Amnion-derived Mesenchymal Stem Cells Research Articles

Hypoxic culture enhances the antimicrobial activity of amnion-derived mesenchymal stem cells, thereby reducing bacterial load and promoting wound healing in diabetic mice

BackgroundConditioned medium from amnion-derived mesenchymal stem cells (AMSCs) enhances wound healing, a process that is further improved under hypoxic culture conditions. Diabetic foot ulcers are difficult to treat and are frequently complicated by a high rate of bacterial infections, mainly Staphylococcus aureus, which can lead to limb amputation and death. Here, we topically applied conditioned medium from AMSCs cultured under hypoxic conditions to S. aureus-infected wounds in diabetic mice to investigate its effect on bacterial counts and wound healing. MethodsWe prepared conditioned medium by culturing AMSCs under 21 % or 1 % O2 and investigated its effects on S. aureus. We infected skin wounds of diabetic mice with S. aureus and treated these with hydrogels containing the conditioned medium to examine its effect on bacterial inhibition and wound healing. ResultsConditioned medium from AMSCs cultured under 1 % O2 contained higher levels of the antimicrobial peptide LL-37. It significantly inhibited S. aureus growth in vitro, reduced bacterial counts in infected wounds, and facilitated wound closure in diabetic mice. ConclusionsHydrogels containing conditioned medium from hypoxically cultured AMSCs inhibited the growth of S. aureus and promoted wound healing in a mouse model of diabetic wounds.

Human Amniotic MSC Response in LPS-Stimulated Ascites from Patients with Cirrhosis: FOXO1 Gene and Th17 Activation in Enhanced Antibacterial Activation.

Spontaneous bacterial peritonitis (SBP) is a severe complication in patients with decompensated liver cirrhosis and is commonly treated with broad spectrum antibiotics. However, the rise of antibiotic resistance requires alternative therapeutic strategies. As recently shown, human amnion-derived mesenchymal stem cells (hA-MSCs) are able, in vitro, to promote bacterial clearance and modulate the immune and inflammatory response in SBP. Our results highlight the upregulation of FOXO1, CXCL5, CXCL6, CCL20, and MAPK13 in hA-MSCs as well as the promotion of bacterial clearance, prompting a shift in the immune response toward a Th17 lymphocyte phenotype after 72 h treatment. In this study, we used an in vitro SBP model and employed omics techniques (next-generation sequencing) to investigate the mechanisms by which hA-MSCs modify the crosstalk between immune cells in LPS-stimulated ascitic fluid. We also validated the data obtained via qRT-PCR, cytofluorimetric analysis, and Luminex assay. These findings provide further support to the hope of using hA-MSCs for the prevention and treatment of infective diseases, such as SBP, offering a viable alternative to antibiotic therapy.

Intravenous Administration of Mesenchymal Stem Cell-Derived Exosome Alleviates Spinal Cord Injury by Regulating Neutrophil Extracellular Trap Formation through Exosomal miR-125a-3p.

Spinal cord injury (SCI) leads to devastating sequelae, demanding effective treatments. Recent advancements have unveiled the role of neutrophil extracellular traps (NETs) produced by infiltrated neutrophils in exacerbating secondary inflammation after SCI, making it a potential target for treatment intervention. Previous research has established that intravenous administration of stem cell-derived exosomes can mitigate injuries. While stem cell-derived exosomes have demonstrated the ability to modulate microglial reactions and enhance blood-brain barrier integrity, their impact on neutrophil deactivation, especially in the context of NETs, remains poorly understood. This study aims to investigate the effects of intravenous administration of MSC-derived exosomes, with a specific focus on NET formation, and to elucidate the associated molecular mechanisms. Exosomes were isolated from the cell supernatants of amnion-derived mesenchymal stem cells using the ultracentrifugation method. Spinal cord injuries were induced in Sprague-Dawley rats (9 weeks old) using a clip injury model, and 100 μg of exosomes in 1 mL of PBS or PBS alone were intravenously administered 24 h post-injury. Motor function was assessed serially for up to 28 days following the injury. On Day 3 and Day 28, spinal cord specimens were analyzed to evaluate the extent of injury and the formation of NETs. Flow cytometry was employed to examine the formation of circulating neutrophil NETs. Exogenous miRNA was electroporated into neutrophil to evaluate the effect of inflammatory NET formation. Finally, the biodistribution of exosomes was assessed using 64Cu-labeled exosomes in animal positron emission tomography (PET). Rats treated with exosomes exhibited a substantial improvement in motor function recovery and a reduction in injury size. Notably, there was a significant decrease in neutrophil infiltration and NET formation within the spinal cord, as well as a reduction in neutrophils forming NETs in the circulation. In vitro investigations indicated that exosomes accumulated in the vicinity of the nuclei of activated neutrophils, and neutrophils electroporated with the miR-125a-3p mimic exhibited a significantly diminished NET formation, while miR-125a-3p inhibitor reversed the effect. PET studies revealed that, although the majority of the transplanted exosomes were sequestered in the liver and spleen, a notably high quantity of exosomes was detected in the damaged spinal cord when compared to normal rats. MSC-derived exosomes play a pivotal role in alleviating spinal cord injury, in part through the deactivation of NET formation via miR-125a-3p.

Intravenous administration of human amnion-derived mesenchymal stem cells improves gait and sensory function in mouse models of spinal cord injury.

Spinal cord injury (SCI) leads to severe disabilities and remains a significant social and economic challenge. Despite advances in medical research, there are still no effective treatments for SCI. Human amnion-derived mesenchymal stem cells (hAMSCs) have shown potential due to their anti-inflammatory and neuroprotective effects. This study evaluates the therapeutic potential of intravenously administered hAMSCs in SCI models. Three days after induction of SCI with forceps calibrated with a 0.2 mm gap, hAMSCs or vehicle were administered intravenously. Up to 4 weeks of SCI induction, motor function was assessed by scores on the Basso Mouse Locomotor Scale (BMS) and the Basso-Beattie-Bresnahan Scale (BBB), and sensory function by hindlimb withdrawal reflex using von Frey filaments. Six weeks after SCI induction, gait function was assessed using three-dimensional motion analysis. Immunohistochemistry, polymerase chain reaction (PCR), flow cytometry, and ELISA assay were performed to clarify the mechanisms of functional improvement. The hAMSC treatment significantly improved sensory response and gait function. In the SCI site, immunohistochemistry showed a reduction in Iba1-positive cells and PCR revealed decreased TNFα and increased BDNF levels in the hAMSC-treated group. In assessing the systemic inflammatory response, hAMSC treatment reduced monocytic bone marrow-derived suppressor cells (M-MDSCs) and Ly6C-positive inflammatory macrophages in the bone marrow by flow cytometry and serum NO levels by ELISA assay. This study demonstrates the therapeutic potential of the hAMSC in SCI, with improvements in gait and sensory functions and reduced inflammation both locally and systemically. The findings support further investigation of the hAMSC as a potential treatment for SCI, focusing on their ability to modulate inflammation and promote neuroprotection.

Human amnion-derived mesenchymal stem cells improve subclinical hypothyroidism by immunocompetence mediating apoptosis inhibition on thyroid cells in aged mice

Subclinical hypothyroidism (SCH) affects 10% of the global population, which is most prevalent in women and the elderly. However, it remains debatable whether the elderly with subclinical hypothyroidism needs thyroxine supplement. Human amnion-derived mesenchymal stem cells (hAMSCs) could play important roles in autoimmune diseases, suggesting that hAMSC be a candidate to regulate the thyroid function of female age-related subclinical hypothyroidism. Herein, we established the model of SCH in the aged female mice. This study was designed to investigate whether human amnion-derived mesenchymal stem cells (hAMSC) could effect on immune regulation, apoptosis inhibition of thyroid cells, thyroid function, blood lipid levels, and heart function. In addition, qualified hAMSCs were intravenously injected into aged female SCH mice via the tail vein on day 0 and day 10. The levels of thyroid hormone and blood lipids as well as cardiac function, serum immunological indexes, and apoptosis of thyroid cells were then analyzed on day 5, 10, 15, and 20; meanwhile, the quantity of Th1, Th2, Th17, and Treg immune cells in peripheral blood was evaluated before and on day 20 post-injection. Our study demonstrated that after hAMSC transplantation, the thyroid functions, blood lipid levels, and heart function indexes of age-related SCH (AR-SCH) mice were significantly improved. Consistent with this, Th1 and Treg cells increased significantly, while Th2 and Th17 cells decreased in peripheral blood. Apoptosis was also suppressed in the thyroid cells. In summary, hAMSC delivery can potentially be a safe and effective therapy for treating SCH in the elderly, improving related complications by immunomodulatory and apoptosis inhibition.

Two Amnion-Derived Mesenchymal Stem-Cells Injections to Osteoarthritic Elbows in Dogs-Pilot Study.

The aim of the study was to investigate the potential of cell-based regenerative therapy for elbow joints affected by osteoarthritis. Interest was focused on two intra-articular applications of amnion-derived mesenchymal stem cells (A-MSCs) to a group of different breeds of dogs with elbow osteoarthritis (13 joints). Two injections were performed 14 days apart. We evaluated synovial fluid biomarkers, such as IFN-γ, IL-6, IL-15, IL-10, MCP-1, TNF-α, and GM-CSF, by multiplex fluorescent micro-bead immunoassay in the treated group of elbows (n = 13) (day 0, day 14, and day 28) and in the control group of elbows (n = 9). Kinematic gait analysis determined the joint range of motion (ROM) before and after each A-MSCs application. Kinematic gait analysis was performed on day 0, day 14, and day 28. Kinematic gait analysis pointed out improvement in the average range of motion of elbow joints from day 0 (38.45 ± 5.74°), day 14 (41.7 ± 6.04°), and day 28 (44.78 ± 4.69°) with statistical significance (p < 0.05) in nine elbows. Correlation analyses proved statistical significance (p < 0.05) in associations between ROM (day 0, day 14, and day 28) and IFN-γ, IL-6, IL-15, MCP-1, TNF-α, and GM-CSF concentrations (day 0, day 14, and day 28). IFN-γ, IL-6, IL-15, MCP-1, GM-CSF, and TNF- α showed negative correlation with ROM at day 0, day 14, and day 28, while IL-10 demonstrated positive correlation with ROM. As a consequence of A-MSC application to the elbow joint, we detected a statistically significant (p < 0.05) decrease in concentration levels between day 0 and day 28 for IFN-γ, IL-6, and TNF-α and statistically significant increase for IL-10. Statistical significance (p < 0.05) was detected in TNF-α, IFN-γ, and GM-CSF concentrations between day 14 and the control group as well as at day 28 and the control group. IL-6 concentrations showed statistical significance (p < 0.05) between day 14 and the control group.

Human amnion-derived mesenchymal stem cells attenuate acute lung injury in two different acute lung injury mice models.

Acute lung injury (ALI) is one of the most common clinical emergencies with limited effective pharmaceutical treatment in the clinic, especially when it progresses to acute respiratory distress syndrome (ARDS). Currently, mesenchymal stem cells (MSCs) exhibit specific superiority for ALI/ARDS treatment. However, stem cells from different sources may result in controversial effects on similar disease conditions. This study aimed to determine the effects of human amnion-derived mesenchymal stem cells (hAMSCs) on two different ALI mice model. The administered hAMSCs effectively accumulated in the lung tissues in all hAMSC-treated groups. Compared with the model and 1% human serum albumin (HSA) groups, high-dose hAMSCs (1.0 × 106 cells) group significantly alleviated alveolar-capillary permeability, oxidative stress, inflammatory factors level and histopathological damage. In addition, the NF-κB signaling pathway is one of the key pathways activated during lipopolysaccharide (LPS) or paraquat (PQ)-induced lung injury. Our results indicated that hAMSCs (1.0 × 106 cells) obviously inhibited the expression of p-IKKα/β, p-IκBα, and p-p65 in the lung tissue (p < 0.05). The high-dose (HD) hAMSC treatment exerted beneficial therapeutic effects on ALI mice models without detectable adverse reactions. The therapeutic effect of hAMSCs might involve NF-κB signaling pathway inhibition. hAMSC treatment is a potential candidate therapy for ALI.

Immunomodulatory amnion-derived mesenchymal stromal cells preserve muscle function in a mouse model of Duchenne muscular dystrophy

BackgroundDuchenne muscular dystrophy (DMD) is an incurable genetic disease characterized by degeneration and necrosis of myofibers, chronic inflammation, and progressive muscle weakness resulting in premature mortality. Immunosuppressive multipotent mesenchymal stromal cell (MSC) therapy could be an option for DMD patients. We focused on amnion-derived mesenchymal stromal cells (AMSCs), a clinically viable cell source owing to their unique characteristics, such as non-invasive isolation, mitotic stability, ethical acceptability, and minimal risk of immune reaction and cancer. We aimed to identify novel immunomodulatory effects of AMSCs on macrophage polarization and their transplantation strategies for the functional recovery of skeletal and cardiac muscles.MethodsWe used flow cytometry to analyze the expression of anti-inflammatory M2 macrophage markers on peripheral blood mononuclear cells (PBMCs) co-cultured with human AMSCs (hAMSCs). hAMSCs were intravenously injected into DMD model mice (mdx mice) to assess the safety and efficacy of therapeutic interventions. hAMSC-treated and untreated mdx mice were monitored using blood tests, histological examinations, spontaneous wheel-running activities, grip strength, and echocardiography.ResultshAMSCs induced M2 macrophage polarization in PBMCs via prostaglandin E2 production. After repeated systemic hAMSC injections, mdx mice exhibited a transient downregulation of serum creatin kinase. Limited mononuclear cell infiltration and a decreased number of centrally nucleated fibers were indicative of regenerated myofibers following degeneration, suggesting an improved histological appearance of the skeletal muscle of hAMSC-treated mdx mice. Upregulated M2 macrophages and altered cytokine/chemokine expressions were observed in the muscles of hAMSC-treated mdx mice. During long-term experiments, a significant decrease in the grip strength in control mdx mice significantly improved in the hAMSC-treated mdx mice. hAMSC-treated mdx mice maintained running activity and enhanced daily running distance. Notably, the treated mice could run longer distances per minute, indicating high running endurance. Left ventricular function in DMD mice improved in hAMSC-treated mdx mice.ConclusionsEarly systemic hAMSC administration in mdx mice ameliorated progressive phenotypes, including pathological inflammation and motor dysfunction, resulting in the long-term improvement of skeletal and cardiac muscle function. The therapeutic effects might be associated with the immunosuppressive properties of hAMSCs via M2 macrophage polarization. This treatment strategy could provide therapeutic benefits to DMD patients.

Circ-100290 Regulates Autophagy-Mediated Angiogenesis of Human Umbilical Vein Endothelial Cells Induced by Conditioned Medium of Human Amniotic Mesenchymal Stem Cells.

Conditioned medium (CM) from human amnion-derived mesenchymal stem cells (hAMSCs) exhibits excellent pro-angiogenic capacity, and circ-100290 participates in this process. Autophagy is involved in the relevant mechanisms of angiogenesis, but it is unclear whether autophagy is related to the pro-angiogenesis effect of hAMSCs. This research sought to determine whether autophagy involved in the process of pro-angiogenesis induced by hAMSCs might be regulated by circ-100290. Upon treatment with CM from hAMSC or 3-methyladenine (3-MA), autophagosomes in human umbilical vein endothelial cells (HUVECs) were observed by transmission electron microscopy. HUVECs' angiogenic ability was evaluated by in vitro assays (transwell, wound healing, tube formation) and an in vivo Matrigel plug assay. Specific small interfering RNAs (siRNA) or inhibitors were used to regulate circ-100290 expression. Additionally, western blot and quantitative reverse transcription-polymerase chain reaction (RT-qPCR) were used to evaluate expression of the following indicators: Beclin-1, LC3-II, matrix metalloproteinase 2 (MMP2), MMP9, vascular endothelial growth factor (VEGF)-A, and endothelial nitric oxide synthase (eNOS). Incubation with hAMSC-CM increased autophagy, angiogenesis and the expressions of VEGF-A and eNOS in HUVECs, all of which were inhibited by 3-MA. Knocking down circ-100290 in hAMSC-CM-treated HUVECs reduced Beclin-1 expression and inhibited autophagy. This resulted in lower angiogenesis in the Matrigel plug assay showing that reduced angiogenesis occurred after circ-100290 silencing in hAMSC-CM-treated HUVECs. Circ-100290 promotes autophagy-mediated angiogenesis in hAMSC-CM-treated HUVECs.

Adipose and amnion-derived mesenchymal stem cells: Extracellular vesicles characterization and implication for reproductive biotechnology

The stem cell-based research for reproductive biotechnology has been widely studied and shows promise for repairing defective tissue or degenerated cells to treat different diseases. The adipose tissue and amniotic membrane have awakened great interest in regenerative medicine and arises as a promising source of mesenchymal stem cells. Both types, adipose and amniotic derived mesenchymal stem cells (AMSCs) are multipotent cells with an enhanced ability to differentiate into multiple lineages.. We aimed to evaluate the effect of basal supplementation of exosomes in cell cultures with canine amniotic mesenchymal stem cells (MSCs). Mesenchymal stem cells derived from canine amniotic and adipose tissue were isolated and cultured performing cell passages until 80–90% confluence was reached. The growth curve was determined and peak cell growth was observed in the second passage. The cells were then characterized and differentiated into adipogenic, chondrogenic and osteogenic lineages. Extracellular vesicles from amnion were isolated using an ultracentrifugation protocol and characterized by nanosight analysis. To evaluate their ability to improve cellular viability in naturally inefficient passages, exosomes were co-cultures to the MSC cells. The results showed a 15–20% increase in the expansion rate of cultures supplemented with vesicles extracted in the first and second passages when compared to the control group. Statistical analysis using the Dunnett test (p ≤ 0.05) corroborated this result, showing a positive correlation between supplementation and expansion rate. These results indicate not only the importance of exosomes in the cell communication process but also the feasibility of the culture supplementation protocol for therapeutic purposes. The potential of the AMSCs for reproductive biotechnology is undoubted, however, their application to repair reproductive disorders and the involved mechanisms remain elusive. The strategies to enable the Adipose Stem Cells and AMSCs application in reproductive biotechnology and optimize their use for tissue regeneration open new venues using exosomes interactions.

Human Adipose- and Amnion-Derived Mesenchymal Stromal Cells Similarly Mitigate Osteoarthritis Progression in the Dunkin Hartley Guinea Pig

Background: Clinical trials are currently underway to investigate the efficacy of intra-articular administration of mesenchymal stromal cells (MSCs) to mitigate osteoarthritis (OA) progression in the knee. Although multiple MSC sources exist, studies have yet to determine whether differences in therapeutic efficacy exist between them. Purpose: To compare the ability of intra-articularly injected adipose-derived MSCs (AD-MSCs) and amnion-derived MSCs (AM-MSCs) to mitigate the progression of knee OA in a small animal model of spontaneous OA, as well as to compare the therapeutic potential of MSCs in hyaluronic acid (HA) and in HA only with saline (OA) controls. Study Design: Controlled laboratory study. Methods: Injections of AD-MSCs or AM-MSCs suspended in HA or HA only were performed in the rear stifle joints of 3-month-old Dunkin Hartley guinea pigs (DHGPs). Repeat injections occurred at 2 and 4 months after the initial injection in each animal. Contralateral limbs received saline injections and served as untreated controls. Subsequently, joints were analyzed for osteoarthritic changes of the cartilage and subchondral bone via histologic and biochemical analyses. To evaluate MSC retention time in the joint space, DHGPs received a single intra-articular injection of fluorescently labeled AD-MSCs or AM-MSCs, and the fluorescence intensity was longitudinally tracked via an in vivo imaging system. Results: No statistically significant differences in outcomes were found when comparing the ability of AD-MSCs and AM-MSCs to mitigate OA. However, the injection of AD-MSCs, AM-MSCs, and HA-only treatments more effectively mitigated cartilage damage compared with that of saline controls by demonstrating higher amounts of cartilage glycosaminoglycan content and improved histological proteoglycan scoring while reducing the percentage of osteophytes present. Conclusion: Intra-articular injection of AD-MSCs, AM-MSCs, or HA only was able to similarly mitigate the progression of cartilage damage and reduce the percentage of osteophytes compared with that of saline controls in the DHGP. However, this study was unable to establish the superiority of AD-MSCs versus AM-MSCs as a treatment to mitigate spontaneous OA. Clinical Relevance: MSCs demonstrate the ability to mitigate the progression of knee OA and thus may be used in a prophylactic approach to delay the need for end-stage treatment strategies.

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

We read with great interest the article entitled “Topical Application of Conditioned Medium from Hypoxically Cultured Amnion-Derived Mesenchymal Stem Cells Promotes Wound Healing in Diabetic Mice,” by Takahashi et al.1 The authors performed an in vivo experiment to demonstrate that hypoxic conditioned medium from amnion-derived mesenchymal stem cells enhanced wound healing in a diabetic mouse model. This study brings new opportunities for the treatment of diabetic wounds. The authors prepared the diabetic mouse model by injecting streptozotocin, which induces acute diabetes. It is well known that diabetes is a multisystemic chronic disease, involving neurological and vascular lesions. The vasculopathy and neuropathy are closely associated with the pathophysiology of the diabetic wound.2 Thus, a disadvantage of using an acute diabetic model is that the translation of the findings to the human setting is uncertain and challenging. To assess the level of inflammation in the diabetic wound, the authors performed quantitative polymerase chain reaction analyses on the expression levels of inflammation-related genes. Due to the posttranscription modification, the data on the transcriptional level do not necessarily represent the translational level. Thus, we suggest performing a Western blot or enzyme-linked immunosorbent assay on the protein level to increase the translation of the results. In their study, the authors isolated mesenchymal stem cells from the amnion. The authors mentioned several advantages of the amnion, including abundant availability, less damage, and fewer ethical concerns. Amnion-derived mesenchymal stem cells enhance diabetic wound healing through the paracrine effect, similar to adipose-derived mesenchymal stem cells.3 Further comparative studies are warranted to confirm the efficacy and safety of the above two mesenchymal stem cells. The hypoxic conditioned medium was made by culturing with serum-free α-minimal essential medium for 48 hours under hypoxia. However, the authors did not detect the composition of the media to demonstrate differences after culturing at different oxygen concentrations. Further study is warranted and would benefit from the comparison on the effect of different culture times on the paracrine function. For the preparation of the medium gel, the conditioned medium was mixed with 7% carboxymethyl cellulose. We wonder if there is the same effect via simple continuous injection of the hypoxic conditioned medium around the wounds. DISCLOSURE The authors have no financial interest to declare in relation to the content of this communication. No funding was received for this work. Ping Jiang, M.D.Ping Cao, M.D.Tianyou HospitalAffiliated to Wuhan University of Science and TechnologyWuhan, People’s Republic of China

Multidisciplinary regenerative treatment and mechanisms for rescuing a severe calciphylaxis patient with human amnion-derived mesenchymal stem cells

Calciphylaxis is a rare disease with severe pain and high-mortality due to cutaneous ischemic necrosis and infection that currently lacks proved effective therapies. The occurrence of calciphylaxis in end stage kidney disease (ESKD) patients is known as calcific uremic arteriolopathy (CUA), which is characterized histologically by dermal microvessel calcification, intimal fibroplasia and microthrombosis. Here we innovatively treated a severe CUA patient with human amnion-derived mesenchymal stem cells (hAMSCs). A 34-year-old uremic woman was presented with progressive, painful malodorous ulcers in buttocks and mummified lower limbs. Skin pathological features supported the diagnosis of calciphylaxis. The patient was refractory to conventional multidisciplinary symptomatic therapies. With the approval of our hospital ethics committee, she was treated with hAMSCs including intravenous and local intramuscular injection, and external application of hAMSC culture supernatant to the wound area. During 15-month follow-up, the patient had regeneration of skin and soft tissues, with improved blood biochemical, inflammatory, mineral and bone metabolic indices and immunoregulation effects. After 15-month hAMSC treatment, the score of pain visual analog scale (VAS) decreased from 10 to 0, Bates-Jensen wound assessment tool (BWAT) score decreased from 65 to 13, and wound-quality of life (Wound-QoL) questionnaire score decreased from 68 to 0. We propose that hAMSC treatment is promising for CUA patients. The therapy is potentially involved in the multiple beneficial effects of inhibiting vascular calcification, stimulating angiogenesis and myogenesis, modulating adverse inflammatory and immunologic responses, promoting re-epithelialization and restoring skin integrity.

Intravenous transplantation of amnion-derived mesenchymal stem cells promotes functional recovery and alleviates intestinal dysfunction after spinal cord injury.

Spinal cord injury (SCI) is often accompanied by gastrointestinal dysfunction due to the disconnection of the spinal autonomic nervous system. Gastrointestinal dysfunction reportedly upregulates intestinal permeability, leading to bacterial translocation of the gut microbiome to the systemic circulation, which further activates systemic inflammation, exacerbating neuronal damage. Mesenchymal stem cells (MSC) reportedly ameliorate SCI. Here, we aimed to investigate their effect on the associated gastrointestinal dysfunction. Human amnion-derived MSC (AMSCs) were intravenously transplanted one day after a rat model of midthoracic SCI. Biodistribution of transplanted cells, behavioral assessment, and histological evaluations of the spinal cord and intestine were conducted to elucidate the therapeutic effect of AMSCs. Bacterial translocation of the gut microbiome was examined by in situ hybridization and bacterial culture of the liver. Systemic inflammations were examined by blood cytokines, infiltrating immune cells in the spinal cord, and the size of the peripheral immune tissue. AMSCs released various neurotrophic factors and were mainly distributed in the liver and lung after transplantation. AMSC-transplanted animals showed smaller spinal damage and better neurological recovery with preserved neuronal tract. AMSCs transplantation ameliorated intestinal dysfunction both morphologically and functionally, which prevented translocation of the gut microbiome to the systemic circulation. Systemic inflammations were decreased in animals receiving AMSCs in the chronic phase. Intravenous AMSC administration during the acute phase of SCI rescues both spinal damage and intestinal dysfunction. Reducing bacterial translocation may contribute to decreasing systemic inflammation.

MO133: Multidisciplinary Regenerative Treatment for Rescuing a Severe Calciphylaxis Patient with Human Amnion-Derived Mesenchymal Stem Cells

Abstract BACKGROUND AND AIMS Calciphylaxis is a rare disease characterized histologically by microvessel calcification and microthrombosis, with high mortality and no proven therapy. Here, we reported multidisciplinary regenerative treatment for rescuing a severe uremic calciphylaxis patient with human amnion-derived mesenchymal stem cells (hAMSCs). METHOD A 34-year-old uremic woman presented progressive skin ischaemia, large areas of painful malodorous ulcers and mummified legs. The patient was diagnosed as calciphylaxis based on clinical manifestations, laboratory examinations and skin pathological features. Because of her refractory to conventional therapy, treatment with hAMSCs was approved. HAMSCs were administered intravenously to the patient at a dose of 1.0 × 106 cells per kilogram of body weight, local intramuscular injection along the wound edge (2.0 × 104 cells/cm2) and external application of the cell culture supernatant on wound surfaces. RESULTS Following up 15 months regularly after hAMSCs treatment, the patient's blood biochemical, inflammatory, mineral and bone metabolic indices improved significantly, with immunoregulation effects, regeneration of skin and soft tissue. Besides, wound healing (Figure 1), pain status and quality of life, evaluated by Pain Visual Analog Scale, Bates-Jensen Wound Assessment and the wound-quality of life questionnaire were also improved significantly (Figure 2). CONCLUSION This case represented the new method of hAMSC regeneration therapy for uremic calciphylaxis with safety and effectiveness, which deserved further investigation. Before hAMSC treatment, the patient had irregular ulcers on the buttocks and lower extremities, some of which were crusted on the surface or had purulent fishy discharge. Skin and soft tissue necrosis can be seen at the ulcerated area of the buttocks, localized deep to the fascia, with purulent secretions on the surface and brown pigmentation of the skin at the edge of the ulcer (Figure 1A and B). Because of her worsening symptoms and signs refractory to conventional therapy, the patient was given intravenous and local intramuscular injections of hAMSCs, supplemented by application of hAMSC culture supernatant to the wound. After 4 months of treatment, the sacrococcygeal wound has reduced size and become shallow with less exudate, some new granulation tissue was visible. The infection was controlled with pain significantly reduced (Figure 1C). After 12 months, most of the sacrococcygeal wound had healed with only a few erosions (Figure1D). Fifteen months later, her skin scarring of the wound was healed without pain (Figure 1E). The pain Visual Analogue Scale (Figure 2A) showed that the pain was unbearable before treatment (10 points) and disappeared after 15 months of hAMSC treatment (0 point). BWAT (Figure 2B) showed that all the 13 indexes got 5 points (worst) before hAMSC treatment, with a total score of 65 points. After 15 months of hAMSC treatment, each index got 1 point (best), and the total score was 13 points. The Wound-QoL questionnaire (Figure 2C) showed that the 17 items were quantified as 4 points (very serious) before treatment, with a total score of 68 points. After 15 months of hAMSC treatment, the score was 0 (none at all), and the total score was 0.

Important role of the SDF-1/CXCR4 axis in the homing of systemically transplanted human amnion-derived mesenchymal stem cells (hAD-MSCs) to ovaries in rats with chemotherapy-induced premature ovarian insufficiency (POI)

BackgroundChemotherapy can induce premature ovarian insufficiency (POI). POI causes multiple sequelae and is currently incurable. As shown in our previous studies, systemically transplanted human amnion-derived mesenchymal stem cells (hAD-MSCs) home to ovaries with chemotherapy-induced POI and subsequently reduce ovarian injury and improve ovarian function in rats with POI. However, the cellular mechanisms that direct the migration and homing of hAD-MSCs to ovaries with chemotherapy-induced POI are incompletely understood. This study investigated the role of the SDF-1/CXCR4 axis in the migration and homing of systemically transplanted hAD-MSCs to ovaries with chemotherapy-induced POI and its relevant downstream signalling pathways.MethodsCXCR4 expression in hAD-MSCs was assessed using Western blotting and immunofluorescence staining. hAD-MSC migration was tested using Transwell migration assays. SDF-1 levels were detected using ELISA. Seventy-two female SD rats were randomly divided into the control, POI, hAD-MSCs and hAD-MSCs + AMD3100 groups. Cyclophosphamide was used to establish rat POI models. For inhibitor treatment, hAD-MSCs were pretreated with AMD3100 before transplantation. PKH26-labeled hAD-MSCs were injected into the tail vein of POI rats 24 h after chemotherapy. After hAD-MSC transplantation, the homing of hAD-MSCs to ovaries and ovarian function and pathological changes were examined. We further investigated the molecular mechanisms by detecting the PI3K/Akt and ERK1/2 signalling pathways.ResultshAD-MSCs expressed CXCR4. SDF-1 induced hAD-MSC migration in vitro. SDF-1 levels in ovaries and serum were significantly increased in rats with chemotherapy-induced POI, and ovaries with POI induced the homing of hAD-MSCs expressing CXCR4. Blocking the SDF-1/CXCR4 axis with AMD3100 significantly reduced the number of hAD-MSCs homing to ovaries with POI and further reduced their efficacy in POI treatment. The binding of SDF-1 to CXCR4 activated the PI3K/Akt signalling pathway, and LY294002 significantly inhibited hAD-MSC migration induced by SDF-1 in vitro. Moreover, inhibition of the PI3K/Akt signalling pathway significantly reduced the number of systemically transplanted hAD-MSCs homing to chemotherapy-induced ovaries in rats with POI.ConclusionsSDF-1/CXCR4 axis partially mediates the migration and homing of systemically transplanted hAD-MSCs to the ovaries of rats with chemotherapy-induced POI, and the PI3K/Akt signalling pathway might be involved in the migration and homing of hAD-MSCs mediated by the SDF-1/CXCR4 axis.

A novel long-term intravenous combined with local treatment with human amnion-derived mesenchymal stem cells for a multidisciplinary rescued uremic calciphylaxis patient and the underlying mechanism.

Calciphylaxis is a rare disease characterized histologically by microvessel calcification and microthrombosis, with high mortality and no proven therapy. Here, we reported a severe uremic calciphylaxis patient with progressive skin ischemia, large areas of painful malodorous ulcers, and mummified legs. Because of the worsening symptoms and signs refractory to conventional therapies, treatment with human amnion-derived mesenchymal stem cells (hAMSCs) was approved. Preclinical release inspections of hAMSCs, efficacy, and safety assessment, including cytokine secretory ability, immunocompetence, tumorigenicity, and genetics analysis in vitro, were introduced. We further performed acute and long-term hAMSC toxicity evaluations in C57BL/6 mice and rats, abnormal immune response tests in C57BL/6 mice, and tumorigenicity tests in neonatal Balbc-nu nude mice. After the preclinical research, the patient was treated with hAMSCs by intravenous and local intramuscular injection and external supernatant application to the ulcers. When followed up to 15 months, the blood-based markers of bone and mineral metabolism improved, with skin soft tissue regeneration and a more favorable profile of peripheral blood mononuclear cells. Skin biopsy after 1-month treatment showed vascular regeneration with mature noncalcified vessels within the dermis, and 20 months later, the re-epithelialization restored the integrity of the damaged site. No infusion or local treatment-related adverse events occurred. Thus, this novel long-term intravenous combined with local treatment with hAMSCs warrants further investigation as a potential regenerative treatment for uremic calciphylaxis due to effects of inhibiting vascular calcification, stimulating angiogenesis and myogenesis, anti-inflammatory and immune modulation, multidifferentiation, re-epithelialization, and restoration of integrity.

Cell therapy using amnion-derived mesenchymal stem cells

This study focused on amnion-derived mesenchymal stem cells (MSCs), which have immune- and inflammation-regulating properties, 1) a large number of stem cells, 2) high proliferative potential, and 3) are non-invasive to harvest. Based on the general research reported in many immune- and inflammation-related disease models, research on their commercial and therapeutic application was conducted. We have successfully manufactured a clinical trial product of amnion MSCs for the first time worldwide (clinical trial product name: AM01) and conducted physician-led clinical trials for acute graft versus host disease and Crohn's disease. Furthermore, CTEX Corporation, the first certified venture from Hyogo College of Medicine, was launched to further accelerate the clinical trial progression to obtain the manufacturing and marketing approval for amnion MSC AM01 to be used as a regenerative medical product at early stage.

Pulsatility is a Predictive Marker of Improved Cardiac Function in Patients with Liquid Matrix-treated Left Ventricular Assist Devices

Objective: Left ventricular assist devices (LVADs) are utilized as a bridge to transplant or as destination therapy for patients with end-stage heart failure. Although cardiac offloading from these devices rarely leads to complete remodeling and functional recovery, the use of mesenchymal cells to modulate heart failure has been explored in recent years due to its intrinsic regenerative properties. Current methods of evaluating cardiac function have too much variability, difficulty of access, or require too frequent follow up to create universal weaning protocols. We hypothesized that the administration of amniotic allograft liquid matrix (LM) containing amnion-derived mesenchymal stem cells (aMSCs) in patients with LVADs could improve left ventricular function and be positively associated with pulsatility. Methods: Flow cytometry, mass spectroscopy, and enzyme-linked immunoassays were used to characterize aMSCs and LM that were administered to 9 patients with LVADs. Results were compared to samples from 7 control patients with LVADs that did not receive aMSC and LM. Results: Patients who received aMSCs and LM (n=9) demonstrated a significant increase in standardized pulsatility at 30 (P = .007), 90 (P = .02), and 180 (P = .05) days post-implant when compared to control patients who did not receive the treatment (n=7). Discussion: We conclude that the use of aMSCs and LM in patients with LVADs could be a promising treatment strategy, and pulsatility can be a reproducible and consistent diagnostic metric to evaluate left ventricular function without intra- or inter-observer variability.

Efficient hepatic differentiation and regeneration potential under xeno-free conditions using mass-producible amnion-derived mesenchymal stem cells

BackgroundAmnion-derived mesenchymal stem cells (AM-MSCs) are an attractive source of stem cell therapy for patients with irreversible liver disease. However, there are obstacles to their use due to low efficiency and xeno-contamination for hepatic differentiation.MethodsWe established an efficient protocol for differentiating AM-MSCs into hepatic progenitor cells (HPCs) by analyzing transcriptome-sequencing data. Furthermore, to generate the xeno-free conditioned differentiation protocol, we replaced fetal bovine serum (FBS) with polyvinyl alcohol (PVA). We investigated the hepatocyte functions with the expression of mRNA and protein, secretion of albumin, and activity of CYP3A4. Finally, to test the transplantable potential of HPCs, we transferred AM-MSCs along with hepatic progenitors after differentiated days 11, 12, and 13 based on the expression of hepatocyte-related genes and mitochondrial function. Further, we established a mouse model of acute liver failure using a thioacetamide (TAA) and cyclophosphamide monohydrate (CTX) and transplanted AM-HPCs in the mouse model through splenic injection.ResultsWe analyzed gene expression from RNA sequencing data in AM-MSCs and detected downregulation of hepatic development-associated genes including GATA6, KIT, AFP, c-MET, FGF2, EGF, and c-JUN, and upregulation of GSK3. Based on this result, we established an efficient hepatic differentiation protocol using the GSK3 inhibitor, CHIR99021. Replacing FBS with PVA resulted in improved differentiation ability, such as upregulation of hepatic maturation markers. The differentiated hepatocyte-like cells (HLCs) not only synthesized and secreted albumin, but also metabolized drugs by the CYP3A4 enzyme. The best time for translation of AM-HPCs was 12 days from the start of differentiation. When the AM-HPCs were transplanted into the liver failure mouse model, they settled in the damaged livers and differentiated into hepatocytes.ConclusionThis study offers an efficient and xeno-free conditioned hepatic differentiation protocol and shows that AM-HPCs could be used as transplantable therapeutic materials. Thus, we suggest that AM-MSC-derived HPCs are promising cells for treating liver disease.