Amniotic Fluid-derived Mesenchymal Stem Cells Research Articles

Tissue-specific populations from amniotic fluid-derived mesenchymal stem cells manifest variant in vitro and in vivo properties.

Amniotic fluid derived mesenchymal stem cells (AFMSCs), shed along the fetal development, exhibit superior multipotency and immunomodulatory properties compared to MSCs derived from other somatic tissues (e.g., bone marrow and fat). However, AFMSCs display heterogeneity due to source ambiguity, making them an underutilized stem cells source for translational clinical trials. Consequently, there is an urgent need to identify a method to purify the AFMSCs for clinical use. We found that the AFMSCs can be categorized into three distinct groups: kidney-specific AFMSCs (AFMSCs-K), lung-specific AFMSCs (AFMSCs-L), and AFMSCs with an undefined tissue source (AFMSCs-X). This classification was based on tissue-specific gene expression pattern of single cell colony. Additionally, we observed that AFMSCs-X, a minority population within the AFMSCs, exhibited the highest multipotency, proliferation, resistance to senescence and immuno-modulation. Our results showed that AFMSCs-X significantly improved survival rates and reduced bacterial colony forming units (CFU) in cecal ligation and puncture (CLP)-induced septic mice. Therefore, our study introduces a novel classification method to enhance the consistency and efficacy of AFMSCs. These subpopulations, originating from different tissue source, may offer a valuable and innovative resource of cells for regenerative medicine purposes.

Conditioned medium from amniotic fluid mesenchymal stem cells could modulate Alzheimer's disease-like changes in human neuroblastoma cell line SY-SY5Y in a paracrine manner

BackgroundAlzheimer's disease is usually diagnosed by significant extracellular deposition of beta-amyloid and intracellular neurofibrillary tangle formation. Here, we investigated the paracrine effect of amniotic fluid-derived mesenchymal stem cells on AD changes in human SH–SY5Y cells. MethodsSH–SY5Y cells were divided into five groups: Control, 0.1 µg/ml LPS, 10 µg/ml LPS, 0.1 µg/ml LPS + conditioned medium, and 10 µg/ml LPS + conditioned medium. Cells were incubated with 0.1% and 10 µg/ml LPS for 48 h, followed by incubation with the conditioned medium of amniotic fluid-derived mesenchymal stem cells for the next 24 h. Beta-amyloid plaques were monitored by Congo-red staining. Survival and apoptosis were assessed by the MTT assay and flow cytometric analysis of Annexin-V. ELISA was used to measure the levels of neprilysin, angiotensin-converting enzyme, and Matrix Metalloproteinase–9. A PCR array was used to measure the expression of genes involved in neurogenesis. ResultsBright-field imaging showed beta-amyloid plaques in the group treated with 10 µg/ml LPS. We found minimal effects in groups receiving 0.1 µg/ml LPS. The data showed that the reduction in the levels of neprilysin, angiotensin-converting enzyme, and Matrix Metalloproteinase–9 in the LPS-treated cells was attenuated after incubation with the stem cell secretome (p < 0.05). Amniotic fluid stem cell secretome increased the viability of LPS-treated SH–SY5Y cells (p 0.05) and was associated with a decrease in apoptotic changes (p < 0.05). We found the modulation of several genes involved in neurogenesis in the 10 µg/ml LPS + conditioned medium group compared to cells treated with 10 µg/ml LPS alone. ConclusionAmniotic fluid stem cell secretion reduces AD-like pathologies in the human neuronal lineage.

Mesenchymal Stromal Cell Therapy Regenerates Rejected Donor Lungs and Reduces Primary Graft Dysfunction Following Lung Transplantation

<h3>Purpose</h3> Given critical shortages of donor lungs coupled with donor utilization rates hovering at a mere 20%, novel therapies are needed to increase the number of organs available for transplantation. Treatments which could regenerate lungs rejected for transplant should be explored to combat waiting list mortality and post-operative complications like primary graft dysfunction (PGD). Term amniotic fluid-derived mesenchymal stromal cells (TAF-MSCs) administered during ex vivo lung perfusion (EVLP) and post transplantation were studied under the hypothesis that the therapy could improve damaged lungs and reduce the incidence of PGD. <h3>Methods</h3> Donor pigs were treated with <i>E. coli-</i>derived lipopolysaccharide to induce acute lung injury as confirmed via blood gas values and histology. Harvested lungs were placed on EVLP for 4 hours, and the left lungs were transplanted. After recipients were monitored for three days, a right pneumectomy was performed and the transplanted left lung alone was evaluated for PGD. The treatment group received intravascular doses of TAF-MSCs during EVLP and at 2 timepoints following transplantation. Half of the lungs were maintained as a non-treated group which underwent the same EVLP and transplantation protocol. <h3>Results</h3> Cell counts of leukocytes were comparable in plasma at the induction of ARDS and in the perfusate during EVLP, however, lymphocytes were significantly decreased in the treated group relative to the non-treated during the first 24 hours following transplantation. Blinded histological scoring by a pathologist demonstrated that the treated lungs were significantly less injured than the non-treated ones and significantly less than biopsies taken at the time of confirmed ARDS. Furthermore, assessment of lung functionality through the PaO₂/FiO₂ ratio showed treated recipients had ratios significantly increased compared to transplanted non-treated recipients and to all lungs at the time of confirmed ARDS. All in the treated group were primary graft dysfunction (PGD) grade 0 on the third day of follow-up, while the non-treated group consisted of one grade 2 and the rest had PGD grade 3. <h3>Conclusion</h3> Delivery of intravascular TAF-MSCs at both EVLP and early during the post-transplantation period enabled recovery of lungs with acute lung injury and decreased the incidence of primary graft dysfunction.

Transamniotic Stem Cell Therapy for Experimental Congenital Diaphragmatic Hernia: Structural, Transcriptional, and Cell Kinetics Analyses in the Nitrofen Model

Purpose: We examined select pulmonary effects and donor cell kinetics after transamniotic stem cell therapy (TRASCET) in a model of congenital diaphragmatic hernia (CDH). Methods: Pregnant dams (n = 58) received nitrofen on gestational day 9.5 (E9) to induce fetal CDH. Fetuses (n = 681) were divided into 4 groups: untreated (n = 99) and 3 groups receiving volume-matched intra-amniotic injections on E17 of either saline (n = 142), luciferase-labeled amniotic fluid-derived mesenchymal stem cells (afMSCs; n = 299), or acellular recombinant luciferase (n = 141). Pulmonary morphometry, quantitative gene expression of pulmonary vascular tone mediators, or screening for labeled afMSCs were performed at term (E22). Statistical comparisons were by Mann-Whitney U-test, nested ANOVA, and Wald test. Results: TRASCET led to significant downregulation of endothelial nitric oxide synthase and endothelin receptor-A expressions compared to both untreated and saline groups (both p < 0.001). TRASCET also led to a significant decrease in arteriole wall thickness compared to the untreated group (p < 0.001) but not the saline group (p = 0.180). Donor afMSCs were identified in the bone marrow and umbilical cord (p = 0.035 and 0.015, respectively, vs. plain luciferase controls). Conclusions: The effects of TRASCET in experimental CDH appear to be centered on the pulmonary vasculature and to derive from circulating donor cells.

Human Amniotic Fluid Stem Cells: General Characteristics and Potential Therapeutic Applications

Introduction: Amniotic fluid contains a mixture of different cell types sloughed from the fetal skin, respiratory, alimentary and urogenital tracts, as well as the amnion membrane. As amniotic fluid develops prior to the process of gastrulation, many cells found in its heterogeneous population do not undergo lineage specialization. Therefore, amniotic fluid-derived mesenchymal stem cells (AF-MSCs) may correspond to a new class of stem cells with properties of intermediate plasticity between pluripotent and adult stem cell types. Compared to mesenchymal stem cells (MSCs) from other sources, such as bone marrow, AF-MSCs have better properties for clinical applications, such as differentiation into the cells of three germ layers, high clonal capacity, ability to form embryoid bodies, expression of pluripotent markers, high self-renewal capacity (over 250 population doublings) with normal karyotype at late passages, long telomere length due to continued telomerase activity, specially non-tumorigenicity, low immunogenicity, anti-inflammatory and immunomodulatory properties.&#x0D; Conclusion: Such features have nominated AF-MSC for a range of clinical applications, including in regenerative medicine. In several studies, these cells have been used to regenerate nerve, lung, and heart tissues. Overall, AF-MSCs are expected to be an ideal source of stem cells for future regenerative medicine and tissue engineering.

Enhancement of transamniotic stem cell therapy for spina bifida by genetic engineering of donor mesenchymal stem cells with an Fgf2 transgene

Background/PurposeWe examined whether engineered overexpression of fibroblast growth factor-2 (Fgf2) in donor mesenchymal stem cells (MSCs) could enhance spina bifida coverage induced by transamniotic stem cell therapy (TRASCET). MethodsPregnant Sprague–Dawley dams (n = 24) exposed to retinoic acid for induction of fetal spina bifida were divided in three groups. An untreated group had no further manipulations. Two groups received volume-matched intra-amniotic injections into all fetuses (n = 157) of either amniotic fluid-derived MSCs (afMSC; n = 85) or afMSCs transduced with an Fgf2 transgene (Fgf2-afMSC; n = 72) on gestational day 17 (term=21–22 days). Defect coverage was categorized at term by histology and pan-cytokeratin immunohistochemistry. Statistical coverage comparisons were by logistic regression. ResultsAmong 84 survivors with isolated spina bifida, 71 had definitive histology. Defect coverage rates in both the afMSC (38.5%) and Fgf2-afMSC (73.3%) groups were statistically significantly higher than in the untreated group (10%; p<0.001 for both). There was a significantly higher coverage rate in the Fgf2-afMSC group compared with the afMSC group (p = 0.025). ConclusionsFgf2 overexpression in donor mesenchymal stem cells increases defect coverage rates in a rodent model of transamniotic stem cell therapy for spina bifida. Genetic engineering of donor cells is a promising strategy for the enhancement of this emerging therapy.

The Effect of Angiotensin II, Retinoic Acid, EGCG, and Vitamin C on the Cardiomyogenic Differentiation Induction of Human Amniotic Fluid-Derived Mesenchymal Stem Cells.

Human amniotic fluid-derived mesenchymal stem cells (AF-MSCs) may be potentially applied in cell therapy or regenerative medicine as a new alternative source of stem cells. They could be particularly valuable in restoring cardiac tissue after myocardial infarction or other cardiovascular diseases. We investigated the potential of biologically active compounds, namely, angiotensin II, retinoic acid (RA), epigallocatechin-3-gallate (EGCG), vitamin C alone, and the combinations of RA, EGCG, and vitamin C with angiotensin II to induce cardiomyogenic differentiation of AF-MSCs. We observed that the upregulated expression of cardiac gene markers (NKX2-5, MYH6, TNNT2, and DES) and cardiac ion channel genes (sodium, calcium, the potassium) as well as the increased levels of Connexin 43 and Nkx2.5 proteins. Extracellular flux analysis, applied for the first time on AF-MSCs induced with biologically active compounds, revealed the switch in AF-MSCs energetic phenotype and enhanced utilization of oxidative phosphorylation for energy production. Moreover, we demonstrated changes in epigenetic marks associated with transcriptionally active (H3K4me3, H3K9ac, and H4hyperAc) or repressed (H3K27me3) chromatin. All in all, we demonstrated that explored biomolecules were able to induce alterations in AF-MSCs at the phenotypic, genetic, protein, metabolic, and epigenetic levels, leading to the formation of cardiomyocyte progenitors that may become functional heart cells in vitro or in vivo.

Initial Mechanistic Screening of Transamniotic Stem Cell Therapy in the Rodent Model of Spina Bifida: Host Bone Marrow and Paracrine Activity

Purpose: Transamniotic stem cell therapy (TRASCET) with mesenchymal stem cells (MSCs) can induce spina bifida coverage with neoskin. We initiated a mechanistic analysis of this host response. Methods: Pregnant dams (n = 28) exposed to retinoic acid to induce fetal spina bifida were divided into an untreated group and 2 groups receiving intra-amniotic injections on gestational day 17 (E17; term = E21–22) of either amniotic fluid-derived MSCs (afMSCs; n = 105) or saline (n = 107). Gene expressions of multiple paracrine and cell clonality markers were quantified at term by RT-qPCR at the defect and fetal bone marrow. Defects were examined histologically for neoskin coverage. Comparisons were by Mann-Whitney U tests and logistic regression. Results: Defect coverage was associated with significant downregulation of both epidermal growth factor (Egf; p = 0.031) and fibroblast growth factor-2 (Fgf-2; p = 0.042) expressions at the defect and with significant downregulation of transforming growth factor-beta-1 (Tgfb-1; p = 0.021) and CD45 (p = 0.028) expressions at the fetal bone marrow. Conclusions: Coverage of experimental spina bifida is associated with local and bone marrow negative feedback of select paracrine factors, as well as increased relative mesenchymal stem cell activity in the bone marrow. Further analyses informed by these findings may lead to strategies of nonsurgical induction of prenatal coverage of spina bifida.

Repair of rat cranial bone defect by using amniotic fluid-derived mesenchymal stem cells in polycaprolactone fibrous scaffolds and platelet-rich plasma

Introduction: Tissue regenerative medicine strategies, as a promising alternative has become of major interest to the reconstruction of critical size bone defects. This study evaluated the effects of the simultaneous application of polycaprolactone (PCL), amniotic fluid mesenchymal stem cells (AF-MSCs) and platelet-rich plasma (PRP) on the repair of rat cranial bone defects. Methods: The AF-MSCs were isolated at the end of the second week of pregnancy in rats. PRP obtained from rat blood and the random PCL fibrous scaffolds were prepared using the electrospinning method. Circular full thickness (5 mm) bone defects were developed on both sides of the parietal bones (animal number=24) and the scaffolds containing AF-MSCs and PRP were implanted in the right lesions. Thereafter, after eight weeks the histological and immunohistochemistry studies were performed to evaluate the bone formation and collagen type I expression. Results: The spindle-shaped mesenchymal stem cells were isolated and the electron microscope images indicated the preparation of a random PCL scaffold. Immunohistochemical findings showed that collagen type I was expressed by AF-MSCs cultured on the scaffold. The results of hematoxylin and eosin (H&E) staining indicated the formation of blood vessels in the presence of PRP. Additionally, immunofluorescence findings suggested that PRP had a positive effect on collagen type I expression. Conclusion: The simultaneous application of fibrous scaffold + AF-MSCs + PRP has positive effects on bone regeneration. This study showed that PRP can affect the formation of new blood vessels in the scaffold transplanted in the bone defect.

The miR-532-3p/Chrdl1 axis regulates the proliferation and migration of amniotic fluid-derived mesenchymal stromal cells

BackgroundAmniotic fluid-derived mesenchymal stromal cells (AFMSCs) are promising stem cells for regeneration medicine. However, AFMSCs isolated at different stages of pregnancy have different biological characteristics, and the therapeutic effects can differ in vivo and in vitro. The mechanisms underlying these differences have not been defined. MethodsBioinformatics analysis of the AFMSC transcriptome identified Chrdl1 as one of the differentially expressed genes. We evaluated the effects of Chrdl1 overexpression or knockdown on the proliferation and migration of AFMSCs. Target prediction was performed using miRanda software to identify the upstream microRNA of Chrdl1. The interaction between Chrdl1 mRNA and its upstream microRNA was evaluated using a dual-luciferase reporter gene assay. ResultsChrdl1 was expressed at lower levels in AFMSCs derived from the early stages of pregnancy. It could suppress AFMSC proliferation and migration. miR-532-3p promoted AFMSC proliferation and migration by targeting the 3’ UTR of Chrdl1 and downregulating its expression.

Congenital diaphragmatic hernia as a potential target for transamniotic stem cell therapy

PurposeWe sought to determine whether TRASCET could impact congenital diaphragmatic hernia (CDH). MethodsTwelve pregnant dams received Nitrofen on gestational day 9.5 (E9; term = 22 days) to induce fetal CDH. Fetuses were divided into three groups: untreated (n = 31) and two groups receiving volume-matched intraamniotic injections of either saline (n = 37) or a suspension of 2 × 106 cells/mL of amniotic fluid-derived mesenchymal stem cells (afMSCs; n = 65) on E17. Animals were euthanized at term. Expression of fibroblast growth factor-10 (FGF-10), vascular endothelial growth factor-A (VEGF-A), and surfactant protein-C (SPC) was quantified by qRT-PCR. Statistical analysis was by the Mann–Whitney U test with Bonferroni adjusted criterion (p ≤ 0.01). ResultsAmong survivors with CDH (n = 27/133), the TRASCET group showed significant downregulation of FGF-10 and VEGF-A gene expressions compared to the untreated (p < 0.001 for both) and saline groups (p = 0.005 and p = 0.004, respectively). SPC expression was higher in the TRASCET group compared to the untreated group (p = 0.01), but not the saline group (p = 0.043). Lung laterality had minimal impact on these comparisons. ConclusionsTransamniotic stem cell therapy affects select processes of lung development in experimental congenital diaphragmatic hernia. Further scrutiny into this novel therapy as a potential component of the prenatal management of this disease is warranted. Level of evidenceN/A (animal and laboratory study).

A comparison between placental and amniotic mesenchymal stem cells in transamniotic stem cell therapy for experimental gastroschisis

PurposeWe compared placental and amniotic fluid-derived mesenchymal stem cells (pMSCs and afMSCs, respectively) in transamniotic stem cell therapy for experimental gastroschisis. MethodsGastroschisis was surgically created in 126 rat fetuses at gestational day 18 (term = 22 days), immediately followed by volume-matched intraamniotic injections of suspensions of afMSCs (n = 32), pMSCs (n = 33), or normal saline (NS) (n = 33). Untreated fetuses served as controls (n = 28). Blinded observers performed computerized measurements of total and segmental (serosa, muscularis, and mucosa) intestinal wall thickness on the herniated bowel at term. Statistical analysis included ANOVA, the Wald test, and Levene's test (p < 0.05). ResultsAmong survivors, there were statistically significant decreases in segmental and total bowel wall thicknesses in both the afMSC and pMSC groups vs. the untreated (p < 0.001 to 0.003) and saline (p < 0.001 to 0.011) groups. There were significant differences between the afMSC and pMSC groups favoring the former in both therapeutic impact and its variability (p < 0.001 to 0.031). Labeled cells were comparably identified within the intestinal wall in the afMSC and pMSC groups. ConclusionsBoth placental and amniotic mesenchymal stem cells can mitigate bowel damage in experimental gastroschisis as agents of transamniotic stem cell therapy. However, amniotic cells lead to improved and more consistent outcomes. Level of evidenceN/A (animal and laboratory study).

Anti-inflammatory effects of mesenchymal stem cell-derived exosomal microRNA-146a-5p and microRNA-548e-5p on human trophoblast cells.

Human umbilical cord mesenchymal stem cells (MSCs) have been reported to improve the migration and invasion of trophoblast cells; however, little is known about whether MSC-derived exosomes and exosomal miRNAs can regulate trophoblast cell properties. In this study, we investigated whether exosomal miRNAs from amniotic fluid-derived MSC (AF-MSC) could regulate the inflammatory response of the human trophoblast cell line HTR8/SVneo. We verified the anti-inflammatory effects of AF-MSCs on lipopolysaccharide (LPS)-induced inflammatory trophoblast cells and found that miR-146a-5p and miR-548e-5p in the AF-MSC-derived exosomes regulate nuclear factor κB, AKT and mitogen-activated protein kinase protein phosphorylation. Furthermore, we found that the transfection of human trophoblast cells with miR-146a-5p and miR-548e-5p inhibitors reduced trophoblast migration (P < 0.05 vs control) and the expression of proliferating cell nuclear antigen, a protein essential for cell proliferation (P < 0.01 vs control). In particular, the miR-548e-5p inhibitor induced apoptosis, while tumor necrosis factor receptor-associated factor 6, a predicted target of miR-146a-5p and miR-548e-5p, was involved in the regulation of oxidative stress in the human trophoblast cells. In a mouse model of LPS-induced preterm birth (PB), miR-146a-5p expression was found to be relatively low in the group in which the effect of AF-MSCs was insignificant. However, this study is limited in that the changes in the expression of some genes in response to AF-MSCs differ between the cell line and mouse model. Collectively, these data show that exosomal miR-146a-5p and miR-548e-5p from AF-MSCs have anti-inflammatory effects on human trophoblast cells and may be novel targets for treating inflammatory diseases and associated problems that occur during pregnancy, such as PB.

Reduction in the Viability of Human Cervical Cancer HeLa Cell Line via Indirect Co-culture With Amniotic Fluid-Derived Mesenchymal Stem Cells

Objectives: This experiment was carried out to evaluate the impacts of unmodified human amniotic fluid-derived mesenchymal stromal/stem cells (hAF-MSCs) on the viability of HeLa cells, as well as the impact of these cells on the expression of common proapoptotic and pro-survival genes in tumor cells by establishing an indirect co-culture system. Materials and Methods: To this end, an indirect co-culture system was established, and hAF-MSCs were co-cultured with HeLa cells at a ratio of 1:2 for five days. The cell viability of co-cultured tumor cells was determined after the incubation period. Then, several parameters were examined, including the gene expression of tumor protein 53 (TP53), BCL2-associated X protein (BAX), B-cell lymphoma 2 (BCL-2), and cyclin-dependent kinase inhibitor 1A (CDKN1A). Finally, gene regulatory networks were analyzed as well. Results: The results of this study confirmed that the co-culture of hAF-MSCs with HeLa cells could decrease the viability of tumor cells. The reduction of HeLa cell viability was accompanied by an increase in BAX, TP53, and CDKN1A while a decrease in BCL2 gene expression. Eventually, the analysis of the regulatory network revealed that the co-culture of Hela ¬cells with hAF-MSCs activated several transcriptional factors and microRNAs which regulated the expression of these genes. Conclusions: In general, hAF-MSCs exerted the inhibitive effects on the growth of HeLa cells, along with alterations in the expression of common pro-apoptotic and pro-survival genes in a timely and concentration-dependent manner.

Combination of Human Amniotic Fluid Derived-Mesenchymal Stem Cells and Nano-hydroxyapatite Scaffold Enhances Bone Regeneration.

BACKGROUND:Human amniotic fluid-derived stem cells (hAF-MSCs) have a high proliferative capacity and osteogenic differentiation potential in vitro. The combination of hAF-MSCs with three-dimensional (3D) scaffold has a promising therapeutic potential in bone tissue engineering and regenerative medicine. Selection of an appropriate scaffold material has a crucial role in a cell supporting and osteoinductivity to induce new bone formation in vivo.AIM:This study aimed to investigate and evaluate the osteogenic potential of the 2nd-trimester hAF-MSCs in combination with the 3D scaffold, 30% Nano-hydroxyapatite chitosan, as a therapeutic application for bone healing in the induced tibia defect in the rabbit.SUBJECT AND METHODS:hAF-MSCs proliferation and culture expansion was done in vitro, and osteogenic differentiation characterisation was performed by Alizarin Red staining after 14 & 28 days. Expression of the surface markers of hAF-MSCs was assessed using Flow Cytometer with the following fluorescein-labelled antibodies: CD34-PE, CD73-APC, CD90-FITC, and HLA-DR-FITC. Ten rabbits were used as an animal model with an induced defect in the tibia to evaluate the therapeutic potential of osteogenic differentiation of hAF-MSCs seeded on 3D scaffold, 30% Nano-hydroxyapatite chitosan. The osteogenic differentiated hAF-MSCs/scaffold composite system applied and fitted in the defect region and non-seeded scaffold was used as control. The histopathological investigation was performed at 2, 3, & 4 weeks post-transplantation and scanning electron microscope (SEM) was assessed at 2 & 4 weeks post-transplantation to evaluate the bone healing potential in the rabbit tibia defect.RESULTS:Culture and expansion of 2nd-trimester hAF-MSCs presented high proliferative and osteogenic potential in vitro. Histopathological examination for the transplanted hAF-MSCs seeded on the 3D scaffold, 30% Nano-hydroxyapatite chitosan, demonstrated new bone formation in the defect site at 2 & 3 weeks post-transplantation as compared to the control (non-seeded scaffold). Interestingly, the scaffold accelerated the osteogenic differentiation of AF-MSCs and showed complete bone healing of the defect site as compared to the control (non-seeded scaffold) at 4 weeks post-transplantation. Furthermore, the SEM analysis confirmed these findings.CONCLUSION:The combination of the 2nd-trimester hAF-MSCs and 3D scaffold, 30% Nano-hydroxyapatite chitosan, have a therapeutic perspective for large bone defect and could be used effectively in bone tissue engineering and regenerative medicine.

Fabrication of biocompatible porous scaffolds based on hydroxyapatite/collagen/chitosan composite for restoration of defected maxillofacial mandible bone

Fabrication of scaffolds from biomaterials for restoration of defected mandible bone has attained increased attention due to limited accessibility of natural bone for grafting. Hydroxyapatite (Ha), collagen type 1 (Col1) and chitosan (Cs) are widely used biomaterials which could be fabricated as a scaffold to overcome the paucity of bone substitutes. Here, rabbit Col1, shrimp Cs and bovine Ha were extracted and characterized with respect to physicochemical properties. Following the biocompatibility, degradability and cytotoxicity tests for Ha, Col1 and Cs a hydroxyapatite/collagen/chitosan (Ha·Col1·Cs) scaffold was fabricated using thermally induced phase separation technique. This scaffold was cross-linked with (1) either glutaraldehyde (GTA), (2) de-hydrothermal treatment (DTH), (3) irradiation (IR) and (4) 2-hydroxyethyl methacrylate (HEMA), resulting in four independent types (Ha·Col1·Cs-GTA, Ha·Col1·Cs-IR, Ha·Col1·Cs-DTH and Ha·Col1·Cs-HEMA). The developed composite scaffolds were porous with 3D interconnected fiber microstructure. However, Ha·Col1·Cs-IR and Ha·Col1·Cs-GTA showed better hydrophilicity and biodegradability. All four scaffolds showed desirable blood biocompatibility without cytotoxicity for brine shrimp. In vitro studies in the presence of human amniotic fluid-derived mesenchymal stem cells revealed that Ha·Col1·Cs-IR and Ha·Col1·Cs-DHT scaffolds were non-cytotoxic and compatible for cell attachment, growth and mineralization. Further, grafting of Ha·Col1·Cs-IR and Ha·Col1·Cs-DHT was performed in a surgically created non-load-bearing rabbit maxillofacial mandible defect model. Histological and radiological observations indicated the restoration of defected bone. Ha·Col1·Cs-IR and Ha·Col1·Cs-DHT could be used as an alternative treatment in bone defects and may contribute to further development of scaffolds for bone tissue engineering.

Therapeutic Potential of Amniotic Fluid Derived Mesenchymal Stem Cells Based on their Differentiation Capacity and Immunomodulatory Properties.

Amniotic Fluid Derived Mesenchymal Stem Cells (AF-MSCs) are adult, fibroblast- like, self-renewable, multipotent stem cells. During the last decade, the therapeutic potential of AF-MSCs, based on their huge differentiation capacity and immunomodulatory characteristics, has been extensively explored in animal models of degenerative and inflammatory diseases. In order to describe molecular mechanisms responsible for the therapeutic effects of AFMSCs, we summarized current knowledge about phenotype, differentiation potential and immunosuppressive properties of AF-MSCs. An extensive literature review was carried out in March 2018 across several databases (MEDLINE, EMBASE, Google Scholar), from 1990 to present. Keywords used in the selection were: "amniotic fluid derived mesenchymal stem cells", "cell-therapy", "degenerative diseases", "inflammatory diseases", "regeneration", "immunosuppression". Studies that emphasized molecular and cellular mechanisms responsible for AF-MSC-based therapy were analyzed in this review. AF-MSCs have huge differentiation and immunosuppressive potential. AF-MSCs are capable of generating cells of mesodermal origin (chondrocytes, osteocytes and adipocytes), neural cells, hepatocytes, alveolar epithelial cells, insulin-producing cells, cardiomyocytes and germ cells. AF-MSCs, in juxtacrine or paracrine manner, regulate proliferation, activation and effector function of immune cells. Due to their huge differentiation capacity and immunosuppressive characteristic, transplantation of AFMSCs showed beneficent effects in animal models of degenerative and inflammatory diseases of nervous, respiratory, urogenital, cardiovascular and gastrointestinal system. Considering the fact that amniotic fluid is obtained through routine prenatal diagnosis, with minimal invasive procedure and without ethical concerns, AF-MSCs represents a valuable source for cell-based therapy of organ-specific or systemic degenerative and inflammatory diseases.

Angiotensin II and TGF-β1 Induce Alterations in Human Amniotic Fluid-Derived Mesenchymal Stem Cells Leading to Cardiomyogenic Differentiation Initiation.

Background and ObjectivesHuman amniotic fluid-derived mesenchymal stem cells (AF-MSCs) may be a valuable source for cardiovascular tissue engineering and cell therapy. The aim of this study is to verify angiotensin II and transforming growth factor-beta 1 (TGF-β1) as potential cardiomyogenic differentiation inducers of AF-MSCs.Methods and ResultsAF-MSCs were obtained from amniocentesis samples from second-trimester pregnant women, isolated and characterized by the expression of cell surface markers (CD44, CD90, CD105 positive; CD34 negative) and pluripotency genes (OCT4, SOX2, NANOG, REX1). Cardiomyogenic differentiation was induced using different concentrations of angiotensin II and TGF-β1. Successful initiation of differentiation was confirmed by alterations in cell morphology, upregulation of cardiac genes-markers NKX2-5, TBX5, GATA4, MYH6, TNNT2, DES and main cardiac ion channels genes (sodium, calcium, potassium) as determined by RT-qPCR. Western blot and immunofluorescence analysis revealed the increased expression of Connexin43, the main component of gap junctions, and Nkx2.5, the early cardiac transcription factor. Induced AF-MSCs switched their phenotype towards more energetic and started utilizing oxidative phosphorylation more than glycolysis for energy production as assessed using Agilent Seahorse XF analyzer. The immune analysis of chromatin-modifying enzymes DNMT1, HDAC1/2 and Polycomb repressive complex 1 and 2 (PRC1/2) proteins BMI1, EZH2 and SUZ12 as well as of modified histones H3 and H4 indicated global chromatin remodeling during the induced differentiation.ConclusionsAngiotensin II and TGF-β1 are efficient cardiomyogenic inducers of human AF-MSCs; they initiate alterations at the gene and protein expression, metabolic and epigenetic levels in stem cells leading towards cardiomyocyte-like phenotype formation.

Survivability of rabbit amniotic fluid-derived mesenchymal stem cells post slow-freezing or vitrification

This work aimed to evaluate the effect of two distinct cryopreservation procedures - conventional slow-freezing and vitrification, on survivability and mesenchymal marker expression stability of rabbit amniotic fluid-derived mesenchymal stem cells (rAF-MSCs). Cells at passage 2 were slowly frozen, using 10% of dimethylsulfoxide, or vitrified, using 40% of ethylene glycol, 0.5 M sucrose and 18% Ficoll 70. After three months storage in liquid nitrogen, viability, chromosomal stability, ultrastructure, surface and intracellular marker expression and differentiation potential of cells were evaluated immediately post-thawing/warming and after additional culture for 48–72 h. Our results showed decreased (P ≤ 0.05) viability of cells post-thawing/warming. However, after additional culture, the viability was similar to those in fresh counterparts in both cryopreserved groups. Increase (P ≤ 0.05) in the population doubling time of vitrified cells was observed, while doubling time of slow-frozen cells remained similar to non-cryopreserved cells. No changes in karyotype (chromosomal numbers) were observed in frozen/vitrified AF-MSCs, and histological staining confirmed similar differentiation potential of fresh and frozen/vitrified cells. Analysis of mesenchymal marker expression by qPCR showed that both cryopreservation approaches significantly affected expression of CD73 and CD90 surface markers. These changes were not detected using flow cytometry. In summary, the conventional slow-freezing and vitrification are reliable and effective approaches for the cryopreservation of rabbit AF-MSCs. Nevertheless, our study confirmed affected expression of some mesenchymal markers following cryopreservation.

The Different Effects of IFN-β and IFN-γ on the Tumor-Suppressive Activity of Human Amniotic Fluid-Derived Mesenchymal Stem Cells.

Current studies have shown that type I or II interferon-modified mesenchymal stem cells have great potential for the application of tumor-targeted therapy, but the underlying mechanism remains largely elusive. Here, we compared the different effects of IFN-β and IFN-γ on the antitumor activity of human amniotic fluid-derived mesenchymal stem cells (AFMSCs) and revealed the potential mechanism. In detail, AFMSCs primed with IFN-β or IFN-β plus IFN-γ, not IFN-γ, inhibited the proliferation of cancer cells in an immunocompetent mouse H460 subcutaneous model, although they all inhibited the proliferation of cancer cells in an immunocompromised mouse H460 subcutaneous model. TRAIL expressed by IFN-β- or IFN-γ-primed AFMSCs specifically exerted the antitumor effect of AFMSCs. AFMSCs primed with IFN-γ highly expressed immunosuppressive molecule IDO1, but IFN-β counteracted the IFN-γ-initiated IDO1 expression. 1-MT (IDO1 inhibitor) decreased TRAIL, but increased IDO1 expression in AFMSCs primed with interferon. As a result, AFMSCs primed with IFN-β or IFN-γ had the antitumor activity, and 1-MT failed to enhance the antitumor effect of IFN-γ-primed AFMSC in vitro and in the immunocompromised mouse H460 subcutaneous model. Furthermore, the expression of TRAIL in AFMSCs was upregulated by apoptotic cancer cells and this positive feedback intensified the antitumor effects of IFNs-primed AFMSCs. The different target gene expression profiles of AFMSCs regulated by IFN-β and IFN-γ determined the different antitumor effects of IFN-β- and IFN-γ-primed AFMSCs on tumor cells. Our finding may help to explore a clinical strategy for cancer intervention by understanding the antitumor mechanisms of MSCs and interferon.