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.

BACKGROUND: Myocardial 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. METHODS: MI/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. RESULTS: Echocardiographic 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. CONCLUSION: Our 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.

The suppression of cell motility through the reduction of FAK activity and expression of cell adhesion proteins by hAMSCs secretome in MDA-MB-231 breast cancer cells.

Breast cancer is a leading cause of death in women worldwide. Cancer therapy based on stem cells is considered as a novel and promising platform. In the present study, we explore the therapeutic effects of human amniotic mesenchymal stromal cells (hAMSCs) through the reduction of focal adhesion kinase (FAK) activity, SHP-2, and cell adhesion proteins such as Paxillin, Vinculin, Fibronectin, Talin, and integrin αvß3 expression in MDA-MB-231 breast cancer cells. For this purpose, we employed a co-culture system using 6-well plate transwell. After 72 h, hAMSCs-treated MDA-MB-231 breast cancer cells, the activity of focal adhesion kinase (FAK) and the expression of SHP-2 and cell adhesion proteins such as Paxillin, Vinculin, Fibronectin, Talin, and integrin αvß3 expression were analyzed using western blot. The shape and migration of cells were also analyzed. Based on our results, a significant reduction in tumor cell motility through downregulation of the tyrosine phosphorylation level of FAK (at Y397 and Y576/577 sites) and cell adhesion expression in MDA-MB-231 breast cancer cells was demonstrated. Our findings indicate that hAMSCS secretome has therapeutic effects on cancer cell migration through downregulation of FAK activity and expression of cell adhesion proteins.

Evaluation of SgK269 expression in colon cancer patients and the effects of hAMSCs secretome on tumor invasion through SgK269/c-Src/p-P130Cas/p-Paxillin/p-ERK1/2 signaling pathway in HT-29 colon cancer cells.

Colon cancer is the fifth leading cause of cancer-related deaths worldwide. Stem cells have unique characteristics and are considered as a novel therapeutic platform for cancer. Sugen Kinase 269 (SgK269) is considered as an oncogenic scaffolding pseudo kinase which governs the rearranging of the cytoskeleton, cellular motility, and invasion. The aim of this study is to evaluate the expression of SgK269 in colon cancer patients and explore the therapeutic effects of human amniotic mesenchymal stromal cells (hAMSCs) on invasion and proliferation of colon cancer cells (HT-29) through analyzing SgK269/c-Src/p-P130Cas/p-Paxillin/p-ERK1/2 signaling pathway. In this regard, we collected 30 samples from colon cancer patients and evaluated SgK269 expression using quantitative real-time PCR (qRT-PCR). Next, we employed a co-culture system using Transwell 6-well plates and after 72 h, tumor growth promotion and invasion were analyzed in hAMSCs-treated HT-29 cells through SgK269/c-Src/p-P130Cas/p-Paxillin/p-ERK1/2/Rac signaling pathway using qRT-PCR, western blot method, MTT assay, wound healing assay, and DAPI staining. Our results showed upregulation of SgK269 in colon cancer patients. Treatment of HT-29 colon cancer cells with hAMSCs secretome can inhibit SgK269/c-Src/p-P130Cas/p-Paxillin/p-ERK1/2/Rac signaling pathway and the resulting suppression of cell invasion and proliferation. Our results suggest that SgK269 is an important target in colon cancer therapy and MSCs secretome may be an effective therapeutic approach to inhibit colon cancer cell invasion and proliferation through SgK269/c-Src/p-P130Cas/p-Paxillin/p-ERK1/2/Rac signaling pathway.

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.

In vitro evaluation of autophagy and cell death induction in Panc1 pancreatic cancer by secretome of hAMSCs through downregulation of p-AKT/p-mTOR and upregulation of p-AMPK/ULK1 signal transduction pathways.

One of the main causes of cancer mortality in the world is pancreatic cancer. Therapies based on stem cells are currently thought to be a hopeful option in the treatment of cancer. Herein, we intend to evaluate the antitumor effects of secretome of human amniotic mesenchymal stromal cells (hAMSCs) on autophagy and cell death induction in Panc1 pancreatic cancer cells. We adopted a co-culture system using Transwell 6-well plates and after 72 h, hAMSCs-treated Panc1 cancer cells were analyzed using quantitative real time PCR (qRT-PCR), flow cytometry, western blot, MTT assay, and DAPI staining. Based on our results, the microtubule-associated protein 1 light chain 3 (LC3) conversion from LC3-I to LC3-II and the upregulation of autophagy-related proteins expression including Beclin1, Atg7, and Atg12 were detected in hAMSCs-treated Panc1 cells. Furthermore, the level of phosphorylated proteins such as Unc-51-like kinase 1 (ULK1), AMP activated protein kinase (AMPK), AKT, and mTOR changed. Apoptotic cell death was also induced via the elevation of Bax and Caspase 3 expression and inhibition of Bcl-2. Our findings showed that secretome of hAMSCs induces autophagy and cell death in Panc1 cancer cells. However, more experiments will be needed to identify more details about the associated mechanisms.

Therapeutic effects of hAMSCs secretome on proliferation of MDA-MB-231 breast cancer cells by the cell cycle arrest in G1/S phase

Background Cancer refers to a disease resulting from the uncontrolled division and growth of abnormal cells. Among different cancer types, breast cancer is considered as one of the most commonly diagnosed cancers. Herein, we explored the therapeutic effects of human amniotic mesenchymal stromal cells (hAMSCs) secretome on breast cancer cells (MDA-MB-231) through analyzing cell cycle progression. Methods We employed a co-culture system using 6-well Transwell plates and after 72 h, the cell cycle progression was evaluated in the hAMSCs-treated MDA-MB-231 cells through analyzing the expressions of RB, CDK4/6, cyclin D, CDK2, cyclin E, p16/INK4a, p21/WAF1/CIP1, and p27/KIP1 using quantitative real-time PCR (qRT-PCR) and western blot method. Cell proliferation, apoptosis, and cell cycle progression were checked using an MTT assay, DAPI staining, and flow cytometry. Results Our results indicated that elevation of RB, p21/WAF1/CIP1, and p27/KIP1 and suppression of RB hyperphosphorylation, p16/INK4a, cyclin E, cyclin D1, CDK2, and CDK4/6 may contribute to inhibiting the proliferation of hAMSCs-treated MDA-MB-231 cells through cell cycle arrest in G1/S phase followed by apoptosis. Conclusion hAMSCs secretome may be an effective approach on breast cancer therapy through the inhibition of cell cycle progression (AU)

Transplantation of Human Amniotic Mesenchymal Stem Cells Up-Regulates Angiogenic Factor Expression to Attenuate Diabetic Kidney Disease in Rats.

Background and Aims: Diabetic kidney disease (DKD) is a prevalent and intractable microvascular complication of diabetes mellitus (DM), the process of which is closely related to abnormal expression of angiogenesis-regulating factors (ARFs). Stem cell transplantation might be a novel strategy for treating DKD. This study aims to explore the effect of transplantation of human amniotic mesenchymal stem cells (hAMSCs) on renal microangiopathy in a type 1 DKD rat model (T1DRM). Methods: Seventy-two rats were randomly divided into three groups, including normal control group, DKD group, and hAMSCs transplantation group. T1DRM was established using a rat tail vein injection of streptozotocin (STZ) (55 mg/kg). hAMSCs were obtained from placental amniotic membranes during cesarean delivery and transplanted at 3 and 4 weeks through penile veins. At 6, 8, and 12 weeks following transplantation, blood glucose levels, renal function, pathological kidney alterations, and the expressions of ARFs' mRNA and protein were analyzed. Results: In T1DRM, transplanted hAMSCs that were homed at the injured site of kidneys increased ARFs' expression and decreased blood glucose levels. Compared to the DKD group, the levels of 24-h urinary protein, serum creatinine, urea, and kidney injury molecule-1 (KIM-1) were reduced in hAMSCs transplantation group. In terms of renal pathology such as the degree of basement membrane thickening, hAMSCs transplantation was also less severe than the DKD group, thereby alleviating kidney injury. Conclusion: hAMSCs transplantation might ameliorate STZ-induced chronic kidney injury through increasing ARFs' expression in kidneys and lowering blood glucose levels.

Therapeutic effects of hAMSCs secretome on proliferation of MDA-MB-231 breast cancer cells by the cell cycle arrest in G1/S phase.

BACKGROUND: Cancer refers to a disease resulting from the uncontrolled division and growth of abnormal cells. Among different cancer types, breast cancer is considered as one of the most commonly diagnosed cancers. Herein, we explored the therapeutic effects of human amniotic mesenchymal stromal cells (hAMSCs) secretome on breast cancer cells (MDA-MB-231) through analyzing cell cycle progression. METHODS: We employed a co-culture system using 6-well Transwell plates and after 72 h, the cell cycle progression was evaluated in the hAMSCs-treated MDA-MB-231 cells through analyzing the expressions of RB, CDK4/6, cyclin D, CDK2, cyclin E, p16/INK4a, p21/WAF1/CIP1, and p27/KIP1 using quantitative real-time PCR (qRT-PCR) and western blot method. Cell proliferation, apoptosis, and cell cycle progression were checked using an MTT assay, DAPI staining, and flow cytometry. RESULTS: Our results indicated that elevation of RB, p21/WAF1/CIP1, and p27/KIP1 and suppression of RB hyperphosphorylation, p16/INK4a, cyclin E, cyclin D1, CDK2, and CDK4/6 may contribute to inhibiting the proliferation of hAMSCs-treated MDA-MB-231 cells through cell cycle arrest in G1/S phase followed by apoptosis. CONCLUSION: hAMSCs secretome may be an effective approach on breast cancer therapy through the inhibition of cell cycle progression.

Downregulation of Pinkbar/pAKT and MMP2/MMP9 Expression in MDA-MB-231 Breast Cancer Cells as Potential Targets in Cancer Therapy by hAMSCs Secretome.

Breast cancer is one of the leading causes of cancer-related deaths among women worldwide. Cancer therapy based on stem cells is considered as a novel and promising platform. In the present study, we explored the therapeutic effects of human amniotic mesenchymal stromal cells (hAMSCs) through Pinkbar (planar intestinal- and kidney-specific BAR domain protein), pAKT, and matrix metalloproteinases including MMP2 and MMP9 on MDA-MB-231 breast cancer cells. For this purpose, we employed a co-culture system using Transwell 6-well plates with a pore size of 0.4 µm. After 72 h, the hAMSCs-treated MDA-MB-231 breast cancer cells, the expression of epidermal growth factor receptor (EGFR), and c-Src (a key mediator in EGFR signaling pathway), Pinkbar, pAKT, MMP2, and MMP9 were analyzed using quantitative real time PCR and western blot methods. Based on 2D and 3D cell culture models, significant reduction of tumor cell growth and motility through downregulation of EGFR, c-Src, Pinkbar, pAKT, MMP2, and MMP9 were found in MDA-MB-231 breast cancer cells. Moreover, induction of cellular apoptosis was also reported. Our finding indicates that the hAMSCS secretome has therapeutic effects on cancer cells. To identify the details of the molecular mechanisms, more experiments will be required.

Construction of biomimetic cell-sheet-engineered periosteum with a double cell sheet to repair calvarial defects of rats.

Background: The periosteum plays a crucial role in the development and injury healing process of bone. The purpose of this study was to construct a biomimetic periosteum with a double cell sheet for bone tissue regeneration. Methods: In vitro, the human amniotic mesenchymal stem cells (hAMSCs) sheet was first fabricated by adding 50 â€‹µg/ml ascorbic acid to the cell sheet induction medium. Characterization of the hAMSCs sheet was tested by general observation, microscopic observation, live/dead staining, scanning electron microscopy (SEM) and hematoxylin and eosin (HE) staining. Afterwards, the osteogenic cell sheet and vascular cell sheet were constructed and evaluated by general observation, alkaline phosphatase (ALP) staining, Alizarin Red S staining, SEM, live/dead staining and CD31 immunofluorescent staining for characterization. Then, we prepared the double cell sheet. In vivo, rat calvarial defect model was introduced to verify the regeneration of bone defects treated by different methods. Calvarial defects (diameter: 4 â€‹mm) were created of Sprague-Dawley rats. The rats were randomly divided into 4 groups: the control group, the osteogenic cell sheet group, the vascular cell sheet group and the double cell sheet group. Macroscopic, micro-CT and histological evaluations of the regenerated bone were performed to assess the treatment results at 8 weeks and 12 weeks after surgery. Results: In vitro, hAMSCs sheet was successfully prepared. The hAMSCs sheet consisted of a large number of live hAMSCs and abundant extracellular matrix (ECM) that secreted by hAMSCs, as evidenced by macroscopic/microscopic observation, live/dead staining, SEM and HE staining. Besides, the osteogenic cell sheet and the vascular cell sheet were successfully prepared, which were verified by general observation, ALP staining, Alizarin Red S staining, SEM and CD31 immunofluorescent staining. In vivo, the macroscopic observation and micro-CT results both demonstrated that the double cell sheet group had better effect on bone regeneration than other groups. In addition, histological assessments indicated that large amounts of new bone had formed in the calvarial defects and more mature collagen in the double cell sheet group. Conclusion: The double cell sheet could promote to repair calvarial defects of rats and accelerate bone regeneration. The translational potential of this article: We successfully constructed a biomimetic cell-sheet-engineered periosteum with a double cell sheet by a simple, low-cost and effective method. This biomimetic periosteum may be a promising therapeutic strategy for the treatment of bone defects, which may be used in clinic in the future.

Small extracellular vesicles derived from Nrf2-overexpressing human amniotic mesenchymal stem cells protect against lipopolysaccharide-induced acute lung injury by inhibiting NLRP3.

BACKGROUND: Acute lung injury (ALI) is a major cause of respiratory failure in critically ill patients that results in significant morbidity and mortality. Recent studies indicate that cell-based therapies may be beneficial in the treatment of ALI. We recently demonstrated that Nrf2-overexpressing human amniotic mesenchymal stem cells (hAMSCs) reduce lung injury, fibrosis and inflammation in lipopolysaccharide (LPS)-challenged mice. Here we tested whether small extracellular vesicles (sEVs) derived from Nrf2-overexpressing hAMSCs (Nrf2-sEVs) could protect against ALI. sEVs were isolated from hAMSCs that overexpressed (Nrf2-sEVs) or silenced (siNrf2-sEVs) Nrf2. We examined the effects of sEVs treatment on lung inflammation in a mouse model of ALI, where LPS was administered intratracheally to mice, and lung tissues and bronchoalveolar lavage fluid (BALF) were analyzed 24 h later. METHODS: Histological analysis, immunofluorescence microscopy, western blotting, RT-PCR and ELISA were used to measure the inflammatory response in the lungs and BALF. RESULTS: We found that sEVs from hAMSCs are protective in ALI and that Nrf2 overexpression promotes protection against lung disease. Nrf2-sEVs significantly reduced lung injury in LPS-challenged mice, which was associated with decreased apoptosis, reduced infiltration of neutrophils and macrophages, and inhibition of pro-inflammatory cytokine expression. We further show that Nrf2-sEVs act by inhibiting the activation of the NLRP3 inflammasome and promoting the polarization of M2 macrophages. CONCLUSION: Our data show that overexpression of Nrf2 protects against LPS-induced lung injury, and indicate that a novel therapeutic strategy using Nrf2-sEVs may be beneficial against ALI.

Systematic comparation of the biological and transcriptomic landscapes of human amniotic mesenchymal stem cells under serum-containing and serum-free conditions.

BACKGROUND: Human amniotic mesenchymal stem cells (hAMSCs) are splendid cell sources for clinical application in the administration of numerous refractory and relapse diseases. Despite the preferable prospect of serum-free (SF) condition for cell product standardization and pathogenic contamination remission, yet the systematic and detailed impact upon hAMSCs at both cellular and transcriptomic levels is largely obscure. METHODS: For the purpose, we preconditioned hAMSCs under serum-containing (SC) and SF medium for 48 h and compared the biological signatures and biofunctions from the view of cell morphology, immunophenotypes, multi-lineage differentiation in vitro, cell vitality, cytokine expression, and immunosuppressive effect upon the subpopulations of T lymphocytes, together with the PI3K-AKT-mTOR signaling reactivation upon cell vitality. Meanwhile, we took advantage of RNA-SEQ and bioinformatic analyses to verify the gene expression profiling and genetic variation spectrum in the indicated hAMSCs. RESULTS: Compared with those maintained in SC medium, hAMSCs pretreated in SF conditions manifested conservation in cell morphology, immunophenotypes, adipogenic differentiation, and immunosuppressive effect upon the proliferation and activation of most of the T cell subpopulations, but with evaluated cytokine expression (e.g., TGF-ß1, IDO1, NOS2) and declined osteogenic differentiation and cell proliferation as well as proapoptotic and apoptotic cells. The declined proliferation in the SF group was efficiently rescued by PI3K-AKT-mTOR signaling reactivation. Notably, hAMSCs cultured in SF and SC conditions revealed similarities in gene expression profiling and variations in genetic mutation at the transcriptome level. Instead, based on the differentially expressed genes and variable shear event analyses, we found those genes were mainly involved in DNA synthesis-, protein metabolism-, and cell vitality-associated biological processes and signaling pathways (e.g., P53, KRAS, PI3K-Akt-mTOR). CONCLUSIONS: Collectively, our data revealed the multifaceted cellular and molecular properties of hAMSCs under SC and SF conditions, which suggested the feasibility of serum-free culture for the preferable preparation of standardized cell products for hAMSC drug development and clinical application.

SPRY4 promotes adipogenic differentiation of human mesenchymal stem cells through the MEK-ERK1/2 signaling pathway.

Obesity is a chronic metabolic disorder characterized by the accumulation of excess fat in the body. Preventing and controlling obesity by inhibiting the adipogenic differentiation of mesenchymal stem cells (MSCs) and thereby avoiding the increase of white adipose tissue is safe and effective. Recent studies have demonstrated that Sprouty proteins (SPRYs) are involved in cell differentiation and related diseases. However, the role and mechanism of SPRY4 in MSC adipogenic differentiation remain to be explored. Here, we found that SPRY4 positively correlates with the adipogenic differentiation of human adipose-derived MSCs (hAMSCs). Via gain- and loss-of-function experiments, we demonstrated that SPRY4 promotes hAMSC adipogenesis both in vitro and in vivo. Mechanistically, SPRY4 functioned by activating the MEK-ERK1/2 pathway. Our findings provide new insights into a critical role for SPRY4 as a regulator of adipogenic differentiation, which may illuminate the underlying mechanisms of obesity and suggest the potential of SPRY4 as a novel treatment option.

Human acellular amniotic membrane scaffolds encapsulating juvenile cartilage fragments accelerate the repair of rabbit osteochondral defects.

AIMS: The purpose of this study was to explore a simple and effective method of preparing human acellular amniotic membrane (HAAM) scaffolds, and explore the effect of HAAM scaffolds with juvenile cartilage fragments (JCFs) on osteochondral defects. METHODS: HAAM scaffolds were constructed via trypsinization from fresh human amniotic membrane (HAM). The characteristics of the HAAM scaffolds were evaluated by haematoxylin and eosin (H&E) staining, picrosirius red staining, type II collagen immunostaining, Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). Human amniotic mesenchymal stem cells (hAMSCs) were isolated, and stemness was verified by multilineage differentiation. Then, third-generation (P3) hAMSCs were seeded on the HAAM scaffolds, and phalloidin staining and SEM were used to detect the growth of hAMSCs on the HAAM scaffolds. Osteochondral defects (diameter: 3.5 mm; depth: 3 mm) were created in the right patellar grooves of 20 New Zealand White rabbits. The rabbits were randomly divided into four groups: the control group (n = 5), the HAAM scaffolds group (n = 5), the JCFs group (n = 5), and the HAAM + JCFs group (n = 5). Macroscopic and histological assessments of the regenerated tissue were evaluated to validate the treatment results at 12 weeks. RESULTS: In vitro, the HAAM scaffolds had a network structure and possessed abundant collagen. The HAAM scaffolds had good cytocompatibility, and hAMSCs grew well on the HAAM scaffolds. In vivo, the macroscopic scores of the HAAM + JCFs group were significantly higher than those of the other groups. In addition, histological assessments demonstrated that large amounts of hyaline-like cartilage formed in the osteochondral defects in the HAAM + JCFs group. Integration with surrounding normal cartilage and regeneration of subchondral bone in the HAAM + JCFs group were better than those in the other groups. CONCLUSION: HAAM scaffolds combined with JCFs promote the regenerative repair of osteochondral defects. Cite this article: Bone Joint Res 2022;11(6):349-361.

Human Amnion-Derived Mesenchymal Stromal/Stem Cells Pre-Conditioning Inhibits Inflammation and Apoptosis of Immune and Parenchymal Cells in an In Vitro Model of Liver Ischemia/Reperfusion.

Ischemia/reperfusion injury (IRI) represents one of the leading causes of primary non-function acute liver transplantation failure. IRI, generated by an interruption of organ blood flow and the subsequent restoration upon transplant, i.e., reperfusion, generates the activation of an inflammatory cascade from the resident Kupffer cells, leading first to neutrophils recruitment and second to apoptosis of the parenchyma. Recently, human mesenchymal stromal/stem cells (hMSCs) and derivatives have been implemented for reducing the damage induced by IRI. Interestingly, sparse data in the literature have described the use of human amnion-derived MSCs (hAMSCs) and, more importantly, no evidence regarding hMSCs priming on liver IRI have been described yet. Thus, our study focused on the definition of an in vitro model of liver IRI to test the effect of primed hAMSCs to reduce IRI damage on immune and hepatic cells. We found that the IFNγ pre-treatment and 3D culture of hAMSCs strongly reduced inflammation induced by M1-differentiated macrophages. Furthermore, primed hAMSCs significantly inhibited parenchymal apoptosis at early timepoints of reperfusion by blocking the activation of caspase 3/7. All together, these data demonstrate that hAMSCs priming significantly overcomes IRI effects in vitro by engaging the possibility of defining the molecular pathways involved in this process.

Potential therapeutic effects of hAMSCs secretome on Panc1 pancreatic cancer cells through downregulation of SgK269, E-cadherin, vimentin, and snail expression.

Pancreatic cancer is one of the leading causes of death from cancer worldwide. The current treatment options for pancreatic cancer are unsuccessful and thereby, finding novel and more effective therapeutic strategies is urgently required. Stem cells-based therapies are currently believed to be a potential promising option in cancer therapy. Herein, we are interested in evaluating the therapeutic effects of human amniotic mesenchymal stromal cells (hAMSCs) secretome on tumor growth suppression and EMT inhibition in Panc1 pancreatic cancer cells using 2D and 3D cell culture models. For this purpose, we employed a co-culture system using 6-well Transwell plates with a pore diameter of 0.4 µm. After 72 h treatment of Panc1 cancer cells with hAMSCs, the expression of c-Src, EGFR, SgK269, E-cadherin, Vimentin, Snail transcriptional factor, Bax, Bcl2, and caspase 3 was analyzed by quantitative real-time PCR (qRT-PCR) and Western blot methods. Our results showed significant reduction in tumor cell growth and motility through downregulation of c-Src, EGFR, SgK269, E-cadherin, Vimentin, and Snail transcriptional factor expression in Panc1 pancreatic cancer cells. The induction of cellular apoptosis was also found. Our finding supports the idea that the secretome from hAMSCS has therapeutic effects on cancer cells.

The Antitumor Activity of hAMSCs Secretome in HT-29 Colon Cancer Cells Through Downregulation of EGFR/c-Src/IRTKS Expression and p38/ERK1/2 Phosphorylation.

Colon cancer is considered as one of the main causes of mortality worldwide. Identifying a novel and more effective platform with fewer side effects is still progress. In various cancer types, Epidermal growth factor receptor (EGFR) and c-Src (a key mediator in EGFR signaling pathway) are the key targets for cancer therapy. Moreover, insulin receptor tyrosine kinase substrate (IRTKS or BAI1-associated protein 2-like 1: BAIAP2L1) is a member of the subfamily of inverse BAR (I-BAR) domain proteins, which mediates cell morphology and movement through regulation of actin polymerization. In this study, we employed a co-culture system using Transwell six-well plates. After 72 h, hAMSCs-treated HT-29 cells, EGFR, c-Src, IRTKS, p38, and ERK1/2 expression were analyzed using quantitative real time PCR (qRT-PCR) and western blot methods. The significant reduction in tumor cell growth and motility through downregulation of EGFR/c-Src/IRTKS expression and p38/ERK1/2 phosphorylation in HT-29 cells was demonstrated based on 2D and 3D cell culture models. The induction of cellular apoptosis was also found. Our results support the idea that the hAMSCS secretome has therapeutic effects on cancer cells. However, further experiments will be required to identify the exact molecular mechanisms.

Management of intrauterine adhesions using human amniotic mesenchymal stromal cells to promote endometrial regeneration and repair through Notch signalling.

Intrauterine adhesions (IUAs) severely hamper women's reproductive functions. Human amniotic mesenchymal stromal cell (hAMSC) transplantation is effective in treating IUAs. Here, we examined the function of Notch signalling in IUA treatment with hAMSC transplantation. Forty-five Sprague-Dawley female rats were randomly divided into the sham operation, IUA, IUA + E2, IUA + hAMSCs and IUA + hAMSCs + E2 groups. After IUA induction in the rats, hAMSCs promoted endometrial regeneration and repair via differentiation into endometrial epithelial cells. In all groups, the expression of key proteins in Notch signalling was detected in the uterus by immunohistochemistry. The results indicated Notch signalling activation in the hAMSCs and hAMSCs + E2 groups. We could also induce hAMSC differentiation to generate endometrial epithelial cells in vitro. Furthermore, the inhibition of Notch signalling using the AdR-dnNotch1 vector suppressed hAMSC differentiation (assessed by epithelial and mesenchymal marker levels), whereas its activation using the AdR-Jagged1 vector increased differentiation. The above findings indicate Notch signalling mediates the differentiation of hAMSCs into endometrial epithelial cells, thus promoting endometrial regeneration and repair; Notch signalling could have an important function in IUA treatment.

Human amniotic mesenchymal stem cells-conditioned medium protects mice from high-fat diet-induced obesity.

BACKGROUND: Obesity is a metabolic disorder syndrome characterized by excessive fat accumulation that is related to many diseases. Human amniotic mesenchymal stem cells (hAMSCs) have a great potential for cell-based therapy due to their characteristics such as pluripotency, low immunogenicity, no tumorigenicity, potent paracrine effects, and no ethical concern. Recently, we observed that both hAMSCs and their conditioned medium (hAMSCs-CM) efficiently repaired skin injury, inhibited hepatocellular carcinoma, and alleviated high-fat diet (HFD)-induced diabetes. However, the effects and the underlying mechanisms of hAMSCs-CM on high-fat diet (HFD)-induced obesity were not explored. METHODS: The characteristics of hAMSCs were confirmed by flow cytometry, RT-PCR, and immunofluorescence. Obese mice were induced by administrating HFD for 15 weeks and simultaneously, the mice were intraperitoneally injected with hAMSCs-CM weekly to evaluate the effects of hAMSCs-CM on HFD-induced obesity. GTT and ITT assays were used to assess the effects of hAMSCs-CM on HFD-induced glucose tolerance and insulin resistance. The lipid accumulation and adipocytes hypertrophy in mouse adipose tissues were determined by histological staining, in which the alterations of blood lipid, liver, and kidney function were also examined. The role of hAMSCs-CM in energy homeostasis was monitored by examining the oxygen consumption (VO2), carbon dioxide production (VCO2), and food and water intake in mice. Furthermore, the expressions of the genes related to glucose metabolism, fatty acid ß oxidation, thermogenesis, adipogenesis, and inflammation were determined by western blot analysis, RT-PCR, and immunofluorescence staining. The roles of hAMSCs-CM in adipogenesis and M1/M2 macrophage polarization were investigated with 3T3-L1 preadipocytes or RAW264.7 cells in vitro. RESULTS: hAMSCs-CM significantly restrained HFD-induced obesity in mice by inhibiting adipogenesis and lipogenesis, promoting energy expenditure, and reducing inflammation. The underlying mechanisms of the anti-obesity of hAMSCs-CM might be involved in inhibiting PPARγ and C/EBPα-mediated lipid synthesis and adipogenesis, promoting GLUT4-mediated glucose metabolism, elevating UCP1/PPARα/PGC1α-regulated energy expenditure, and enhancing STAT3-ARG1-mediated M2-type macrophage polarization. CONCLUSION: Our studies demonstrated that hAMSCs significantly alleviated HFD-induced obesity through their paracrine effects. Obviously, our results open up an attractive therapeutic modality for the prevention and treatment of obesity and other metabolic disorders clinically. The cytokines, exosomes, or micro-vesicles secreted from hAMSCs significantly inhibited HFD-induced obesity in mice by inhibiting lipid production and adipogenesis, promoting energy consumption, and reducing inflammation.

POSTN Promotes the Proliferation of Spermatogonial Cells by Activating the Wnt/ß-Catenin Signaling Pathway.

The self-renewal of spermatogonial cells (SCs) provides the foundation for life-long spermatogenesis. To date, only a few growth factors have been used for the culture of SCs in vitro, and how to enhance proliferation capacity of SCs in vitro needs further research. This study aimed to explore the effects of periostin (POSTN) on the proliferation of human SCs. GC-1 spg cells were cultured in a medium with POSTN, cell proliferation was evaluated by MTS analysis and EdU assay, and the Wnt/ß-catenin signaling pathway was examined. Thereafter, the proliferations of human SC were detected using immunofluorescence and RT-PCR. In this study, we found that CM secreted by human amniotic mesenchymal stem cells (hAMSCs) could enhance the proliferation capacity of mouse GC-1 spg cells. Label-free mass spectrometry and ELISA analysis demonstrated that high level of POSTN was secreted by hAMSCs. MTS and EdU staining showed that POSTN increased GC-1 spg cell proliferation, whereas CM from POSTN-silenced hAMSCs suppressed cell proliferation capacity. Then POSTN was found to activate the Wnt/ß-catenin signaling pathway to regulate the proliferation of GC-1 spg cells. XAV-939, a Wnt/ß-catenin inhibitor, partially reversed the effects of POSTN on GC-1 spg cell proliferation. We then analyzed human SCs and found that POSTN promoted human SC proliferation in vitro. These findings provide insights regarding the role of POSTN in regulating SC proliferation via the Wnt/ß-catenin signaling pathway and suggest that POSTN may serve as a cytokine for male infertility therapy.