Amnion-derived Mesenchymal Stem Cells Research Articles

TIPE2-modified human amnion-derived mesenchymal stem cells promote the efficacy of allogeneic heart transplantation through inducing immune tolerance.

BackgroundImmune rejection of heart transplantation has been regarded as the biggest challenge encountered by a patient suffering from end-stage heart disease. The transplantation of human amnion-derived mesenchymal stem cells (hAD-MSCs) has exhibited promising application prospects in organ transplantation. However, its persistent unsatisfactory tolerance has limited the widespread application of this technology. We aim to investigate the role of tumor necrosis factor-α-induced protein-8 like-2 (TIPE2)-mediated hAD-MSCs in immune tolerance in heart transplantation and its molecular regulatory mechanisms.MethodsThis project detected the effect of TIPE2 on immune tolerance by constructing an allogeneic heart transplantation mouse model through which TIPE2-overexpressed hAD-MSCs were injected into recipients. The fluorescence distribution of TIPE2-hAD-MSCs in mice was observed by a small animal in vivo imaging system. Pathological changes of the transplanted heart were detected by hematoxylin and eosin (HE) staining. Flow cytometry was performed to detect the content of cardiac lymphocytes. The expression of immune-induced related factors was measured by quantitative real-time PCR (qRT-PCR) and western blot assays.ResultsTIPE2-hAD-MSCs protected myocardial tissue structures, reduced the spleen and thymus indexes in recipient mice, minimized the content of cardiac lymphocytes, reduced expressions of ERK, p38, and IFN-γ, and elevated expressions of both IL-10 and TGF-β, markedly improving the survival time and survival rates of recipient mice.ConclusionsTIPE2-hAD-MSCs induce immune tolerance and improve the survival rates of allogeneic heart transplantation in mice. This study is expected to offer an ideal source and target of cells for organ transplantation.

Human amnion-derived mesenchymal stem cells improved the reproductive function of age-related diminished ovarian reserve in mice through Ampk/FoxO3a signaling pathway

BackgroundAge-related diminished ovarian reserve (AR-DOR) reduced the quality of oocytes, resulting in decreased female fertility. Aging is tightly related to abnormal distribution and function of mitochondria, while mitophagy is a major process to maintain normal quality and quantity of mitochondria in cells, especially in oocytes which containing a large number of mitochondria to meet the demand of energy production during oocyte maturation and subsequent embryonic development. Ampk/FoxO3a signaling is crucial in the regulation of mitophagy. It is reported mesenchymal stem cells (MSCs) can improve ovarian function. Here we aim to explore if human amnion-derived mesenchymal stem cells (hAMSCs) are effective in improving ovarian function in AR-DOR mice and whether Ampk/FoxO3a signaling is involved.MethodsThe AR-DOR model mice were established by 32-week-old mice with 3–8 litters, significantly low serum sex hormone levels and follicle counts. The old mice were divided into 5 treatment groups: normal saline (NS, control), 1% human serum albumin (HSA, resolver), low dose (LD, 5.0 × 106cells/kg), middle dose (MD, 7.5 × 106cells/kg), and high dose (HD, 10.0 × 106cells/kg). The prepared hAMSCs were injected through tail vein. Serum sex hormone level, follicle counts, fertilization rate, gestation rate, little size, apoptosis of granulosa and stromal cells, expression level of Sod2, Ampk, and ratio of phosphorylated FoxO3a to total FoxO3a in ovaries were examined.ResultsOur results show that after hAMSC transplantation, the ovarian function in AR-DOR mice was significantly improved, meanwhile the apoptosis of granulosa and stromal cells in the ovaries was significantly repressed, the expression level of Ampk and the ratio of phosphorylated FoxO3a to total FoxO3a both were significantly increased, meanwhile increased Sod2 expression was also observed.ConclusionOur results demonstrate hAMSC transplantation via tail-injection can improve ovarian function of AR-DOR mice through Ampk/FoxO3a signaling pathway.

Topical Application of Conditioned Medium from Hypoxically Cultured Amnion-Derived Mesenchymal Stem Cells Promotes Wound Healing in Diabetic Mice.

Mesenchymal stem cells or their conditioned medium improve chronic wound healing, and their effect is enhanced by hypoxia. Diabetic foot ulcers are chronic wounds characterized by abnormal and delayed healing, which frequently require amputation. The authors evaluated the effect of topical application of conditioned medium from hypoxically cultured amnion-derived mesenchymal stem cells on wound healing in diabetic mice. Amnion-derived mesenchymal stem cells were cultured under 21% oxygen to prepare normoxic conditioned medium and under 1% oxygen to prepare hypoxic conditioned medium. Hydrogels containing standard medium, normoxic conditioned medium, or hypoxic conditioned medium were topically applied to excisional wounds of mice with streptozotocin-induced diabetes. Ulcer tissues were harvested on day 9; immunohistochemical and quantitative polymerase chain reaction analyses were performed to analyze angiogenesis, inflammatory cell infiltration, and expression levels of inflammation-related genes. Hypoxic conditioned medium significantly enhanced wound closure, increased capillary density and epithelization, and reduced macrophage infiltration. It also tended to reduce the infiltration of neutrophils and enhance the infiltration of regulatory T cells; it showed a tendency to downregulate the expression of the inflammation-related genes interleukin-1β, interleukin-6, chemokine ligand 1, and chemokine ligand 2. Normoxic conditioned medium exhibited similar effects, although they were of lesser magnitude than those of hypoxic conditioned medium. Hydrogels containing hypoxically cultured, amnion-derived mesenchymal stem cell conditioned medium accelerated wound healing in diabetic mice by enhancing angiogenesis, accelerating epithelization, and suppressing inflammation. Therefore, topical application of amnion mesenchymal stem cell-derived hypoxic conditioned medium could be a novel treatment for diabetic foot ulcers.

Human amnion-derived mesenchymal stem cells attenuate xenogeneic graft-versus-host disease by preventing T cell activation and proliferation

Acute graft-versus-host disease (GVHD) is characterized by severe tissue damage that is a life-threatening complication of allogeneic hematopoietic stem cell transplantation. Due to their immunosuppressive properties, mesenchymal stem cells (MSC) have been increasingly examined for the treatment of immune-related diseases. We aimed to assess the immunosuppressive effects of human amnion-derived MSC (AMSC) in a xenogeneic GVHD NOD/Shi-scid IL2rγnull mouse model using human peripheral blood mononuclear cells (PBMC). Additionally, we used human bone marrow-derived MSC (BMSC) as comparative controls to determine differences in immunomodulatory functions depending on the MSC origin. Administration of AMSC significantly prolonged survival, and reduced human tumor necrosis factor-α (TNF-α) concentration and percentage of programmed cell death protein-1 receptor (PD-1)+CD8+ T cell populations compared with in GVHD control mice. Furthermore, colonic inflammation score and percentage of human CD8+ T cell populations in AMSC-treated mice were significantly lower than in GVHD control and BMSC-treated mice. Interestingly, gene expression and protein secretion of the PD-1 ligands were higher in AMSC than in BMSC. These findings are the first to demonstrate that AMSC exhibit marked immunosuppression and delay acute GVHD progression by preventing T cell activation and proliferation via the PD-1 pathway.

Human amnion-derived mesenchymal stem cells promote osteogenic differentiation of lipopolysaccharide-induced human bone marrow mesenchymal stem cells via ANRIL/miR-125a/APC axis

Background and aimPeriodontitis is a chronic inflammatory disease inducing the absorption of alveolar bone and leading to tooth loss. Human amnion-derived mesenchymal stem cells (HAMSCs) have been used for studying inflammatory processes. This study aimed to explore the role of long noncoding RNA (lncRNA) antisense noncoding RNA in the INK4 locus (ANRIL) in HAMSC-driven osteogenesis in lipopolysaccharide (LPS)-induced human bone marrow mesenchymal stem cells (HBMSCs).MethodsThe cells were incubated with a co-culture system. Reactive oxygen species (ROS) level and superoxide dismutase (SOD) activity were used to detect the oxidative stress level. Flow cytometry was performed to determine cell proliferation. The alkaline phosphatase (ALP) activity, Alizarin red assay, cell transfection, and rat mandibular defect model were used to evaluate the osteogenic differentiation. Quantitative real-time reverse transcription–polymerase chain reaction (RT-PCR), Western blot analysis, dual-luciferase reporter assay, and immunofluorescence staining were used to evaluate the molecular mechanisms.ResultsThis study showed that HAMSCs promoted the osteogenesis of LPS-induced HBMSCs, while the ANRIL level in HBMSCs decreased during co-culture. ANRIL had no significant influence on the proliferation of LPS-induced HBMSCs. However, its overexpression inhibited the HAMSC-driven osteogenesis in vivo and in vitro, whereas its knockdown reversed these effects. Mechanistically, this study found that downregulating ANRIL led to the overexpression of microRNA-125a (miR-125a), and further contributed to the competitive binding of miR-125a and adenomatous polyposis coli (APC), thus significantly activating the Wnt/β-catenin pathway.ConclusionThe study indicated that HAMSCs promoted the osteogenic differentiation of LPS-induced HBMSCs via the ANRIL/miR-125a/APC axis, and offered a novel approach for periodontitis therapy.

Human Amnion-Derived Mesenchymal Stromal Cells in Cirrhotic Patients with Refractory Ascites: A Possible Anti-Inflammatory Therapy for Preventing Spontaneous Bacterial Peritonitis

Cirrhosis is associated with dysregulated immune cell activation and immune dysfunction. These conditions modify gut flora, facilitate bacterial translocation, and increase susceptibility to bacterial peritonitis and consequent systemic infections by dramatically affecting long-term patient survival. Human amnion-derived mesenchymal stromal cells (hA-MSCs) exert immunomodulatory potential benefit, and have the ability to modulate their actions, especially in situations requiring immune activation through mechanisms not fully understood. In this study, we aimed to investigate, in vitro, the immunostimulant or immunosuppressive effects of hA-MSCs on cellular components of ascitic fluid obtained from cirrhotic patients with refractory ascites. We found that hA-MSCs viability is not affected by ascitic fluid and, interestingly, hA-MSCs diminished the pro-inflammatory cytokine production, and promoted anti-inflammatory M2 macrophage polarization. Moreover, we found that there was no simultaneous significant decrease in the M1-like component, allowing a continual phagocytosis activity of macrophages and NK cells to restore a physiological condition. These data highlight the plasticity of hA-MSCs’ immunomodulatory capacity, and pave the way to further understanding their role in conditions such as spontaneous bacterial peritonitis.Graphical abstract

Amnion-Derived Mesenchymal Stem Cell Exosomes-Mediated Autophagy Promotes the Survival of Trophoblasts Under Hypoxia Through mTOR Pathway by the Downregulation of EZH2.

Human amnion-derived mesenchymal stem cells (AD-MSCs) have been reported as a promising effective treatment to repair tissue. Trophoblast dysfunction during pregnancy is significantly involved in the pathogenesis of preeclampsia (PE). To understand how AD-MSCs regulated trophoblast function, we treated trophoblasts with AD-MSC-derived exosomes under hypoxic conditions. The treatment markedly enhanced the trophoblast proliferation and autophagy. Furthermore, significant decrease of EZH2 levels and inactivation of mTOR signaling were observed in AD-MSC exosomes-treated trophoblasts. Consistent with these findings, overexpression of EZH2 activated the mTOR signaling in trophoblasts, and reduced the autophagy and survival of trophoblasts, even in the presence of AD-MSC-derived exosomes. In addition, EZH2 inhibition exhibited the same trophoblast autophagy-promoting effect as induced by AD-MSC-derived exosomes, also accompanied by the inactivation of mTOR signaling. Importantly, when EZH2 was overexpressed in trophoblasts treated with PQR620, a specific mTOR signaling inhibitor, the autophagy and proliferation in trophoblasts were decreased. Studies on human placental explants also confirmed our findings by showing that the expression levels of EZH2 and mTOR were decreased while the autophagy-associated protein level was increased by AD-MSC-derived exosome treatment. In summary, our results suggest that EZH2-dependent mTOR signaling inactivation mediated by AD-MSC-derived exosomes is a prerequisite for autophagy augmentation in hypoxic trophoblasts.

Circular RNA-ABCB10 promotes angiogenesis induced by conditioned medium from human amnion-derived mesenchymal stem cells via the microRNA-29b-3p/vascular endothelial growth factor A axis.

The powerful ability of human amnion-derived mesenchymal stem cells (hAMSCs) to promote angiogenesis suggests that they may facilitate angiogenesis-associated therapeutic strategies. However, the molecular mechanisms underlying hAMSC-induced angiogenesis remain largely unknown. The present study results suggested that enhanced migration and tube formation in human umbilical vein endothelial cells (HUVECs) was induced by conditioned medium from hAMSCs (hAMSC-CM). In addition, culture with this conditioned medium resulted in the increased expression of circular RNA ATP binding cassette subfamily B member 10 (circ-ABCB10) and vascular endothelial growth factor A (VEGFA). In the present study genes related to thecirc-ABCB10/microRNA (miR)-29b-3p/VEGFA pathway were predicted using bioinformatics software, and further investigated using in vitro luciferase reporter assays. Loss-of-function assays were performed using small interfering RNAs (siRNAs). The results suggested that siRNA-silencing of circ-ABCB10 in HUVECs weakened migration and tube formation of HUVECs following hAMSC-CM treatment and reduced the levels of VEGFA expression. Treatment with an miR-29b-3p inhibitor could largely rescue these effects in HUVECs, following circ-ABCB10 silencing. The present study results suggest that the circ-ABCB10/miR-29b-3p/VEGFA pathway may be involved in the pro-angiogenic role of hAMSC-CM in HUVECs.

Human amnion-derived mesenchymal stem cells promote osteogenic differentiation of human bone marrow mesenchymal stem cells via H19/miR-675/APC axis.

Bone volume inadequacy is an emerging clinical problem impairing the feasibility and longevity of dental implants. Human bone marrow mesenchymal stem cells (HBMSCs) have been widely used in bone remodeling and regeneration. This study examined the effect of long noncoding RNAs (lncRNAs)-H19 on the human amnion-derived mesenchymal stem cells (HAMSCs)-droved osteogenesis in HBMSCs. HAMSCs and HBMSCs were isolated from abandoned amniotic membrane samples and bone marrow. The coculture system was conducted using transwells, and H19 level was measured by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). The mechanism was further verified. We here discovered that osteogenesis of HBMSCs was induced by HAMSCs, while H19 level in HAMSCs was increased during coculturing. H19 had no significant effect on the proliferative behaviors of HBMSCs, while its overexpression of H19 in HAMSCs led to the upregulated osteogenesis of HBMSCs in vivo and in vitro; whereas its knockdown reversed these effects. Mechanistically, H19 promoted miR-675 expression and contributed to the competitively bounding of miR-675 and Adenomatous polyposis coli (APC), thus significantly activating the Wnt/β-catenin pathway. The results suggested that HAMSCs promote osteogenic differentiation of HBMSCs via H19/miR-675/APC pathway, and supply a potential target for the therapeutic treatment of bone-destructive diseases.

Placenta-derived mesenchymal stem cells enhance human liver organoid maturation: translational implications for liver regeneration

Background & Aim The global prevalence of end-stage liver disease (ESLD) has reached very high levels, and, to date, the best treatment option is orthotopic liver transplantation (1). However, due to the complexity of this procedure and the limited numbers of healthy organ donors, new therapeutic options are being sought. Liver progenitor and multipotent stem cells offer potential cell sources that could be used clinically (2). Different studies have reported therapeutic effects of transplanted mesenchymal stem cells (MSCs) on ESLD (3). Indeed, an improvement of liver function has been achieved in patients with liver cirrhosis after autologous MSCs injection (4). Based on our recent work where we showed that a three-dimensional (3D) culture method is effective to improve MSCs paracrine activity (5), in this work, we used a human liver organoid system to study if the paracrine signal secreted by human amnion-derived MSCs (hAMSCs) affect liver stem/progenitor cells to differentiate (Fig. 1). Epithelial organoids recapitulate multiple aspects of real organs, making them promising models of organ function to model and treat human disease (6). Methods, Results & Conclusion Our data show that the expanded human liver organoids had distinct traits in the ALB production, Krt19 and HNF1β expression when compared to the reference cell line HepG2 cells. On liver organoids, we tested conventional differentiation medium (DM) conditioned by both 2D (2D CM) and 3D (3D CM) hAMSCs cultures observing that ALB, NTCP (markers of mature hepatocytes), and LGR5 (stem cell marker) expression were significantly upregulated by 3D CM compared with both 2D CM and conventional DM. Furthermore, CYP3A4 expression (marker of mature hepatocytes) was upregulated in both 2D and 3D CM. These data were also confirmed by the over-production/activity of both ALB and CYP3A4 observed in organoids grown with 3D CM. Liver epithelial dysfunctional repair play a role in the development of liver disease, and, effective repair likely requires the normal functioning of liver stem/progenitor cells. Herein, we showed that hAMSCs, principally grown in 3D cultures, has the potential to increase hepatic stem/progenitor cell differentiation, demonstrating that soluble factors secreted by hAMSCs are likely responsible for the response. This work revealing a potential approach to improve cell transplantation with the ultimate goal being efficient liver regeneration.

Intratracheal Transplantation of Amnion-Derived Mesenchymal Stem Cells Ameliorates Hyperoxia-Induced Neonatal Hyperoxic Lung Injury via Aminoacyl-Peptide Hydrolase.

Background and ObjectivesBronchopulmonary dysplasia (BPD) has major effects in premature infants. Although previous literature has indicated that mesenchymal stem cells (MSCs) can alleviate lung pathology in BPD newborns and improve the survival rate, few research have been done investigating significantly differentially expressed genes in the lungs before and after MSCs therapy. The aim of this study is to identify differentially expressed genes in lung tissues before and after hAD-MSC treatment.Methods and ResultsHuman amnion-derived MSCs (hAD-MSCs) were cultured and met the MSCs criteria for cell phenotype and multidirectional differentiation. Then we confirmed the size of hAD-MSCs-EXOs and their expressed markers. An intratracheal drip of living cells showed the strongest effect on NHLI compared to cellular secretions or exosomes, both in terms of ameliorating pulmonary edema and reducing inflammatory cell infiltration. Through gene chip hybridization, PCR, and western blotting, acylaminoacyl-peptide hydrolase (APEH) expression was found to be significantly decreased under hyperoxia, and significantly increased after hAD-MSC treatment.ConclusionsThe intratracheal transplantation of hAD-MSCs ameliorated NHLI in neonatal rats through APEH.

In Vitro Regeneration of Decellularized Pig Esophagus Using Human Amniotic Stem Cells.

Decellularization of esophagus was studied using three different protocols. The sodium deoxycholate/DNase-I (SDC/DNase-I) method was the most successful as evidenced by histology and DNA quantification of the acellular scaffolds. Acellular scaffolds were further analyzed and compared with native tissue by histology, quantitative analysis of DNA, and extracellular matrix (ECM) proteins. Histologically, the SDC/DNase-I protocol effectively produced scaffold with preserved structural architecture similar to native tissue architecture devoid of any cell nucleus. ECM proteins, such as collagen, elastin, and glycosaminoglycans were present even after detergent-enzymatic decellularization. Immunohistochemical analysis of acellular scaffold showed weak expression of Gal 1, 3 Gal epitope compared with native tissue. For performing recellularization, human amnion-derived mesenchymal stem cells (MSCs) and epithelial cells were seeded onto acellular esophagus in a perfusion–rotation bioreactor. In recellularized esophagus, immunohistochemistry showed infiltration of MSCs from adventitia into the muscularis externa and differentiation of MSCs into the smooth muscle actin and few endothelial cells (CD31). Our study demonstrates successful preparation and characterization of a decellularized esophagus with reduced load of Gal 1, 3 Gal epitope with preserved architecture and ECM proteins similar to native tissue. Upon subsequent recellularization, xenogeneic acellular esophagus also supported stem cell growth and partial differentiation of stem cells. Hence, the current study offers the hope for preparing a tissue-engineered esophagus in vitro which can be transplanted further into pigs for further in vivo evaluation.

Effect of ‘in air’ freezing on post-thaw recovery of Callithrix jacchus mesenchymal stromal cells and properties of 3D collagen-hydroxyapatite scaffolds

Through enabling an efficient supply of cells and tissues in the health sector on demand, cryopreservation is increasingly becoming one of the mainstream technologies in rapid translation and commercialization of regenerative medicine research. Cryopreservation of tissue-engineered constructs (TECs) is an emerging trend that requires the development of practically competitive biobanking technologies. In our previous studies, we demonstrated that conventional slow-freezing using dimethyl sulfoxide (Me2SO) does not provide sufficient protection of mesenchymal stromal cells (MSCs) frozen in 3D collagen-hydroxyapatite scaffolds. After simple modifications to a cryopreservation protocol, we report on significantly improved cryopreservation of TECs.Porous 3D scaffolds were fabricated using freeze-drying of a mineralized collagen suspension and following chemical crosslinking. Amnion-derived MSCs from common marmoset monkey Callithrix jacchus were seeded onto scaffolds in static conditions. Cell-seeded scaffolds were subjected to 24 h pre-treatment with 100 mM sucrose and slow freezing in 10% Me2SO/20% FBS alone or supplemented with 300 mM sucrose. Scaffolds were frozen ‘in air’ and thawed using a two-step procedure. Diverse analytical methods were used for the interpretation of cryopreservation outcome for both cell-seeded and cell-free scaffolds. In both groups, cells exhibited their typical shape and well-preserved cell-cell and cell-matrix contacts after thawing. Moreover, viability test 24 h post-thaw demonstrated that application of sucrose in the cryoprotective solution preserves a significantly greater portion of sucrose-pretreated cells (more than 80%) in comparison to Me2SO alone (60%).No differences in overall protein structure and porosity of frozen scaffolds were revealed whereas their compressive stress was lower than in the control group. In conclusion, this approach holds promise for the cryopreservation of ‘ready-to-use’ TECs.

Effects of Human Amnion-Derived Mesenchymal Stem Cell (hAD-MSC) Transplantation In Situ on Primary Ovarian Insufficiency in SD Rats.

Human amnion-derived mesenchymal stem cell (hAD-MSC) transplantation can repair ovarian injury and improve ovarian function in rats with chemotherapy-induced primary ovarian insufficiency (POI). However, ensuring that stem cells home to the ovary to improve their effects on ovarian injury is challenging. This research aimed to directly inject ovarian tissue with hAD-MSCs and improve the homing of stem cells to the ovary. The animals were divided into POI, hAD-MSC (tail vein) treatment, hAD-MSC (in situ) treatment, and control groups. POI rat models were established by intraperitoneal injection of cyclophosphamide (CTX) and busulfan (BUS). The hAD-MSCs isolated from the amnion were injected into the tail vein or ovary of POI rats. The estrous cycle, serum sex hormone levels, follicle counts, ovarian pathological changes, and proteome of the ovaries were evaluated. hAD-MSCs were successfully isolated and cultured from the amnion. Both hAD-MSC (tail vein) and hAD-MSC (in situ) transplantation increased body weight, improved the AMH levels and follicle numbers, and reduced reproductive organ injuries in POI rats. Transplantation of hAD-MSCs (in situ) upregulated 24 proteins and downregulated 4 proteins. Both hAD-MSC (tail vein) and hAD-MSC (in situ) transplantations can repair ovarian injury and improve ovarian function in rats with chemotherapy-induced POI. The paracrine proteome of hAD-MSCs in the ovarian microenvironment can protect against chemotherapy-induced damage by reducing apoptosis and promoting angiogenesis, cell proliferation, and gene expression.

Circ-100290 Positively Regulates Angiogenesis Induced by Conditioned Medium of Human Amnion-Derived Mesenchymal Stem Cells Through miR-449a/eNOS and miR-449a/VEGFA Axes.

The powerful pro-angiogenic capacity of human amnion-derived mesenchymal stem cells (hAMSCs) could be a valuable therapeutic angiogenesis strategy for bone regeneration. However, the molecular mechanisms underlying this process remain largely unknown. Herein, we report upregulated expression of circular RNA 100290 (circ-100290) and an enhanced angiogenic phenotype of human umbilical vein endothelial cells (HUVECs) incubated with conditioned medium from hAMSCs (hAMSC-CM), whereas downregulation of circ-100290 reversed the pro-angiogenic capacity of HUVECs induced by hAMSC-CM. Circ-100290/microRNA 449a (miR-449a)/endothelial nitric oxide synthase (eNOS) and circ-100290/miR-449a/vascular endothelial growth factor A (VEGFA) axes were predicted by a bioinformatics method and subsequently verified by luciferase reporter assays in vitro. Gain- or loss-of-function assays were then performed using small interfering RNAs (siRNAs) targeting circ-100290, or a plasmid overexpressing circ-100290. As expected, downregulation of circ-100290 in HUVECs led to weakened tube formation and migration of HUVECs following hAMSC-CM treatment, along with decreased expression of eNOS and VEGFA. In contrast, upregulation of circ-100290 led to enhanced tube formation and migration of HUVECs following hAMSC-CM treatment, along with increased expression of eNOS and VEGFA. Furthermore, a miR-449a inhibitor could largely rescue the effect of circ-100290 silencing on HUVECs, whereas a miR-449a mimic could significantly rescue the effect of overexpressing circ-100290 on HUVECs. Functional assays using eNOS or VEGF receptor inhibitors indicated eNOS and VEGFA may be important targets of miR-449a. Finally, a Matrigel plug assay revealed weakened angiogenesis when circ-100290 was silenced in HUVECs, but enhanced angiogenesis when circ-100290 was overexpressed in vivo. Our results suggest that circ-100290 might function via miR-449a/eNOS and miR-449a/VEGFA axes in the pro-angiogenic role of hAMSC-CM on HUVECs.

P2569Self-adhesive bi-layered dressing incorporating amnion-derived mesenchymal stromal cells for the treatment of heart failure: a pre-clinical proof of concept study

Abstract Introduction Mesenchymal stromal cell (MSC) transplantation is a promising treatment to promote myocardial repair. Among various sources, the amnion has an advantage in mass production of high-quality MSCs due to its large initial cell-yield and prenatal nature of isolated cells. In addition to the powerful tissue-repair potential, amnion-derived MSCs (AMSCs) exhibit a robust immunomodulative ability, enabling allogeneic transplantation without immunosuppressive reagents. We here report a novel bioengineering technique to deliver AMSCs for myocardial repair by epicardial placement of self-adhesive, bi-layered, AMSC-incorporating dressings (AMSC-dressing), which is fabricable on-site (Figure A). Methods and results AMSC-dressing was fabricated by spreading AMSC suspension on the inner layer of a fibrin sealant film, composed of fibrinogen and thrombin. Due to the resulting adhesive AMSC-fibrin complex, the AMSC-dressing firmly adhered to the heart surface without the need for suture or additional glue. The outer collagen layer of the film facilitated the easy handling and also protected the AMSC-fibrin complex from external damage. We applied a 1 cm2 dressing containing 0, 1, 2 or 4 millions of rat AMSCs to a rat ischemic cardiomyopathy model (4 weeks post coronary artery ligation). Intramyocardial (IM) injection of 4 millions of AMSCs and sham treatment were also conducted. Echocardiography and catheterization consistently demonstrated that AMSC-dressing therapy improved cardiac function and reduced heart dilatation in a dose-dependent manner compared to the sham control. Furthermore, this therapeutic effect exceeded that of IM injection (Figure B). Histological analyses revealed that AMSC-dressing therapy resulted in augmented myocardial tissue repair (increased neovascularization, attenuated pathological fibrosis and reduced cardiomyocyte hypertrophy) compared to IM injection and sham groups. These effects were associated with increased upregulation of a range of tissue repair-related genes including Il10, Cxcl12, Igf1, Timp1, Hif1a, Tgfb, Mmp2, Hgf, Fgf2 and Vegf. Of note, it was elucidated that both initial retention and subsequent survival of donor AMSCs were enhanced by the dressing technique compared to IM injection. In addition, in vitro studies demonstrated that culturing in a fibrin glue not only enhanced upregulation of tissue-repair genes of AMSCs but also improved their survival against environmental stress through activating the Akt/PI3K cell-survival pathway. Conclusion AMSC-dressing therapy enhanced both quantity and quality of donor cell engraftment, leading to the augmented therapeutic efficacy, compared to the current method. Furthermore, this technique is user-friendly and requires no specialized equipment at the treating hospital, highlighting its great potential to be a widely-adopted, standard treatment for heart failure. Further development of this advanced cell therapy towards clinical application is justified. Acknowledgement/Funding British Heart Foundation, Heart Research UK, Japan Agency for Medical Research and Development, Kaneka Corporation

Human amnion-derived mesenchymal stem cells enhance the osteogenic differentiation of human adipose-derived stem cells by promoting adiponectin excretion via the APPL1-ERK1/2 signaling pathway.

Human adipose-derived stem cells (HASCs) represent pluripotent cells capable of differentiating into the bone tissue. Meanwhile, human amnion-derived mesenchymal stem cells (HAMSCs) could cause mesenchymal stem cells to differentiate into the bone tissue. This work assessed the osteogenic effects exerted by HAMSCs on the potential of HASCs to form bone cells. Cell growth was evaluated flow-cytometrically. Differentiation into osteoblasts and mineral formation were assessed by chromogenic alkaline phosphatase activity substrate assay and Alizarin red S staining. Adiponectin (APN), the adipocytokine secreted by adipocytes, was evaluated by enzyme-linked immunosorbent assay. In this study, HAMSCs concentration-dependently induced growth, osteoblastic differentiation, and APN excretion in HASCs. Mechanistically, immunofluorescence and immunoblot revealed HAMSCs promoted cytosolic translocation of leucine zipper motif (APPL1) from the nucleus and induced extracellular signaling-regulated kinase 1/2 (ERK1/2) phosphorylation in HASCs. Furthermore, HAMSC effects were markedly blunted by pretreatment with APPL1 siRNA and U0126, an ERK1/2 signaling inhibitor with high selectivity. These results suggested that APN excretion is not suppressed by APPL1 knockdown in HASCs, but by ERK1/2 inhibition. These findings collectively indicate that HAMSCs induce the osteogenesis of HASCs by promoting APN excretion through APPL1-ERK1/2 activation.

Effect of Mother’s Age and Pathology on Functional Behavior of Amniotic Mesenchymal Stromal Cells—Hints for Bone Regeneration

Human amnion-derived mesenchymal stromal cells (hAMSCs) are used increasingly in regenerative medicine applications, including dentistry. The aim of this study was to evaluate if hAMSCs from aged and pathological mothers could be affected in their phenotype and functional behavior. hAMSCs were isolated from placentas of women aged younger than 40 years (Group 1, n = 7), older than 40 years (Group 2, n = 6), and with pre-eclampsia (Group 3, n = 5). Cell yield and viability were assessed at isolation (p0). Cell proliferation was evaluated from p0 to p5. Passage 2 was used to determine the phenotype, the differentiation capacity, and the adhesion to machined and sandblasted titanium disks. hAMSCs recovered from Group 3 were fewer than in Group 1. Viability and doubling time were not different among the three groups. Percentages of CD29+ cells were significantly lower in Group 3, while percentages of CD73+ cells were significantly lower in Groups 2 and 3 as compared with Group 1. hAMSCs from Group 2 showed a significant lower differentiation capacity towards chondrogenic and osteogenic lineages. hAMSCs from Group 3 adhered less to titanium surfaces. In conclusion, pathology can affect hAMSCs in phenotype and functional behavior and may alter bone regeneration capacities.

Safety and efficacy of amnion-derived mesenchymal stem cells (AM01) in patients with steroid-refractory acute graft-versus-host disease after allogeneic haematopoietic stem cell transplantation: a study protocol for a phase I/II Japanese trial

IntroductionRegenerative medicine and cell therapies have been gaining much attention among clinicians. Therapeutic infusion of mesenchymal stromal cells (MSCs) is now a leading investigational strategy for the treatment of acute...

A comparative study of HAMSCs/HBMSCs transwell and mixed coculture systems.

Our previous studies indicated that a coculture system containing human amnion-derived mesenchymal stem cells (HAMSCs) and human bone marrow mesenchymal stem cells (HBMSCs) has the potential of application for bone regeneration. However, there is currently no enough comparative investigation between HAMSCs/HBMSCs transwell and mixed coculture systems. This study aimed to assess the phenotype and mechanisms regulated by indirect and direct coculture systems, respectively. Two in vitro models were employed with HAMSCs and HBMSCs at a ratio of 3:1, and then were analyzed by a series of processes, including flow cytometry, alkaline phosphatase (ALP) substrate assays, Alizarin red S staining, quantitative reverse transcription polymerase chain reaction (RT-qPCR), and Western blot analysis. We found that cell proliferation, ALP activity, mineralized matrix formation, and osteoblast-related mRNA expression were accelerated in transwell coculture system compared with mixed coculture system. Conditioned medium from transwell coculture system achieved an elevated level of vascular endothelial growth factor and induced more vascular structures in human umbilical vein endothelial cells than those of mixed coculture system. Moreover, we observed that transwell coculture system, promoted osteogenesis and angiogenesis by maintaining stemness through extracellular regulated protein kinases 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) signaling pathway. U0126, a selective inhibitor of ERK1/2 MAPK signaling, significantly suppressed maintaining of the stemness-based effects on transwell coculture system. Taken together, our results compared the merits of two different models and clarified the role of HAMSCs/HBMSCs transwell coculture system in the development of bone tissue engineering. © 2019 IUBMB Life, 2019.