Amniotic Mesenchymal Stem Cells Research Articles

Transfersomal serum loading amniotic mesenchymal stem cells metabolite products with hyaluronic acid addition for skin regeneration in UV aging-induced mice.

Amniotic Mesenchymal Stem Cells Metabolite Products (AMSC-MP) contain growth factors that benefit human health. This study aims to evaluate the use of transfersomal serum (Trans) with hyaluronic acid (HA) addition to deliver large molecules of AMSC-MP for skin regeneration. Trans is composed of L-α-phosphatidylcholine and surfactants, i.e., sodium cholate (SC) or stearylamine (SA), at the weight ratio of 85:15, prepared by the thin film method with or without HA addition. The results showed that HA addition increased the particle size of Trans-SA and Trans-SC, from 261.9 ± 1.9 and 105.3 ± 0.9 nm respectively, to 317.7 ± 9.1 and 144.3 ± 0.8 nm for Trans-SA-HA and Trans-SC-HA. In contrast, no significant changes in the zeta potential occurred. The relative deformability indexes of Trans-SA, Trans-SA-HA, Trans-SC, and Trans-SC-HA compared to liposome were 0.43 ± 0.09, 0.46 ± 0.09, 1.58 ± 0.17, and 1.40 ± 0.17 respectively. The addition of HA successfully increases the in vivo skin hydration, collagen density, and number of fibroblast cells, reflecting the capacity for skin regeneration in UV-induced aged mice. Furthermore, no erythema or skin rash was observed at the 24-hour post-topical application sites. AMSC-MP transfersomal serum with HA addition successfully enhanced skin regeneration and proved safe during the in vivo study using UV aging-induced mice models, thereby enabling its potential use as skin-aging therapy.

Conditioned Medium from Human Amniotic Membrane-Derived Mesenchymal Stem Cells Modulates Inflammatory and Myofibrotic Factors in Vivo

Background: Heart failure (HF) is a prevalent diagnosis with a significant mortality rate. Various therapeutic approaches exist for treating HF, and human adipose-derived mesenchymal stem cells-conditioned medium (hAMSCs-CM) therapy has emerged as a promising option. Despite its potential efficacy, the precise mechanism of action underlying hAMSCs-CM treatment remains unclear. To address this knowledge gap, we conducted a novel animal study to investigate the mechanism of action of hAMSCs-CM in an HF model, with a specific focus on transforming growth factor-β (TGF-β)/galectin-3, monocyte chemoattractant protein-1 (MCP1), B-type natriuretic peptide (BNP), and aldosterone (ALD). Methods: Forty adult male Wistar rats were divided into 4 groups: control, HF, culture medium, and CM. All rats, except those in the control group, received an injection of isoproterenol to induce an animal model of HF. The CM group was administered the CM, while those in the culture medium group received standard culture media. Subsequently, serum levels of fibrotic factors, including TGF-β/galectin-3, MCP1, BNP, and ALD, were measured using ELISA. Statistical analysis was performed using one-way analysis of variance and the Tukey test. Results: Serum levels of TGF-β/galectin-3, MCP1, BNP, and ALD were significantly elevated in the HF, CM, and culture medium groups compared with the control group (P<0.001). Additionally, these fibrotic factors were significantly reduced in the CM group compared with the HF group (P<0.001). Notably, CM therapy could not restore TGF-β/galectin-3, MCP1, BNP, or ALD levels to the normal range observed in the control group. Conclusion: Our findings indicate that hAMSCs-CM modulates the expression of inflammatory and fibrotic cytokines, such as TGF-β/galectin-3, MCP1, BNP, and ALD, in isoproterenol-induced HF in male rats. These results contribute to a better understanding of the therapeutic mechanisms underlying hAMSCs-CM treatment for HF.

Development of liposomal amniotic mesenchymal stem cell metabolite products (AMSC-MP) loaded-scaffold for in vitro osteogenesis induction

The use of scaffolds with good biocompatibility and mechanical properties largely determines the success of bone defect therapy intended to improve quality-adjusted life years. Furthermore, amniotic mesenchymal stem cell metabolite product (AMSC-MP) is required to accelerate bone regeneration with sustained release required to maximize the effectiveness of therapy. This study aims to evaluate the effect of different phospholipids on the osteogenesis capacity of AMSC-MP liposome loaded into scaffold. The thin layer hydration method was employed to create the AMSC-MP liposomes which had different lipid components: L-α-phosphatidylcholine (PC) an unsaturated or hydrogenated soybean phosphatidylcholine (HSPC), a saturated phospholipid, with different types of charged phospholipids i.e., 1,2-dioleoyltrimethylammoniumpropane (DOTAP) or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), which were cationic and non-ionic lipids respectively.AMSC-MP liposomes were embedded in the scaffold during a 24-hour incubation period. The results indicated that the particle sizes of liposomes prepared with saturated phospholipids, HS-DOTAP (132.35 ± 2.75 nm) and HS-DOTAP (152.85 ± 5.86 nm) were larger than those with saturated phospholipids, namely PC-DOTAP (139.355 ± 2.75 nm) and PC-DOTAP (91.85 ± 0.07 nm). The use of DOTAP in AMSC-liposomes resulted in positively charged vesicles i.e., +5 mV for HSPC-DOTAP and + 10 mV for PC-DOTAP. Moreover, the results of an MTT viability test conducted on 7f2 cells and MSC indicated that all the formulas demonstrate good in vitro biocompatibility. Formulations containing DOPE produced high cellular uptake values and had superior sustain release properties compared to the others. This result correlates positively with cell attachment to the liposome scaffold which was higher than the others. Moreover, DOPE-containing formulations increased RUNX2 expression, indicating enhanced in vitro osteogenesis capacity. Therefore, Liposome AMSC-MP loaded scaffold demonstrates high potential for use in bone regeneration therapy.

RGDSP-functionalized peptide hydrogel stimulates growth factor secretion via integrin αv/PI3K/AKT axis for improved wound healing by human amniotic mesenchymal stem cells.

The wound healing process involves communication among growth factors, cytokines, signaling pathways, and cells in the extracellular matrix, with growth factors acting as key regulators. Although stem cells can promote wound healing by secreting diverse growth factors, their therapeutic potential is hindered by poor survival and engraftment. Mimicking the stem cell-matrix interactions can improve stem cell survival, regulate their fate, and even enhance their paracrine effects. This study investigated the use of composite RGDmix hydrogel, which can support the survival and proliferation of human amniotic mesenchymal stem cells (hAMSCs), and effectively increase the expression of various growth factors, thereby promoting wound re-epithelialization, angiogenesis, and epidermal maturation. At last, the specific role of integrin αv and PI3K/AKT signaling pathways in the secretion of growth factors were examined by silencing them in vitro and in vivo. Results suggested that the RGDmix hydrogel improved the secretion of growth factors by hAMSCs through the RGDSP/integrin αv/PI3K/AKT axis, thereby enhancing the therapeutic effect in wound healing.

MiR-16a-5p antagonizes FGF-2 in ligamentogenic differentiation of MSC: a new therapeutic perspective for tendon regeneration

With the increasing demand for exercise, the population of patients with ankle sprain to anterior talofibular ligament injury has the characteristics of a large base and high requirements for returning to sports, and how to promote the repair of damaged ligaments from a microscopic perspective is an urgent problem to be solved. In many studies, human amniotic mesenchymal stem cells have strong differentiation ability, and can be induced to continuously differentiate into ligament cells to achieve the purpose of repairing damaged ligaments. Human amniotic stem cells were extracted and cultured from human amniotic tissues, evaluated by cell identification and other techniques, and evaluated into ligament differentiation by toluidine blue, alizarin red, oil red O staining and detection of ligament cell differentiation, protein detection by Western blot, mRNA level by qPCR, and finally, the targeted binding relationship between miR-16a-5p and mRNA FGF2 was verified by double luciferase reporter assay. The expression of collagen type 1 (COL 1), collagen type 3 (COL3), SCX and MKX was increased by overexpression of mRNA FGF2, respectively, and miR-16a-5p had a targeted effect on FGF2 and regulated the ligamentous differentiation of human amniotic mesenchymal stem cells. We found that the regulatory effect of overexpressed mRNA FGF2 on mesenchymal stem cells could be inhibited by up-regulation of miR-16a-5p, while the knockdown of FGF2 could reverse the regulatory effect of miR-16a-5p inhibition on ligament-forming differentiation of human amniotic mesenchymal stem cells. In this study, we discovered the existence of the miR-16a-5p–FGF2 axis in human amniotic mesenchymal stem cells, and the differentiation of human amniotic mesenchymal stem cells into ligamentous cells can be regulated by regulating various links in this axis.

Downregulating LKB1 in bone marrow mesenchymal stem cells could inhibit CD4+ T cell proliferation via the PD-1/PD-L1 signaling pathway

BackgroundOur previous research has shown that LKB1 in amniotic mesenchymal stem cells (MSCs) serves as a vital regulator of regulatory T cell differentiation and T cell proliferation, which may have a similar role in bone marrow MSCs (BMMSCs). Therefore, we investigated the role of LKB1 in BMMSCs for regulating CD4+ T cell proliferation in the bone micro-environment of AML. MethodsRT-PCR was used to assessed LKB1 expression in BMMSCs derived from AML patients and healthy controls. Subsequently, LKB1 was knocked down in the BMMSCs line HS-5 (HS-5-LKB1KD). Co-cultures in vitro were established to analyze the effect of HS-5-LKB1KD on CD4+ T cell. Flow cytometry was employed to measure PD-L1 and CD4+ T cell proliferation levels. Western blot was utilized to detect related proteins. ResultsThe expression of LKB1 in BMMSCs derived from AML patients was decreased. Knockdown of LKB1 in HS-5 resulted in upregulation of PD-L1 expression. Co-culture of peripheral blood CD4+ T cell with HS-5-LKB1KD exhibited reduced CD4+ T cell proliferation compared to co-culture with HS-5-LKB1con. Furthermore, blocking PD-L1 in the co-culture conditions could restore the reduced CD4+ T cell proliferation. Additionally, it was found that upregulation of the Wnt signaling pathway-related proteins following LKB1 knockdown in HS-5, indicating that downregulating LKB1 could promote PD-L1 expression through activation of the Wnt signaling pathway. ConclusionsThe decreased expression of LKB1 in BMMSCs may activate the Wnt signaling pathway, leading to increased PD-L1 expression. This inhibited CD4+ T cell proliferation, which might lead to impaired anti-tumor immunity in AML patients and promote AML progression.

Steroidogenic differentiation of human amniotic membrane-derived mesenchymal stem cells into a progesterone-/androgen-producing cell lineage by SF-1 and an estrogen-producing cell lineage by WT1-KTS.

Sex steroid hormones, primarily synthesized by gonadal somatic cells, are pivotal for sexual development and reproduction. Mice studies have shown that two transcription factors, steroidogenic factor 1 (SF-1) and Wilms' tumor 1 (WT1), are involved in gonadal development. However, their role in human gonadal somatic differentiation remains unclear. We therefore aimed to investigate the roles of SF-1 and WT1 in human gonadal steroidogenic cell differentiation. Using a transient lentivirus-mediated gene expression system, we assessed the effects of SF-1 and WT1 expression on the steroidogenic potential of human amniotic membrane-derived mesenchymal stem cells (hAmMSCs). SF-1 and WT1-KTS, a splice variant of WT1, played distinct roles in human steroidogenic differentiation of hAmMSCs. SF-1 induced hAmMSC differentiation into progesterone- and androgen-producing cell lineages, whereas WT1-KTS promoted hAmMSC differentiation into estrogen-producing cell lineages. Our findings revealed that SF-1 and WT1-KTS play important roles in human gonadal steroidogenic cell differentiation, especially during ovarian development. These findings may pave the way for future studies on human ovarian differentiation and development.

Dissolving microneedle patches for delivery of amniotic mesenchymal stem cell metabolite products for skin regeneration in UV-aging induced mice

Microneedles offer a promising solution to enhancing dermal delivery of amniotic mesenchymal stem cell metabolite product (AMSC-MP), which contains hydrophilic protein components with high molecular weight, for the purposes of skin rejuvenation and improving human health. This study aimed to evaluate the physicochemical characteristics and in vivo efficacy of AMSC-MP-loaded microneedle patches for effectively regenerating skin tissues in UV-aging induced mice. Dissolving microneedle patches, composed of polyvinyl alcohol with an MW of 9–10 kDa and polyvinylpyrrolidone with an MW of 56 kDa, were fabricated using the double-casting method at three AMSC-MP concentrations: i.e., 30 % (MN30), 25 % (MN25), and 20 % (MN20). The microneedles patches were then evaluated for morphological, mechanical resistance, and insertion properties. An ex vivo release study was also conducted using the Franz cell method, and in vivo efficacy and irritation were then determined through collagen density scores, fibroblast cell counts, and skin irritation studies of UV-aging induced mice. The AMSC-MP microneedles displayed a pyramidal shape with 500 µm sharp tips. Mechanical testing revealed that MN30 achieved its deepest insertion into Parafilm® M (447.44 ± 37.21 µm), while MN25 achieved its deepest insertion into full-thickness porcine skin (717.92 ± 25.40 µm). The study revealed a controlled EGF release for up to 24 h, with MN20 exhibiting the highest deposition (55.94 ± 12.34 %). These findings demonstrate the successful penetration of microneedles through the stratum corneum and viable epidermis. Collagen density scores and fibroblast cell counts were significantly higher in all microneedle formulations than the control, with MN30 having the highest values. Inflammatory cell counts indicated minimal presence suggesting non-irritation in the in vivo study. Dissolving microneedle patches exhibited favorable characteristics and efficiently delivered AMSC-MP with minimal potential for irritation, providing potential technology for delivering biological anti-aging agents for the purposes of fostering skin regeneration.

Amniotic fluid-derived mesenchymal stem cells reduce inflammation and improve lung function following transplantation in a porcine model

BackgroundLung transplantation is hindered by low donor lung utilization rates. Infectious complications are reasons to decline donor grafts due to fear of post-transplant primary graft dysfunction. Mesenchymal stem cells are a promising therapy currently investigated in treating lung injury. Full-term amniotic fluid-derived lung-specific mesenchymal stem cell treatment may regenerate damaged lungs. These cells have previously demonstrated inflammatory mediation in other respiratory diseases, and we hypothesized that treatment would improve donor lung quality and post-operative outcomes. MethodsIn a transplantation model, donor pigs were stratified to either the treated or the non-treated group. Acute respiratory distress syndrome was induced in donor pigs and harvested lungs were placed on ex vivo lung perfusion before transplantation. Treatment consisted of three doses of 2x106 cells/kg: one during ex vivo lung perfusion and two after transplantation. Donors and recipients were assessed on clinically relevant parameters and recipients were followed for 3 days before evaluation for primary graft dysfunction (PGD). ResultsRepeated injection of the cell treatment showed reductions in inflammation seen through lowered immune cell counts, reduced histology signs of inflammation, and decreased cytokines in the plasma and bronchoalveolar lavage fluid. Treated recipients showed improved pulmonary function, including increased PaO2/FiO2 ratios and reduced incidence of PGD. ConclusionsRepeated injection of lung-specific cell treatment during EVLP and post-transplant was associated with improved function of previously damaged lungs. Cell treatment may be considered as a potential therapy to increase the number of lungs available for transplantation and the improvement of post-operative outcomes.

A novel tissue-engineered corneal epithelium based on ultra-thin amniotic membrane and mesenchymal stem cells

Currently, in vitro cultured corneal epithelial transplantation is effective in treating limbal stem cell dysfunction (LSCD). Selecting carriers is crucial for constructing the corneal epithelium through tissue engineering. In this study, the traditional amniotic membrane (AM) was modified, and mesenchymal stem cells (MSCs) were inoculated into the ultra-thin amniotic membrane (UAM) stroma to construct a novel UAM-MSC tissue-engineered corneal epithelial carrier, that could effectively simulate the limbal stem cells (LSCs) microenvironment. The structure of different carriers cultured tissue-engineered corneal epithelium and the managed rabbit LSCD model corneas were observed through hematoxylin–eosin staining. Cell phenotypes were evaluated through fluorescence staining, Western blotting, and RT-qPCR. Additionally, cell junction genes and expression markers related to anti-neovascularization were evaluated using RT-qPCR. Corneal epithelium cell junctions were observed via an electron microscope. The tissue-engineered corneal epithelium culture medium was analyzed through mass spectrometry. Tissue-engineered corneal epithelial cells expanded by LSCs on UAM-MSCs had good transparency. Simultaneously, progenitor cell (K14, PNCA, p63) and corneal epithelial (PAX6) gene expression in tissue-engineered corneal epithelium constructed using UAM-MSCs was higher than that in corneal epithelial cells amplified by UAM and de-epithelialized amniotic membrane. Electron microscopy revealed that corneal epithelial cells grafted with UAM-MSCs were closely connected. In conclusion, the UAM-MSCs vector we constructed could better simulate the limbal microenvironment; the cultured tissue-engineered corneal epithelium had better transparency, anti-neovascularization properties, closer intercellular connections, and closer resemblance to the natural corneal epithelial tissue phenotype.

Influence of the Surface Topography of Titanium Dental Implants on the Behavior of Human Amniotic Stem Cells.

The dental implant surface plays a crucial role in osseointegration. The topography and physicochemical properties will affect the cellular functions. In this research, four distinct titanium surfaces have been studied: machined acting (MACH), acid etched (AE), grit blasting (GBLAST), and a combination of grit blasting and subsequent acid etching (GBLAST + AE). Human amniotic mesenchymal (hAMSCs) and epithelial stem cells (hAECs) isolated from the amniotic membrane have attractive stem-cell properties. They were cultured on titanium surfaces to analyze their impact on biological behavior. The surface roughness, microhardness, wettability, and surface energy were analyzed using interferometric microscopy, Vickers indentation, and drop-sessile techniques. The GBLAST and GBLAST + AE surfaces showed higher roughness, reduced hydrophilicity, and lower surface energy with significant differences. Increased microhardness values for GBLAST and GBLAST + AE implants were attributed to surface compression. Cell viability was higher for hAMSCs, particularly on GBLAST and GBLAST + AE surfaces. Alkaline phosphatase activity enhanced in hAMSCs cultured on GBLAST and GBLAST + AE surfaces, while hAECs showed no mineralization signals. Osteogenic gene expression was upregulated in hAMSCs on GBLAST surfaces. Moreover, α2 and β1 integrin expression enhanced in hAMSCs, suggesting a surface-integrin interaction. Consequently, hAMSCs would tend toward osteoblastic differentiation on grit-blasted surfaces conducive to osseointegration, a phenomenon not observed in hAECs.

A novel induced pluripotent stem cell model of Schwann cell differentiation reveals NF2 - related gene regulatory networks of the extracellular matrix.

Schwann cells are vital to development and maintenance of the peripheral nervous system and their dysfunction has been implicated in a range of neurological and neoplastic disorders, including NF2 -related schwannomatosis. We developed a novel human induced pluripotent stem cell (hiPSC) model to study Schwann cell differentiation in health and disease. We performed transcriptomic, immunofluorescence, and morphological analysis of hiPSC derived Schwann cell precursors (SPCs) and terminally differentiated Schwann cells (SCs) representing distinct stages of development. To validate our findings, we performed integrated, cross-species analyses across multiple external datasets at bulk and single cell resolution. Our hiPSC model of Schwann cell development shared overlapping gene expression signatures with human amniotic mesenchymal stem cell (hAMSCs) derived SCs and in vivo mouse models, but also revealed unique features that may reflect species-specific aspects of Schwann cell biology. Moreover, we identified gene co-expression modules that are dynamically regulated during hiPSC to SC differentiation associated with ear and neural development, cell fate determination, the NF2 gene, and extracellular matrix (ECM) organization. By cross-referencing results between multiple datasets, we identified new genes potentially associated with NF2 expression. Our hiPSC model further provides a tractable platform for studying Schwann cell development in the context of human disease.

Amniotic miracle: Investigating the unique development and applications of amniotic membrane in wound healing.

The perfect repair of damaged skin has always been a constant goal for scientists; however, the repair and reconstruction of skin is still a major problem and challenge in injury and burns medicine. Human amniotic membrane (hAM), with its good mechanical properties and anti-inflammatory, antioxidant and antimicrobial benefits, containing growth factors that promote wound healing, has evolved over the last few decades from simple skin sheets to high-tech dressings, such as being made into nanocomposites, hydrogels, powders, and electrostatically spun scaffolds. This paper aims to explore the historical development, applications, trends, and research hotspots of hAM in wound healing. We examined 2660 publications indexed in the Web of Science Core Collection (WoSCC) from January 1, 1975 to July 12, 2023. Utilizing bibliometric methods, we employed VOSviewer, CiteSpace, and R-bibliometrix to characterize general information, identify development trends, and highlight research hotspots. Subsequently, we identified a collection of high-quality English articles focusing on the roles of human amniotic epithelial stem cells (hAESCs), human amniotic mesenchymal stem cells (hAMSCs), and amniotic membrane (AM) scaffolds in regenerative medicine and tissue engineering. Bibliometric analysis identified Udice-French Research Universities as the most productive affiliation and Tseng S.C.G. as the most prolific author. Keyword analysis, historical direct quotations network, and thematic analysis helped us review the historical and major themes in this field. Our examination included the knowledge structure, global status, trends, and research hotspots regarding the application of hAM in wound healing. Our findings indicate that contemporary research emphasizes the preparation and application of products derived from hAM. Notably, both hAM and the cells isolated from it - hADSCs and hAESCs are prominent and promising areas of research in regenerative medicine and tissue engineering. This research delivers a comprehensive understanding of the knowledge frameworks, global dynamics, emerging patterns, and primary research foci in the realm of hAM applications for wound healing. The field is rapidly evolving, and our findings offer valuable insights for researchers. Future research outcomes are anticipated to be applied in clinical practice, enhancing methods for disease prevention, diagnosis, and treatment.

Arthrospira platensis and Gracilaria gracilis algae extracts as biological inducers for human amniotic mesenchymal stem cells (hAMSC)

Arthrospira platensis and Gracilaria gracilis algae extracts as biological inducers for human amniotic mesenchymal stem cells (hAMSC)

Human amniotic mesenchymal stem cell-islet organoids enhance the efficiency of islet engraftment in a mouse diabetes model

AimsDespite islet transplantation has proved a great potential to become the standard therapy for type 1 diabetes mellitus (T1DM), this approach remains limited by ischemia, hypoxia, and poor revascularization in early post-transplant period as well as inflammation and life-long host immune rejection. Here, we investigate the potential and mechanism of human amniotic mesenchymal stem cells (hAMSCs)-islet organoid to improve the efficiency of islet engraftment in immunocompetent T1DM mice. Main methodsWe generated the hAMSC-islet organoid structure through culturing the mixture of hAMSCs and islets on 3-dimensional-agarose microwells. Flow cytometry, whole-body fluorescent imaging, immunofluorescence, Calcein-AM/PI staining, ELISA, and qPCR were used to assess the potential and mechanism of shielding hAMSCs to improve the efficiency of islet transplantation. Key findingsTransplant of hAMSC-islet organoids results in remarkably better glycemic control, an enhanced glucose tolerance, and a higher β cell mass in vivo compared with control islets. Our results show that hAMSCs shielding provides an immune privileged microenvironment for islets and promotes graft revascularization in vivo. In addition, hAMSC-islet organoids show higher viability and reduced dysfunction after exposure to hypoxia and inflammatory cytokines in vitro. Finally, our results show that shielding with hAMSCs leads to the activation of PKA-CREB-IRS2-PI3K and PKA-PDX1 signaling pathways, up-regulation of SIL1 mRNA levels, and down-regulation of MT1 mRNA levels in β cells, which ultimately promotes the synthesis, folding and secretion of insulin, respectively. SignificancehAMSC-islet organoids can evidently increase the efficiency of islet engraftment and might develop into a promising alternative for the clinical treatment of T1DM.

Beneficial effects of bioactive peptides extracted from Spirulina platensis and Gracilaria gracilis algae on bone regeneration/osteogenic differentiation of mesenchymal stem cells

Mesenchymal stem cells are used in the treatment of many diseases, particularly in the repair of bone injuries. Algae with various medicinal applications are considered important natural resources. There is limited research on the effects of bioactive peptides from algae extraction on mesenchymal stem cells. In this study the impact of bioactive proteins, protein lysates and peptide fractions (<3, <30 and <50 kDa) isolated from two algae species, Spirulina platensis and Gracilaria gracilis on the proliferation and osteogenic differentiation of human amniotic mesenchymal stem cells (hAMSCs) was investigated. The proteins were extracted ant hydrolyzed with trypsin enzyme to create peptides, which were then separated by ultrafiltration. hAMSCs were exposed to different concentrations of bioactive compounds (100, 300, 500 and 700 µg/ml) for varying time periods. Cell proliferation was assessed using the with 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and to evaluate differentiation into bone tissue, the amount of mineral deposition was measured with alizarin red staining, and alkaline phosphatase enzyme activity was determined using a colorimetric method. The expression of Runx2, Osteocalcin, and β-Catenin genes expression was analyzed using RT-qPCR on days 7, 14 and 21 post-treatment. The results indicated that the <3 kDa peptide fraction of S. platensis and G. gracilis had no cytotoxic effects, increased cell proliferation at a concentration of 300 μg/ml, and enhanced the expression of osteogenic marker genes, alkaline phosphatase enzyme a activity, and calcium deposition in the extracellular matrix. In general, fractions that show positive effects on hAMSC differentiation have the potential to treat bone defects and promote osteoregeneration.

Investigating the indirect therapeutic effect of hAMSCs utilizing a novel scaffold (PGS-co-PCL/PGC/PPy/Gelatin) in myocardial ischemia-reperfusion-induced renal failure in male Wistar rats

BackgroundMyocardial ischemia-reperfusion (MI/R) occurs due to temporary or permanent interruptions in the coronary and circulatory system, indirectly affecting kidney function through reduced cardiac output for metabolic needs. In this study, the aim was to explore the indirect effects of using human amniotic membrane mesenchymal stem cells (hAMSCs) with the PGS-co-PCL/PGC/PPy/Gelatin scaffold in male rats with renal failure induced by miocardial ischemia-reperfusion. MethodsMI/R injury was induced in 48 male Wistar rats through left anterior descending artery ligation, divided into four groups (n=12); control group, cell group, scaffold group, and celss+scaffold group. Evaluations were conducted at two and thirty days post MI/R injury, encompassing echocardiography, biochemical, inflammatory markers analysis, and histological assessment. ResultsEchocardiographic findings exhibited notable enhancement in ejection fraction, fractional shortening, and stroke volume of treated groups compared to controls after 30 days (P< 0.05). Serum creatinine (P< 0.001) and urea (P< 0.05) levels significantly decreased in the scaffold+cells group) compared to the control group. The treated cells+ scaffold group displayed improved kidney structure, evidenced by larger glomeruli and reduced Bowman's space compared to the control group (P< 0.01). Immunohistochemical analysis indicated reduced TNF-α protein in the scaffold+ cells group (P< 0.05) in contrast to the control group (P< 0.05). Inflammatory factors IL-6, TNF-α, and AKT gene expression in renal tissues were improved in scaffold+ cells-treated animals. ConclusionOur research proposes the combination of hAMSCs and the PGS-co-PCL/PGC/PPy/Gelatin scaffold in MI/R injured rats appears to enhance renal function and reduce kidney inflammation by improving cardiac output.

Human amnion mesenchymal stem cells promote endometrial repair via paracrine, preferentially than transdifferentiation

BackgroundIntrauterine adhesion (IUA) is one of the most severe causes of infertility in women of childbearing age with injured endometrium secondary to uterine performance. Stem cell therapy is effective in treating damaged endometrium. The current reports mainly focus on the therapeutic effects of stem cells through paracrine or transdifferentiation, respectively. This study investigates whether paracrine or transdifferentiation occurs preferentially in treating IUA.MethodsHuman amniotic mesenchymal stem cells (hAMSCs) and transformed human endometrial stromal cells (THESCs) induced by transforming growth factor beta (TGF-β1) were co-cultured in vitro. The mRNA and protein expression levels of Fibronectin (FN), Collagen I, Cytokeratin19 (CK19), E-cadherin (E-cad) and Vimentin were detected by Quantitative real-time polymerase chain reaction (qPCR), Western blotting (WB) and Immunohistochemical staining (IHC). The Sprague-Dawley (SD) rats were used to establish the IUA model. hAMSCs, hAMSCs-conditional medium (hAMSCs-CM), and GFP-labeled hAMSCs were injected into intrauterine, respectively. The fibrotic area of the endometrium was evaluated by Masson staining. The number of endometrium glands was detected by hematoxylin and eosin (H&E). GFP-labeled hAMSCs were traced by immunofluorescence (IF). hAMSCs, combined with PPCNg (hAMSCs/PPCNg), were injected into the vagina, which was compared with intrauterine injection.ResultsqPCR and WB revealed that FN and Collagen I levels in IUA-THESCs decreased significantly after co-culturing with hAMSCs. Moreover, CK19, E-cad, and Vimentin expressions in hAMSCs showed no significant difference after co-culture for 2 days. 6 days after co-culture, CK19, E-cad and Vimentin expressions in hAMSCs were significantly changed. Histological assays showed increased endometrial glands and a remarkable decrease in the fibrotic area in the hAMSCs and hAMSCs-CM groups. However, these changes were not statistically different between the two groups. In vivo, fluorescence imaging revealed that GFP-hAMSCs were localized in the endometrial stroma and gradually underwent apoptosis. The effect of hAMSCs by vaginal injection was comparable to that by intrauterine injection assessed by H&E staining, MASSON staining and IHC.ConclusionsOur data demonstrated that hAMSCs promoted endometrial repair via paracrine, preferentially than transdifferentiation.

DNMT1 driven by mouse amniotic fluid mesenchymal stem cell exosomes improved corneal cryoinjury via inducing microRNA-33 promoter DNA hypermethylation modification in corneal epithelium cells.

Severe corneal cryoinjury can cause permanent corneal swelling and bullous keratopathy, one of the main reason for loss of sight. Mouse amniotic fluid mesenchymal stem cells (mAF-MSCs) can repair corneal damage caused by freezing; however, whether the exosomes derived from mAF-MSCs have the same repair effect is unknown. In this study, the mAF-MSC-exosomes were transplanted into the eyeballs of corneal cryoinjured mice. Histopathological examination showed that the mAF-MSC-exosomes improved the corneal structure and status of corneal epithelial cells in corneal cryoinjured mice. RRBS-sequencing showed that compared with the control group, four genes (Rpl13-ps6, miR-33, Hymai, and Plagl1), underwent DNA hypermethylation modification after mAF-MSC-exosomes treatment. The result of FISH indicated that miR-33-3p hybridization signals were enhanced in corneal epithelial cells from mice treated with mAF-MSC-exosomes. Semi-quantitative PCR and western blotting indicated that mAF-MSC-exosomes contained high levels of DNMT1 mRNA and protein. Additionally, luciferase report assays indicated that miR-33-3p overexpression in NIH-3T3 mouse embryonic fibroblast cells inhibited the activity of luciferase carrying a sequence from the 3' untranslated region of Bcl6. Moreover, BCL6 mRNA and protein levels in corneal tissues from mice treated with mAF-MSC-exosomes were higher than those in the control group. Therefore, our results suggested that mAF-MSC-exosomes could repair corneal cryoinjury by releasing DNMT1, which induced hypermethylation of the miR-33 promoter in corneal epithelial cells. Consequent downregulated miR-33 transcription upregulated Bcl6 expression, ultimately achieving the repair of corneal cryoinjury in mice.

The improvement of inflammatory infiltration and pregnancy outcome in mice with recurrent spontaneous abortion by human amniotic mesenchymal stem cells.

Recurrent spontaneous abortion is thought to be mostly triggered by immune-related causes. Mesenchymal stem cells, which exhibit the traits of multi-directional differentiation capacity and low immunogenicity, have recently been recommended as a viable treatment for spontaneous abortion-prone mice to increase the success of pregnancy. Amniotic membrane tissue is a byproduct of pregnancy and delivery that has a wide range of potential uses due to its easy access to raw materials and little ethical constraints. To construct an abortion-prone mouse model for this investigation, CBA/J female mice were coupled with male DBA/2 mice, while CBA/J female mice were paired with male BALB/c mice as a control. The identical volume of human amniotic mesenchymal stem cells or phosphate buffer was injected intraperitoneally on the 4.5th day of pregnancy. CBA/J female mice were sacrificed by cervical dislocation on the 13.5th day of pregnancy, the embryo absorption rate was calculated, and the uterus, decidua tissues and placenta were gathered for examination. Through detection, it was discovered that human amniotic mesenchymal stem cells significantly increased the expression of interleukin 10 and transforming growth factor beta, while they significantly decreased the expression of interleukin 1 beta and interleukin 6, improved vascular formation and angiogenesis, and minimized the embryo absorption rate and inflammatory cell infiltration in the recurrent spontaneous abortion + human amniotic mesenchymal stem cells group. In any case, human amniotic mesenchymal stem cells regulate inflammatory factors and cell balance at the maternal-fetal interface, which result in a reduction in the rate of embryo absorption and inflammatory infiltration and provide an innovative perspective to the clinical therapy of recurrent spontaneous abortion.