Neuronal cell differentiation of mesenchymal stem cells originating from canine amniotic fluid

The amniotic fluid contains mesenchymal stem cells (MSCs) and can be readily available for tissue engineering. Regenerative treatments such as tissue engineering, cell therapy, and transplantation show potential in clinical trials of degenerative diseases. Disease presentation and clinical responses in the Canis familiaris not only are physiologically similar to human compared with other traditional mammalian models but is also a suitable model for human diseases. The aim of this study was to investigate whether canine amniotic-fluid-derived mesenchymal stem cells (cAF-MSCs) can differentiate into neural precursor cells in vitro when exposed to neural induction reagent. During neural differentiation, cAF-MSCs progressively acquire neuron-like morphology. Messenger RNA (mRNA) expression levels of neural-specific genes, such as NEFL, NSE, and TUBB3 (βIII-tubulin) dramatically increased in the differentiated cAF-MSCs after induction. In addition, protein expression levels of nestin, βIII-tubulin, and tyrosine hydroxylase remarkably increased in differentiated cAF-MSCs. This study demonstrates that cAF-MSCs have great potential for neural precursor differentiation in vitro. Therefore, amniotic fluid may be a suitable alternative source of stem cells and can be applied to cell therapy in neurodegenerative diseases.     

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]     

Multipotent mesenchymal stem cells from amniotic fluid originate neural precursors with functional voltage-gated sodium channels

Background aimsAmniotic fluid (AF) contains stem cells with high proliferative and differentiative potential that might be an attractive source of multipotent stem cells. We investigated whether human AF contains mesenchymal stem cells (MSC) and evaluated their phenotypic characteristics and differentiation potential in vitro.MethodsAF was harvested during routine pre-natal amniocentesis at 14–16 weeks of pregnancy. AF sample pellets were plated in α-minimum essential medium (MEM) with 10% fetal bovine serum (FBS). We evaluated cellular growth, immunophenotype, stemness markers and differentiative potential during in vitro expansion. Neural progenitor maintenance medium (NPMM), a medium normally used for the growth and maintenance of neural stem cells, containing hFGF, hEGF and NSF-1, was used for neural induction.ResultsTwenty-seven AF samples were collected and primary cells, obtained from samples containing more than 6 mL AF, had MSC characteristics. AF MSC showed high proliferative potential, were positive for CD90, CD105, CD29, CD44, CD73 and CD166, showed Oct-4 and Nanog molecular and protein expression, and differentiated into osteoblasts, adypocytes and chondrocytes. The NPMM-cultured cells expressed neural markers and increased Na⁺ channel density and channel inactivation rate, making the tetrodotoxin (TTX)-sensitive channels more kinetically similar to native neuronal voltage-gated Na⁺ channels.ConclusionsThese data suggest that AF is an important multipotent stem cell source with a high proliferative potential able to originate potential precursors of functional neurons.     

Mesenchymal stem cells can be obtained from the human saphena vein

Mesenchymal stem cells (MSC) can be isolated from many sites adults and the fetus. Cells with osteoblastic, chondrogenic, leiomiogenic and stromogenic potentials have been obtained from the bovine artery wall, and we now show that MSC can be isolated also from the adult human vein wall. Cells detached from internal surface of the saphenous vein are cultured in vitro for 2-3 weeks and replated weekly. The culture forms a semi-confluent layer of spindle-shaped cells that are CD13(+), CD29(+), CD44(+), CD34(-), CD45(-), CD14(-), CD133(-), CD31(-), CD33(-), CD54(+), CD106(-), CD90(+), KDR(-), cadherin-5-, HLA class I(+) and HLA-DR- and differentiate in vitro into osteoblasts, chondrocytes and adipocytes. Gene expression, when compared with seven other normal tissues, shows strong similarity with MSC obtained from other sources. Three genes more expressed in saphenous MSC than in the other two MSC are related to angiogenesis, and the expression of two of them is shared by endothelial cells. These results demonstrate that the human vein wall contains mesenchymal cells with morphologic features, immunophenotypic markers, gene expression profile and differentiation potential that are similar to MSC obtained from the bone marrow and from the umbilical vein.     

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.     

Neurogenic differentiation of amniotic fluid stem cells

In 2003, human amniotic fluid has been shown to contain stem cells expressing Oct-4, a marker for pluripotency. This finding initiated a rapidly growing and very promising new stem cell research field. Since then, amniotic fluid stem (AFS) cells have been demonstrated to harbour the potential to differentiate into any of the three germ layers and to form three-dimensional aggregates, so-called embryoid bodies, known as the principal step in the differentiation of pluripotent stem cells. Marker selection and minimal dilution approaches allow the establishment of monoclonal AFS cell lineages with high proliferation potential. AFS cells have a lower risk for tumour development and do not raise the ethical issues of embryonic stem cells. Compared to induced pluripotent stem cells, AFS cells do not need exogenic treatment to induce pluripotency, are chromosomal stable and do not harbour the epigenetic memory and accumulated somatic mutations of specific differentiated source cells. Compared to adult stem cells, AFS can be grown in larger quantities and show higher differentiation potential. Accordingly, in the recent past, AFS became increasingly accepted as an optimal tool for basic research and probably also for specific cell-based therapies. Here, we review the current knowledge on the neurogenic differentiation potential of AFS cells.     

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。     

Culture of human amniotic fluid stem cells in 3D collagen matrix

Most of the researchers attribute amniotic fluid stem cells (AF SCs) to mesenchymal stem cells (MSCs). However, AF SCs express both mesenchymal and epithelial markers, which distinguishes them from postnatal MSCs. Cultivation in the three-dimensional (3D) matrix provides a different look at the nature of the cells. We showed that in 3D collagen gel AF SCs form epithelial structures (tubules and cysts). The active contraction of the gel during the first days of cultivation, which is characteristic of mesenchymal cells, does not occur. Electron microscopic study showed that adherent junctions typical to epithelial cells are formed between AF SCs. On the other hand, during culturing in the gel AF SCs continue to express MSCs markers. Thus, AF SCs may be not true mesenchymal cells because they can display properties of epithelial cells. Perhaps these cells undergo epithelial-mesenchymal transition, a process which actively takes place during embryogenesis.     

Stem cells in human amniotic fluid

Amniotic fluid (AF) contains a heterogeneous population of cells of fetal origin in which stem cells are present. These cells are characterized by the expression of mesenchymal (CD73, CD90, CD105) and neural (Nestin, β3-tubulin, NEFH) markers, and also some markers of pluripotency (Oct4, Nanog), and they are capable of differentiating into diverse derivatives in vitro. We have shown that epithelial markers (Keratin 19, Keratin 18, and p63) are expressed in AF stem cells simultaneously with mesenchymal ones. During cloning, colonies of cells with fibroblastoid and epithelioid cells are formed. The status and differentiation potential of stem cells from AF have been discussed.     

Transplanted human amniotic membrane-derived mesenchymal stem cells ameliorate carbon tetrachloride-induced liver cirrhosis in mouse

Background

Human amniotic membrane-derived mesenchymal stem cells (hAMCs) have the potential to reduce heart and lung fibrosis, but whether could reduce liver fibrosis remains largely unknown.

Methodology/Principal Findings

Hepatic cirrhosis model was established by infusion of CCl₄ (1 ml/kg body weight twice a week for 8 weeks) in immunocompetent C57Bl/6J mice. hAMCs, isolated from term delivered placenta, were infused into the spleen at 4 weeks after mice were challenged with CCl₄. Control mice received only saline infusion. Animals were sacrificed at 4 weeks post-transplantation. Blood analysis was performed to evaluate alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Histological analysis of the livers for fibrosis, hepatic stellate cells activation, hepatocyte apoptosis, proliferation and senescence were performed. The donor cell engraftment was assessed using immunofluorescence and polymerase chain reaction. The areas of hepatic fibrosis were reduced (6.2%±2.1 vs. control 9.6%±1.7, p<0.05) and liver function parameters (ALT 539.6±545.1 U/dl, AST 589.7±342.8 U/dl,vs. control ALT 139.1±138.3 U/dl, p<0.05 and AST 212.3±110.7 U/dl, p<0.01) were markedly ameliorated in the hAMCs group compared to control group. The transplantation of hAMCs into liver-fibrotic mice suppressed activation of hepatic stellate cells, decreased hepatocyte apoptosis and promoted liver regeneration. More interesting, hepatocyte senescence was depressed significantly in hAMCs group compared to control group. Immunofluorescence and polymerase chain reaction revealed that hAMCs engraftment into host livers and expressed the hepatocyte-specific markers, human albumin and α-fetoproteinran.

Conclusions/Significance

The transplantation of hAMCs significantly decreased the fibrosis formation and progression of CCl₄-induced cirrhosis, providing a new approach for the treatment of fibrotic liver disease.     

Clonal amniotic fluid-derived stem cells express characteristics of both mesenchymal and neural stem cells

Recent evidence has shown that amniotic fluid may be a novel source of fetal stem cells for therapeutic transplantation. We previously developed a two-stage culture protocol to isolate a population of amniotic fluid-derived mesenchymal stem cells (AFMSCs) from second-trimester amniocentesis. AFMSCs maintain the capacity to differentiate into multiple mesenchymal lineages and neuron-like cells. It is unclear whether amniotic fluid contains heterogeneous populations of stem cells or a subpopulation of primitive stem cells that are similar to marrow stromal cells showing the behavior of neural progenitors. In this study, we showed a subpopulation of amniotic fluid-derived stem cells (AF-SCs) at the single-cell level by limiting dilution. We found that NANOG- and POU5F1 (also known as OCT4)-expressing cells still existed in the expanded single cell-derived AF-SCs. Aside from the common mesenchymal characteristics, these clonal AF-SCs also exhibit multiple phenotypes of neural-derived cells such as NES, TUBB3, NEFH, NEUNA60, GALC, and GFAP expressions both before and after neural induction. Most importantly, HPLC analysis showed the evidence of dopamine release in the extract of dopaminergic-induced clonal AF-SCs. The results of this study suggest that besides being an easily accessible and expandable source of fetal stem cells, amniotic fluid will provide a promising source of neural progenitor cells that may be used in future cellular therapies for neurodegenerative diseases and nervous system injuries.     

Growth factor combination for chondrogenic induction from human mesenchymal stem cell

During the last decade, many strategies for cartilage engineering have been emerging. Stem cell induction is one of the possible approaches for cartilage engineering. The mesenchymal stem cells (MSCs) with their pluripotency and availability have been demonstrated to be an attractive cell source. It needs the stimulation with cell growth factors to make the multipluripotent MSCs differentiate into chondrogenic lineage. We have shown particular patterns of in vitro chondrogenesis induction on human bone marrow MSCs (hBMSCs) by cycling the growth factors. The pellet cultures of hBMSCs were prepared for chondrogenic induction. Growth factors: TGF-beta3, BMP-6, and IGF-1 were used in combination for cell induction. Gene expression, histology, immunohistology, and real-time PCR methods were measured on days 21 after cell induction. As shown by histology and immunohistology, the induced cells have shown the feature of chondrocytes in their morphology and extracellular matrix in both inducing patterns of combination and cycling induction. Moreover, the real-time PCR assay has shown the expression of gene markers of chondrogenesis, collagen type II and aggrecan. This study has demonstrated that cartilage tissue can be created from bone marrow mesenchymal stem cells. Interestingly, the combined growth factors TGF-beta3 and BMP-6 or TGF-beta3 and IGF-1 were more effective for chondrogenesis induction as shown by the real-time PCR assay. The combination of these growth factors may be the important key for in vitro chondrogenesis induction.     

Multilineage potential research of Beijing duck amniotic mesenchymal stem cells

Amnion, which is usually discarded as medical waste, is considered as abundant sources for mesenchymal stem cells. In human and veterinary medicine, the multipotency of mesenchymal stem cells derived from amnion (AMSCs) together with their plasticity, self-renewal, low immunogenicity and nontumorigenicity characteristics make AMSCs a promising candidate cell for cell-based therapies and tissue engineering. However, up till now, the multipotential characteristics and therapeutic potential of AMSCs on preclinical studies remain uncertain. In this work, we successfully obtained AMSCs from Beijing duck embryos in vitro, and also attempted to detect their biological characteristics. The isolated AMSCs were phenotypically identified, the growth kinetics and karyotype were tested. Also, the cells were positive for MSCs-related markers (CD29, CD71, CD105, CD166, Vimentin and Fibronection), while the expression of CD34 and CD45 were undetectable. Additionally, AMSCs also expressed the pluripotent marker gene OCT4. Particularly, when appropriately induced, AMSCs could be induced to trans-differentiate into adipocytes, osteoblasts, chondrocytes and neurocytes in vitro. Together, these results demonstrated that the isolated AMSCs maintained their stemness and proliferation in vitro, which may be useful for future cell therapy in regenerative medicine.     

Adult human RPE can be activated into a multipotent stem cell that produces mesenchymal derivatives

The retinal pigment epithelium (RPE) is a monolayer of cells underlying and supporting the neural retina. It begins as a plastic tissue, capable, in some species, of generating lens and retina, but differentiates early in development and remains normally nonproliferative throughout life. Here we show that a subpopulation of adult human RPE cells can be activated in vitro to a self-renewing cell, the retinal pigment epithelial stem cell (RPESC) that loses RPE markers, proliferates extensively, and can redifferentiate into stable cobblestone RPE monolayers. Clonal studies demonstrate that RPESCs are multipotent and in defined conditions can generate both neural and mesenchymal progeny. This plasticity may explain human pathologies in which mesenchymal fates are seen in the eye, for example in proliferative vitroretinopathy (PVR) and phthisis bulbi. This study establishes the RPESC as an accessible, human CNS-derived multipotent stem cell, useful for the study of fate choice, replacement therapy, and disease modeling.     

One-step derivation of cardiomyocytes and mesenchymal stem cells from human pluripotent stem cells

Cardiomyocytes (CMs) and mesenchymal stem cells (MSCs) are important cell types for cardiac repair post myocardial infarction. Here we proved that both CMs and MSCs can be simultaneously generated from human induced pluripotent stem cells (hiPSCs) via a pro-mesoderm differentiation strategy. Two hiPSC lines, hiPSC (1) and hiPSC (2) were generated from human dermal fibroblasts using OCT-4, SOX-2, KLF-4, c-Myc via retroviral-based reprogramming. H9 human embryonic stem cells (hESCs) served as control. CMs and MSCs were co-generated from hiPSCs and hESCs via embryoid body-dependent cardiac differentiation protocol involving a serum-free and insulin-depleted medium containing a p38 MAPK inhibitor, SB 203580. Comparing to bone marrow and umbilical cord blood-derived MSCs, hiPSC-derived MSCs (iMSCs) expressed common MSC markers and were capable of adipogenesis, osteogenesis and chondrogenesis. Moreover, iMSCs continuously proliferated for more than 32 population doublings without cellular senescence and showed superior pro-angiogenic and wound healing properties. In summary, we generated a large number of homogenous MSCs in conjunction with CMs in a low-cost and efficient one step manner. Functionally competent CMs and MSCs co-generated from hiPSCs may be useful for autologous cardiac repair.     

Cell sourcing for bone tissue engineering: amniotic fluid stem cells have a delayed, robust differentiation compared to mesenchymal stem cells

Cell based therapies for bone regeneration are an exciting emerging technology, but the availability of osteogenic cells is limited and an ideal cell source has not been identified. Amniotic fluid-derived stem cells (AFS) and bone-marrow derived mesenchymal stem cells (MSCs) were compared to determine their osteogenic differentiation capacity in both 2D and 3D environments. In 2D culture, the AFS cells produced more mineralized matrix but delayed peaks in osteogenic markers. Cells were also cultured on 3D scaffolds constructed of poly-ε-caprolactone for 15 weeks. MSCs differentiated more quickly than AFS cells on 3D scaffolds, but mineralized matrix production slowed considerably after 5 weeks. In contrast, the rate of AFS cell mineralization continued to increase out to 15 weeks, at which time AFS constructs contained 5-fold more mineralized matrix than MSC constructs. Therefore, cell source should be taken into consideration when used for cell therapy, as the MSCs would be a good choice for immediate matrix production, but the AFS cells would continue robust mineralization for an extended period of time. This study demonstrates that stem cell source can dramatically influence the magnitude and rate of osteogenic differentiation in vitro.