression of neural cell-specific genes was examined before and after in vitro induction of cellular differentiation. We found that neuron specific enolase, neurofilament-medium, beta-tubulin isotype III, and glial fibrillary acidic protein (GFAP) showed significantly increased expression following the induction of differentiation. In addition, immunostaining demonstrated that neuron specific enolase, GFAP and myelin basic protein (MBP) were present in HADFIL cells following the induction of differentiation, although one of the HADFIL cell populations showed a lower expression of GFAP and MBP. These results indicate that HADFIL cell populations have the potential to differentiate into neural cells. Although further studies are necessary to determine whether such in vitro-differentiated cells can function in vivo as neural cells, these amniotic cell populations might be of value in therapeutic applications that require human neural cells.     

Tissue engineering for neurodegenerative diseases using human amniotic membrane and umbilical cord

Regenerative medicine, based on the use of stem cells, scaffolds and growth factors, has the potential to be a good approach for restoring damaged tissues of the central nervous system. This study investigated the use of human amniotic mesenchymal stem cells (hAMSC), human amniotic epithelial stem cells (hAESC), and human Wharton’s jelly mesenchymal stem cells (hWJMSC) derived from human umbilical cord as a source of stem cells, and the potential of the human amniotic membrane (HAM) as a scaffold and/or source of growth factors to promote nerve regeneration. The hAMSC and hAESC obtained from HAM and the hWJMSC from umbilical cords were cultured in induction medium to obtain neural-like cells. The morphological differentiation of hAMSC, hAESC and hWJMSC into neural-like cells was evident after 4–5 days, when they acquired an elongated and multipolar shape, and at 21 days, when they expressed neural and glial markers. On other way, the HAM was completely decellularized without affecting the components of the basement membrane or the matrix. Subsequently, hAMSC, hAESC and hWJMSC differentiated into neural-like cells were seeded onto the decellularized HAM, maintaining their morphology. Finally, conditioned media from the HAM allowed proliferation of hAMSC, hAESC and hWJMSC differentiated to neural-like cells. Both HAM and umbilical cord are biomaterials with great potential for use in regenerative medicine for the treatment of neurodegenerative diseases.     

Potential of embryonic stem cells

Embryonic stem (ES) cells are pluripotent cell lines established from undifferentiated embryonic cells characterized by nearly unlimited self-renewal and differentiation capacity. During differentiation in vitro, ES cells were found to be able to develop into specialized somatic cells types and to recapitulate processes of early embryonic development. These properties allow to use ES cells as model system for studying early embryonic development by gain- or loss-of-function approaches, or to investigate the effects of drugs and environmental factors on differentiation and cell function in embryotoxicity and pharmacology. Now, ES cells derived of human blastocysts may be used for the generation of somatic precursor or differentiated cells in cell and tissue therapy. The review presents data of mouse ES cell differentiation and gives an outlook on future perspectives and problems of using human ES cells in regenerative medicine.     

Progenitor Cells for Regenerative Medicine and Consequences of ART and Cloning-Associated Epimutations

The “holy grail” of regenerative medicine is the identification of an undifferentiated progenitor cell that is pluripotent, patient specific, and ethically unambiguous. Such a progenitor cell must also be able to differentiate into functional, transplantable tissue, while avoiding the risks of immune rejection. With reports detailing aberrant genomic imprinting associated with assisted reproductive technologies (ART) and reproductive cloning, the idea that human embryonic stem cells (hESCs) derived from surplus in vitro fertilized embryos or nuclear transfer ESCs (ntESCs) harvested from cloned embryos may harbor dangerous epigenetic errors has gained attention. Various progenitor cell sources have been proposed for human therapy, from hESCs to ntESCs, and from adult stem cells to induced pluripotent stem cells (iPS and piPS cells). This review highlights the advantages and disadvantages of each of these technologies, with particular emphasis on epigenetic stability.     

Comparative proteomic analysis of human mesenchymal and embryonic stem cells: Towards the definition of a mesenchymal stem cell proteomic signature

Mesenchymal stem cells (MSC) are adult multipotential progenitors which have a high potential in regenerative medicine. They can be isolated from different tissues throughout the body and their homogeneity in terms of phenotype and differentiation capacities is a real concern. To address this issue, we conducted a 2‐DE gel analysis of mesenchymal stem cells isolated from bone marrow (BM), adipose tissue, synovial membrane and umbilical vein wall. We confirmed that BM and adipose tissue derived cells were very similar, which argue for their interchangeable use for cell therapy. We also compared human mesenchymal to embryonic stem cells and showed that umbilical vein wall stem cells, a neo‐natal cell type, were closer to BM cells than to embryonic stem cells. Based on these proteomic data, we could propose a panel of proteins which were the basis for the definition of a mesenchymal stem cell proteomic signature.     

Characterization of laminin isoforms in human amnion

Epithelial cells of the human amnion have been reported to possess similar functions to many types of cells, such as hepatocytes, neurons, and pancreatic beta-cells. We reported previously that one of the hepatocyte-like functions of human amniotic epithelial cells was reinforced by the presence of basement membrane components. Laminin is one of the main components of the basement membrane; it critically contributes to cell differentiation. Laminin has several heterotrimer isoforms composed of an alpha-, a beta-, and a gamma-chain, and each type of chain has several types of subunit chains: alpha1-5, beta1-3, and gamma1-3. In this study, we characterized the laminin subunit chains in human amnion. Laminin is produced and secreted from adjacent epithelial cells, and therefore, the gene expression of laminin subunit chains in human amniotic epithelial cells was investigated by RT-PCR. Their localization was examined by immunohistochemical staining of frozen sections. The findings suggested that the basement membrane of the human amnion contains a broad spectrum of laminin isoforms, laminin-2, -4, -5, -6, -7, -10, -11. These findings will provide clues not only for understanding the physiological roles of the amnion and hAECs, but also for applying this tissue as a source of donor cells for cell transplantation therapy.     

Supportive properties of basement membrane layer of human amniotic membrane enable development of tissue engineering applications

Human amniotic membrane (HAM) has been widely used as a natural scaffold in tissue engineering due to many of its unique biological properties such as providing growth factors, cytokines and tissue inhibitors of metalloproteinases. This study aimed at finding the most suitable and supportive layer of HAM as a delivery system for autologous or allogeneic cell transplantation. Three different layers of HAM were examined including basement membrane, epithelial and stromal layers. In order to prepare the basement membrane, de-epithelialization was performed using 0.5 M NaOH and its efficiency was investigated by histological stainings, DNA quantification, biomechanical testing and electron microscopy. Adipose-derived stromal cells (ASCs) and a human immortalized keratinocyte cell line (HaCaT) were seeded on the three different layers of HAM and cultured for 3 weeks. The potential of the three different layers of HAM to support the attachment and viability of cells were then monitored by histology, electron microscopy and (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Moreover, mechanical strengths of the basement membrane were assessed before and after cell culture. The results indicated that the integrity of extra cellular matrix (ECM) components was preserved after de-epithelialization and resulted in producing an intact basement amniotic membrane (BAM). Moreover, all three layers of HAM could support the attachment and proliferation of cells with no visible cytotoxic effects. However, the growth and viability of both cell types on the BAM were significantly higher than the other two layers. We conclude that growth stimulating effectors of BAM and its increased mechanical strength after culturing of ASCs, besides lack of immunogenicity make it an ideal model for delivering allogeneic cells and tissue engineering applications.     

Isolation and Characterization of Canine Amniotic Membrane-Derived Multipotent Stem Cells

Recent studies have shown that amniotic membrane tissue is a rich source of stem cells in humans. In clinical applications, the amniotic membrane tissue had therapeutic effects on wound healing and corneal surface reconstruction. Here, we successfully isolated and identified multipotent stem cells (MSCs) from canine amniotic membrane tissue. We cultured the canine amniotic membrane-derived multipotent stem cells (cAM-MSCs) in low glucose DMEM medium. cAM-MSCs have a fibroblast-like shape and adhere to tissue culture plastic. We characterized the immunophenotype of cAM-MSCs by flow cytometry and measured cell proliferation by the cumulative population doubling level (CPDL). We performed differentiation studies for the detection of trilineage multipotent ability, under the appropriate culture conditions. Taken together, our results show that cAM-MSCs could be a rich source of stem cells in dogs. Furthermore, cAM-MSCs may be useful as a cell therapy application for veterinary regenerative medicine.     

Establishment and characterization of a pluripotent stem cell line derived from human amniotic membranes and initiation of germ layers in vitro

OBJECTIVES: Pluripotent stem cells are proposed to be used in regenerative therapy and may exist in the human amniotic membrane. The present article is aimed at establishing a pluripotent stem cell line from human placenta. METHODS: HAM-1 (stem cell line derived from human amniotic membranes) was established by the colonial cloning technique using aMEM culture medium containing 10 ng/ml of EGF, 10 ng/ml of hLIF and 10% fetal bovine serum. RESULTS: HAM-1 cells appeared to maintain a normal karyotype indefinitely in vitro and expressed markers characteristic of stem cells from mice and human, namely alkaline phosphatase. Also, these cells contributed to the formation of chimeric mouse embryoid bodies and gave rise to cells of all germ layers in vitro. CONCLUSIONS: This study demonstrates that human amniotic membranes derived stem cells have a wide developmental capability and might be utilized to regenerate different types of cells or tissues for transplantation therapy.     

Calcium channels and their role in regenerative medicine

Stem cells hold indefinite self-renewable capability that can be differentiated into all desired cell types.Based on their plasticity potential,they are divided into totipotent(morula stage cells),pluripotent(embryonic stem cells),multipotent(hematopoietic stem cells,multipotent adult progenitor stem cells,and mesenchymal stem cells[MSCs]),and unipotent(progenitor cells that differentiate into a single lineage)cells.Though bone marrow is the primary source of multipotent stem cells in adults,other tissues such as adipose tissues,placenta,amniotic fluid,umbilical cord blood,periodontal ligament,and dental pulp also harbor stem cells that can be used for regenerative therapy.In addition,induced pluripotent stem cells also exhibit fundamental properties of self-renewal and differentiation into specialized cells,and thus could be another source for regenerative medicine.Several diseases including neurodegenerative diseases,cardiovascular diseases,autoimmune diseases,virus infection(also coronavirus disease 2019)have limited success with conventional medicine,and stem cell transplantation is assumed to be the best therapy to treat these disorders.Importantly,MSCs,are by far the best for regenerative medicine due to their limited immune modulation and adequate tissue repair.Moreover,MSCs have the potential to migrate towards the damaged area,which is regulated by various factors and signaling processes.Recent studies have shown that extracellular calcium(Ca²⁺)promotes the proliferation of MSCs,and thus can assist in transplantation therapy.Ca²⁺signaling is a highly adaptable intracellular signal that contains several components such as cell-surface receptors,Ca²⁺channels/pumps/exchangers,Ca²⁺buffers,and Ca²⁺sensors,which together are essential for the appropriate functioning of stem cells and thus modulate their proliferative and regenerative capacity,which will be discussed in this review.     

Paracrine Effect of Mesenchymal Stem Cells Derived from Human Adipose Tissue in Bone Regeneration

Mesenchymal stem cell (MSC) transplantation has proved to be a promising strategy in cell therapy and regenerative medicine. Although their mechanism of action is not completely clear, it has been suggested that their therapeutic activity may be mediated by a paracrine effect. The main goal of this study was to evaluate by radiographic, morphometric and histological analysis the ability of mesenchymal stem cells derived from human adipose tissue (Ad-MSC) and their conditioned medium (CM), to repair surgical bone lesions using an in vivo model (rabbit mandibles). The results demonstrated that both, Ad-MSC and CM, induce bone regeneration in surgically created lesions in rabbit''s jaws, suggesting that Ad-MSC improve the process of bone regeneration mainly by releasing paracrine factors. The evidence of the paracrine effect of MSC on bone regeneration has a major impact on regenerative medicine, and the use of their CM can address some issues and difficulties related to cell transplants. In particular, CM can be easily stored and transported, and is easier to handle by medical personnel during clinical procedures.     

Mesenchymal stromal cells from umbilical cord blood

Mesenchymal Stromal Cells (MSC) are key candidates for cellular therapies. Although most therapeutic applications have focused on adult bone marrow derived MSC, increasing evidence suggests that MSC are present within a wide range of tissues. Umbilical cord blood (CB) has been proven to be a valuable source of hematopoietic stem cells, but its therapeutic potential extends beyond the hematopoietic component suggesting regenerative potential in solid organs as well. There is evidence that other stem or progenitor populations, such as MSC, exist in CB which might be responsible for these effects. Many different stem and progenitor cell populations have been postulated with potential ranging from embryonic like to lineage-committed progenitor cells. Based on the confusing data, this review focuses on a human CB derived, plastic adherent fibroblastoid population expressing similar characteristics to bone marrow derived MSC. It concentrates especially on concepts of isolation and expansion, comparing the phenotype with bone marrow derived MSC, describing the differentiation capacity and finally in the last the therapeutic potential with regard to regenerative medicine, stromal support, immune modulation and gene therapy.