Amniotic Membrane-derived Stem Cells Research Articles

Human amniotic membrane-derived stem cells down-regulation of proinflammatory cytokines in CDK of the experimental model

Chronic kidney disease (CKD) is viewed as quite possibly one of the most well-known renal problems that is treated with dialysis or renal transplant. Amniotic membrane stem cell (AMSC) injections may be able to improve kidney function, according to recent research. As a result, the current study looked into how intra-kidney injection of human amniotic membrane-derived stem cells (hAMCs) affected inflammatory cytokines like IL-10 and IL-17 in mice using the CKD model. Twenty male wild mice were arranged into four gatherings: control, CKD, culture medium infusion bunch, and hAMCs infusion bunch. Following two months, blood tests were taken from the creatures, and the articulation levels of interleukins 10 and 17 were estimated by the ELISA method. The outcomes showed that infusion of hAMCs into male mice with CKD caused down-guideline of IL-17 fiery cytokine and over-articulation of IL-10 calming cytokine. In light of the aftereffects of this review and past ones, we presumed that hAMCs could be viewed as one of the up-and-comers in later examinations on decreasing irritation in CKD treatment by changing a few provocative cytokines.

Explant culture and multilineage differentiation of amniotic membrane derived stem cells

Amniotic membrane derived stem cells (AMSCs) are reported to have a comparatively higher potency than multipotent stem cells. These cells are shown to have low immunogenicity and no teratogenicity. Among various conventional methods of isolation using enzymes, explant culture method is believed to be an easy and cost-effective way to harvest stem cells. The purpose of this study was to isolate AMSCs from amniotic membrane of rats and to characterize them for multilineage differentiation, including generation of neurospheres to use them later in in-vivo experiments. Amniotic membranes were collected from Wistar rats on 17th day of pregnancy. After processing of the tissues, AMSCs were isolated by the explant culture method and continued to grow until 10th passage. The doubling time was estimated and the cells were analyzed for growth curve parameters at passages 5 and 9. The osteogenic and adipogenic differentiation studies were carried out from the same cells after 3rd passage. Neurospheres generation from AMSCs was performed using neurogenic induction media. The cells were further assessed for their mesenchymal, haemopoietic, and neurogenic marker expressions by immunofluorescence staining and PCR analysis The study suggests that AMSCs isolated through explant culture are reliable stem cells which could generate neurospheres under proper induction conditions and could be a potential candidate to be used on in-vivo neural degeneration models.

Treatment of Chronic Spinal Cord Injury in Dogs Using Amniotic Membrane-Derived Stem Cells: Preliminary Results.

IntroductionIntervertebral disc diseases (IVDD) represent the majority of neurological attendance and responsible for the most cases of paralysis in dogs. Treatments currently used do not show satisfactory results in patients with more severe and chronic neurological manifestations.MethodsTo promote nerve and muscular recovery, as well as improve quality of life, we aimed to create a double-blind test method, associating spinal decompression surgery and allogeneic transplantation of amniotic membrane-derived stem cells (AMSCs) in dogs with chronic IVDD. Cells were characterized as fetal mesenchymal cells and safe for application. Eight animals completed the experiment: stem cell applications were made in four animals that had previously undergone an unsuccessful surgical procedure (“SC group”, n = 4); two animals were submitted to surgery, followed by applications of stem cells (“Surgery + SC”, n = 2); two other animals were submitted to surgery, followed by the application of saline solution (“Surgery + placebo”, n = 2). During the surgical procedure, a topical application was performed on the lesion and after fifteen and forty-five days another two applications were made via epidural. Animals were monitored biweekly and reassessed three months after surgery, by functional tests and magnetic resonance exams.ResultsSome animals presented significant neurological improvement, such as the recovery of nociception and ability to remain on station. Despite the need further studies, until the present moment, cell therapy has been feasible and has no harmful effects on animals.ConclusionThe protocol of preclinical trial showed the association with decompressive surgery and cell transplantation in dogs with thoracolumbar IVDD proved feasible, and it was possible to observe neurological improvement after treatment. No tissue improvement through MRI was found. The double-blind test guaranteed reliability of the evaluations and results obtained that, even with a small sample size, generated satisfactory results for the animals and owners.

Distribution of amniotic stem cells in human term amnion membrane

Amnion membrane studies related to miscarriage have been conducted in the field of obstetrics and gynecology. However, the distribution of stem cells within the amnion and the differences in the properties of each type of stem cells are still not well understood. We address this gap in knowledge in the present study where we morphologically classified the amnion membrane, and we clarified the distribution of stem cells here to identify functionally different amniotic membrane-derived stem cells. The amnion can be divided into a site that is continuous with the umbilical cord (region A), a site that adheres to the placenta (region B), and a site that is located opposite the placenta (region C). We found that human amnion epithelial stem cells (HAECs) that strongly express stem cell markers were abundant in area A. HAEC not only expressesed stem cell-specific surface markers TRA-1-60, Tra-1-81, SSEA4, SSEA3, but was also OCT-3/4 positive and had alkaline phosphatase activity. Human amniotic mesenchymal stem cells expressed KLF-A, OCTA, Oct3/4, c-MYC and Sox2 which is transcription factor. Especially, in regions A and B they have expressed CD73, and the higher expression of BCRP which is drug excretion transporter protein than the other parts. These data suggest that different types of stem cells may have existed in different area. The understanding the relation with characteristics of the stem cells in each area and function would allow for the efficient harvest of suitable HAE and HAM stem cells as using tool for regenerative medicine.

Chorionic and amniotic membrane-derived stem cells have distinct, and gestational diabetes mellitus independent, proliferative, differentiation, and immunomodulatory capacities.

Placental membrane-derived mesenchymal stem cells (MSCs), with the advantages of being non-invasive and having fewer ethical issues, are a promising source for cell therapy. Gestational diabetes (GDM) alters the uterine environment and may affect the therapeutic potential of MSCs derived from placenta. Therefore, we evaluated the biological properties of amniotic (AMSCs) and chorionic membrane MSCs (CMSCs) from human GDM placenta in order to explore their therapeutic potential. In comparison of GDM-/Healthy- CMSCs and AMSCs, the immunophenotypes and typical stellate morphology of MSC were similar in CMSCs irrespective of disease state while the MSC morphology in GDM-AMSCs was less evident. GDM- and Healthy- CMSCs displayed an enhanced proliferation rate and tri-lineage differentiation capacity compared with AMSCs. Notably, GDM-CMSCs had a significantly increased adipogenic ability than Healthy-CMSCs accompanied by increased transcriptional responsiveness of PPARγ and ADIPOQ induction. The secretome effect of Healthy- and GDM- CMSCs/AMSCs by using conditioned media and coculture experiments, suggests that GDM- and Healthy- CMSCs provided an equivalent immunoregulatory effect on suppressing T-cells activation but a reduced effect of GDM-CMSCs on macrophage regulation. However, Healthy- and GDM- CMSCs displayed a superior immunomodulatory capacity in regulation of both T-cells and macrophages than AMSCs. In summary, we highlight the importance of the maternal GDM intrauterine environment during pregnancy and its impact on CMSCs/AMSCs proliferation ability, CMSCs adipogenic potential, and macrophage regulatory capacity.

Silk Sponges Ornamented with a Placenta-Derived Extracellular Matrix Augment Full-Thickness Cutaneous Wound Healing by Stimulating Neovascularization and Cellular Migration.

Regeneration of full-thickness wounds without scar formation is a multifaceted process, which depends on in situ dynamic interactions between the tissue-engineered skin substitutes and a newly formed reparative tissue. However, the majority of the tissue-engineered skin substitutes used so far in full-thickness wound healing cannot mimic the natural extracellular matrix (ECM) complexity and thus are incapable of providing a suitable niche for endogenous tissue repair. Herein, we demonstrated a simple approach to fabricate porous hybrid ECM sponges (HEMS) using a placental ECM and silk fibroin for full-thickness wound healing. HEMS with retained cytokines/growth factors provided a noncytotoxic environment in vitro for human foreskin fibroblasts (HFFs), human epidermal keratinocytes (HEKs), and human amniotic membrane-derived stem cells to adhere, infiltrate, and proliferate. Interestingly, HEMS-conditioned media accelerated the migration of HFFs and HEKs owing to the presence of cytokines/growth factors. Also, the ex vivo chick chorioallantoic membrane assay of HEMS demonstrated its excellent vascularization potential by inducing and supporting blood vessels. Additionally, HEMS when subcutaneously implanted demonstrated no severe immune response to the host. Furthermore, HEMS implanted in full-thickness wounds in a rat model showed augmented healing progression with well-organized epidermal-dermal junctions via pronounced angiogenesis, accelerated migration of HFFs/HEKs, enhanced granulation tissue formation, and early re-epithelialization. Taken together, these findings show that porous HEMS ornamented with cytokines/growth factors having superior physicomechanical properties may be an appropriate skin substitute for full-thickness cutaneous wounds.

Oleoyl-Chitosan-Based Nanofiber Mats Impregnated with Amniotic Membrane Derived Stem Cells for Accelerated Full-Thickness Excisional Wound Healing.

Wound healing management is a major challenge for critical full-thickness skin wounds. Development of nanofibrous scaffolds with tunable wettability, degradation, and biocompatibility are highly desirable. Herein, we demonstrated synthesis of oleoyl chitosan (OC) by grafting monounsaturated fatty acid residue, C18 oleoyl chain, to the backbone of chitosan molecule and blending with gelatin to form the nanofiber mats. The physicochemical properties of the nanofiber mats revealed mechanical strength, moderate surface wettability, and suitable degradation rate. The nanofibrous mats showed excellent in vitro cytocompatibility with human amniotic membrane-derived stem cells (HAMSCs) in terms of enhanced adhesion and proliferation owing to biomimetic nanoarchitecture and chemical cues. Furthermore, the fabricated nanofiber was implanted with and without preseeded HAMSCs in the full-thickness wound to evaluate the skin wound healing efficacy in a rat model. Histological and immunohistochemical studies were conducted to evaluate the plausible changes of tissue architecture and expression of molecular markers involved in wound healing process. Both acellular and HAMSCs incorporated cellular nanofibers promoted wound contraction remarkably with superior skin tissue regeneration in terms of enhanced collagen synthesis, re-epithelialization and initiation of epithelial cells stratification compared to control group.

Amniotic membrane-derived stem cells help repair osteochondral defect in a weight-bearing area in rabbits.

Our study evaluated the use of amniotic membrane-derived stem cells for repairing osteochondral defects in a weight-bearing area in rabbits. Twenty-four 3-month-old male or female New Zealand white rabbits were selected. The rabbits were randomly divided into 3 groups of eight, according to the treatment received for an experimentally inflicted femoral medial malleolus lesion, group I received a human acellular amniotic membrane seeded with bone marrow-derived mesenchymal stem cells (HAAM-BMSCs) implant; group II received a simple HAAM implant and the control group received no experimental lesion or treatment. The rabbits were sacrificed at 12 and 24 weeks after the procedures (4 rabbits in each time-point) and the cartilage repair status in each animal was evaluated under the microscope. The tissue of the HAAM-BMSCs group grew well covering an area in the visual field that was significantly larger than that of the HAAM group (p<0.05). The percentage of collagen II-positive area in the HAAM-BMSC group was significantly higher than that in HAAM group (p<0.05). The number of chondrocytes determined by toluidine blue staining was higher in the HAAM-BMSC group than that in the HAAM group (p<0.05). The Wakitani scores of the HAAM and HAAM-BMSC groups were significantly higher (worse) than those of the normal control group (p<0.05), but the score in the HAAM-BMSC group was significantly lower than that in the HAAM group (p<0.05). The Wakitani scores in the HAAM-BMSC group were not different between the two time-points taken. Based on our findings, the amniotic membrane-derived stem cells had a good therapeutic effect in repairing the osteochondral defects in the weight-bearing area, and the number of chondrocytes in the injured area was increased significantly, which accelerated the repair of the damaged tissue in rabbits.

Fetal Membranes-Derived Stem Cells Microenvironment.

Recently, the regenerative medicine has been trying to congregate different areas such as tissue engineering and cellular therapy, in order to offer effective treatments to overcome several human and veterinary medical problems. In this regard, fetal membranes have been proposed as a powerful source for obtainment of multipotent stem cells with low immunogenicity, anti-inflammatory properties and nontumorigenicity properties for the treatment of several diseases, including replacing cells lost due to tissue injuries or degenerative diseases. Morpho-physiological data have shown that fetal membranes, especially the yolk sac and amnion play different functions according to the gestational period, which are direct related to the features of the microenvironment that their cells are subject. The characteristics of the microenvironment affect or controls important cellular events involved with proliferation, division and maintenance of the undifferentiated stage or differentiation, especially acting on the extracellular matrix components. Considering the importance of the microenvironment and the diversity of embryonic and fetal membrane-derived stem cells, this chapter will addressed advances in the isolation, phenotyping, characteristics of the microenvironment, and applications of yolk sac and amniotic membrane-derived stem cells for human and veterinary regenerative medicine.

Characterization of teratogenic potential and gene expression in canine and feline amniotic membrane‐derived stem cells

The biosafety of innovative procedures that utilize stem cells in regenerative medicine has been addressed in several studies. Previous work has showed no tumour formation following the use of feline and human amniotic membrane-derived stem cells (AMSCs). In contrast, tumour formation was observed when canine AMSCs were utilized. These findings suggested that feline and human, but not canine, AMSCs are suitable for cell transplantation trials. This study aimed to further evaluate the feasibility of utilizing canine AMSCs for transplantation purposes as well as for felines. We tested teratoma formation following cell injection into BALB/c nude mice and then assessed expression of haematopoietic, mesenchymal, tumorigenic, pluripotency and cellular regulation markers using flow cytometry and qPCR. The use of canine AMSCs did not result in macroscopic tumour formation as determined 60days after transplantation. The immunophenotypic characterization by flow cytometry revealed expression of mesenchymal markers (CD73 and CD90) and expression of the pluripotent marker OCT4 and SOX2. Quantitative PCR analysis revealed that there were no differences in the patterns of gene expression (CD34, CD73, OCT4, CD30 and P53) between canine and feline AMSCs, with the exception of the expression of SOX2 and CD90.

Investigating the potential of human placenta-derived extracellular matrix sponges coupled with amniotic membrane-derived stem cells for osteochondral tissue engineering.

Osteochondral injuries are challenging to repair due to their complex tissue anatomy and restricted self-repairing ability associated with a limited blood supply. Osteochondral tissue engineering is an important clinical aspect of the management and treatment of cartilage and underlying bone. In the present study, we fabricated human placenta-derived extracellular matrix sponges (PEMS) for repair of osteochondral tissue through a decellularization process. There were no significant cellular components present in the PEMS; hematoxylin & eosin/DAPI staining, DNA quantification and agarose gel electrophoresis were used to evaluate the extent of decellularization. Moreover, no significant alteration to the collagen and glycosaminoglycan (native extracellular matrix) content of the PEMS was observed. PEMS in vitro provided a non-cytotoxic environment rich in bioactive cues for human amniotic membrane-derived stem cells (HAMSCs) to proliferate in and differentiate into chondrogenic and osteogenic lineages under induction. Histological analysis at 28 days after the PEMS were subcutaneously implanted demonstrated no severe immune response in the host and supported the formation of blood vessels. To assess the osteochondral tissue repair ability of PEMS, cell-free PEMS (CFP) and cell-seeded PEMS (CSP) were implanted at osteochondral defect sites in a rabbit model. Histological scores indicated that osteochondral regeneration was more successful in the defects filled with CSP compared to those filled with CFP and empty defects (ED) after 60 days of implantation. In summary, a naturally derived biocompatible scaffold composed of extracellular matrix from human placenta has been successfully developed for osteochondral tissue engineering.

Therapeutic efficacy of amniotic membrane stem cells and adipose tissue stem cells in rats with chemically induced ovarian failure

The present study was conducted to compare between the therapeutic efficacies of human amniotic membrane-derived stem cells (hAM-MSCs) vs. adipose tissue derived stem cells (AD-MSCs) in cyclophosphamide (CTX)-induced ovarian failure in rats. Forty-eight adult female rats were included in the study; 10 rats were used as control group. Thirty-eight rats were injected with CTX to induce ovarian failure and divided into four groups: ovarian failure (IOF) (IOF group), IOF+phosphate buffer saline (PBS group), IOF+hAM-MSCs group and IOF+AD-MSCs group. Serum levels of FSH and estradiol (E2) were assessed. Histopathological examination of the ovarian tissues was performed and quantitative gene expressions of Oct-4, Stra8 and integrin beta-1 genes were conducted by quantitative real time PCR. Results showed that IOF and IOF+PBS rat groups exhibited decreased ovarian follicles, increased interstitial fibrosis with significant decrease of serum E2, significant increase serum FSH level and significant down-regulation of Stra8 and integrin beta-1. In hAM-MSCs and AD-MSCs rat groups, there were increased follicles and corpora with evident the presence of oocytes, significant increase in serum E2, significant decrease in serum FSH levels (in hAM-MSCs treated group only) and significant up-regulation of the three studied genes with higher levels in hAM-MSCs treated rats group when compared to AD-MSCs treated rats group. In Conclusion, administration of either hAM-derived MSCs or AD-MSCs exerts a significant therapeutic efficacy in chemotherapy induced ovarian insult in rats. hAM-MSCs exert higher therapeutic efficacy as compared to AD-MSCs.

Neuronal differentiation of mouse amnion membrane derived stem cells in response to neonatal brain conditioned medium.

Background: Amniotic membrane derived stem cells (AMSCs) have considerable advantages to use in regenerative medicine and their anti- inflammatory effects, growth factor secretion and differentiation potential make them suitable candidates for stem cell therapy of nervous system. The developing and neonatal brain contains a spectrum of growth factors to direct development of endogenous and donor cells. Using an in vitro model system, we investigated the plasticity and potential of mouse AMSCs to differentiate into neural cells in response to neonatal mice brain extracted medium. Methods: Mouse amniotic membrane stem cells were isolated from embryos, cultured in presence of medium derived from neonatal mouse brain medium and immunohistochemistry and flow cytometry analyses were used to explore the neural differentiation of them. Results: Isolated amnion membrane stem cells showed high rate of viability and proliferation and presented neural characters in the presence of neonatal brain extracted medium such morphological changes and Nestin and Map-2 expression. Conclusion: In conclusion, results from this study showed that amnion membrane derives stem cells are potent stem cells to respond to environmental signals promoting them to neural fate.

Amniotic membrane-derived stem cells: immunomodulatory properties and potential clinical application.

Epithelial and mesenchymal cells isolated from the amniotic membrane (AM) possess stem cell characteristics, differentiation potential toward lineages of different germ layers, and immunomodulatory properties. While their expansion and differentiation potential have been well studied and characterized, knowledge about their immunomodulatory properties and the mechanisms involved is still incomplete. These mechanisms have been evaluated on various target cells of the innate and the adaptive system and in animal models of different inflammatory diseases. Some results have evidenced that the immunomodulatory effect of AM-derived cells is dependent on cell-cell contact, but many of them have demonstrated that these properties are mediated through the secretion of suppressive molecules. In this review, we present an update on the described immunomodulatory properties of the derived amniotic cells and some of the proposed involved mechanisms. Furthermore, we describe some assays in animal models of different inflammatory diseases which reveal the potential use of these cells to treat such diseases.

Differentiation into Hair Cells from AM‐Derived Stem Cells

Objective: Placenta, umbilical cord, and blood are alternative sources of stem cells. We designed an experiment to isolate and culture stem cells from human amniotic membrane. And we made a plan to induce auditory hair cells and neurons from the stem cells of amniotic membrane. Method: Human amniotic membrane was obtained.) We dissociated amniotic membrane with trypsin and transferred it to culture medium which contained 10ï¼… fetal bovine serum and 1ï¼… penicillin-streptomycin. We sub cultured it until the third generation was used for further experimental procedures. For differentiation into neural progenitor cells, we cultured amniotic membrane stem cells in EGF (epidermal growth factor) and β-FGF (fibrous growth factor) and for differentiation into hair cells and neurons, we cultured in neurobasal medium. Results: We can isolate and culture stem cells from human amniotic membrane. By flow cytometric analysis of stem cells from human amniotic membrane, they showed positive responses at mesenchymal stem cell marker (CD 73, CD90, CD105) and negative responses at hematopoietic stem cell marker (CD34, CD14, CD19, HLA). The results of culture at different culture medium showed a differentiation to neural progenitor cells, hair cells, and neuron, and we confirmed by immunofluorescence staining and RT-PCR. Conclusion: We demonstrated that amniotic membrane–derived stem cells could be differentiated into neuronal progenitor cells, and this could lead to the differentiation into hair cells and neurons.

Amniotic membrane-derived stem cells and pancreatic islet-cell differentiation

Amniotic membrane-derived stem cells and pancreatic islet-cell differentiation

Ex Vivo Characteristics of Human Amniotic Membrane-Derived Stem Cells

Cells were isolated from four human amniotic membranes, and their biological characteristics analyzed during ex vivo expansion. Morphologically homogenous populations of fibroblast-like cells were obtained from the second or third passage. Under the appropriate culture conditions, these human amniotic membrane-derived mesenchymal cells (HAM) were shown to differentiate into adipocytes, osteocytes, chondrocytes and neuronal cells, as visualized by Oil Red O, von Kossa, alcian blue, anti-Neu N, and anti-Gal C antibody staining, respectively. Immunophenotype analysis of HAM cells revealed the presence of antigens for SSEA-3, SSEA-4, collagen type-I, -II, -III, -IV, -XII, fibronectin, alpha-SMA, vimentin, desmin, cytokeratin18 (CK18), HCAM-1, fibroblast surface protein, and human leukocyte antigen (HLA) ABC. ICAM-1 protein was weakly detectable, and proteins of TRA-1-60, VCAM-1, von Willebrand factor, PECAM-1, and HLA DR were not detected. HAM cells reached senescence after 14.5+/-0.9 passages, over a period of 146.8+/-8.9 days, and underwent an average of 36.9 4.7 population doublings. RT-PCR analysis showed that all four HAM cell lines consistently expressed genes of Oct-4, Rex-1, SCF, NCAM, nestin, BMP-4, GATA-4, HNF-4alpha, vimentin, and CK18, regardless of the passage number. The genes of Brachyury, FGF-5, Pax-6, and BMP2 were never expressed. Strikingly, alpha-fetoprotein (alphaFP), HLA ABC, and HLA DR genes were expressed in an earlier passage but not expressed in later passages. Telomerase activity of two HAM lines was discernable upon the third passage. These observations strongly suggest that HAM might be immune-privileged and, thus, advantageous as therapeutic cells.