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.

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.

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.

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.

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.

Non-Ionizing Radiation for Cardiac Human Amniotic Mesenchymal Stromal Cell Commitment: A Physical Strategy in Regenerative Medicine.

Cell therapy is an innovative strategy for tissue repair, since adult stem cells could have limited regenerative ability as in the case of myocardial damage. This leads to a local contractile dysfunction due to scar formation. For these reasons, refining strategy approaches for "in vitro" stem cell commitment, preparatory to the "in vivo" stem cell differentiation, is imperative. In this work, we isolated and characterized at molecular and cellular level, human Amniotic Mesenchymal Stromal Cells (hAMSCs) and exposed them to a physical Extremely Low Frequency Electromagnetic Field (ELF-EMF) stimulus and to a chemical Nitric Oxide treatment. Physically exposed cells showed a decrease of cell proliferation and no change in metabolic activity, cell vitality and apoptotic rate. An increase in the mRNA expression of cardiac and angiogenic differentiation markers, confirmed at the translational level, was also highlighted in exposed cells. Our data, for the first time, provide evidence that physical ELF-EMF stimulus (7 Hz, 2.5 µT), similarly to the chemical treatment, is able to trigger hAMSC cardiac commitment. More importantly, we also observed that only the physical stimulus is able to induce both types of commitments contemporarily (cardiac and angiogenic), suggesting its potential use to obtain a better regenerative response in cell-therapy protocols.

Regulation of PPARγ and CIDEC expression by adenovirus 36 in adipocyte differentiation.

This study is to investigate the role of adenovirus 36 (Ad36) in regulating expression of peroxisome proliferator-activated receptor γ (PPARγ) and cell death-inducing DFFA-like effector c (CIDEC) in Ad36-induced adipocyte differentiation. Human adipose-derived mesenchymal stem cells (hAMSCs) were isolated and cultured, and then infected with Ad36. Ad36-induced adipocytes were identified using quantitative real-time PCR and Oil red O staining. The expression levels of PPARγ and CIDEC in Ad36-induced adipocytes were determined by quantitative real-time PCR and Western blot analysis. Glucose uptake and intracellular triglyceride content were also determined in these induced cells. Our results from the Oil red O staining showed that Ad36 induced the differentiation of hAMSCs into human adipocytes in vitro. Moreover, the medium glucose concentration was significantly decreased, while the intracellular triglyceride content was significantly increased, in the Ad36-induced adipocytes, compared with the control group. Furthermore, our results showed that, the mRNA and protein expression levels of PPARγ and CIDEC were significantly upregulated in Ad36-induced adipocytes, in a time-dependent manner. On the other hand, compared with the control group, the CIDEC expression was downregulated when the Ad36-induced adipocytes were treated with the PPARγ inhibitor, GW9662. Ad36 could upregulate the expression level of CIDEC through increasing PPARγ expression during the adipocyte differentiation process.

Biological and biomechanical analysis of two types of mesenchymal stem cells for intervention in chemotherapy-induced ovarian dysfunction.

PURPOSE: The study aim was to investigate the biological and biomechanical features of a chemotherapy-induced ovarian dysfunction (CIOD) rat model after intervention with human umbilical cord mesenchymal stem cells (UC-MSCs) and human amniotic mesenchymal stem cells (h-AMSCs), thus providing a biological and biomechanical research basis for clinical treatment. METHODS: The serum levels of estradiol (E2), follicle-stimulating hormone (FSH), luteinizing hormone (LH), and vascular endothelial growth factor (VEGF) in the rat CIOD models were evaluated on the 14th, 30th, 60th, and 90th day of intervention with h-AMSCs and UC-MSCs. In addition, the ovaries in each group were sampled on the 14th and 90th day of intervention for tissue morphology and tensile testing. RESULTS: The serum levels of E2, LH, and VEGF in the h-AMSC and UC-MSC groups were greater than in the model group, but the serum FSH level was less than in the model group, and the differences were significant (P < 0.05); the maximum tensile stress and maximum strain in h-AMSC and UC-MSC groups were significantly greater than in the model group (P < 0.05). CONCLUSIONS: UC-MSC and h-AMSC intervention restored damaged ovarian morphology, elasticity, and toughness to a certain extent, and ovarian function showed some recovery.

Arctigenin Inhibits Adipogenesis by Inducing AMPK Activation and Reduces Weight Gain in High-Fat Diet-Induced Obese Mice.

Although arctigenin (ARC) has been reported to have some pharmacological effects such as anti-inflammation, anti-cancer, and antioxidant, there have been no reports on the anti-obesity effect of ARC. The aim of this study is to investigate whether ARC has an anti-obesity effect and mediates the AMP-activated protein kinase (AMPK) pathway. We investigated the anti-adipogenic effect of ARC using 3T3-L1 pre-adipocytes and human adipose tissue-derived mesenchymal stem cells (hAMSCs). In high-fat diet (HFD)-induced obese mice, whether ARC can inhibit weight gain was investigated. We found that ARC reduced weight gain, fat pad weight, and triglycerides in HFD-induced obese mice. ARC also inhibited the expression of peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (C/EBPα) in in vitro and in vivo. Furthermore, ARC induced the AMPK activation resulting in down-modulation of adipogenesis-related factors including PPARγ, C/EBPα, fatty acid synthase, adipocyte fatty acid-binding protein, and lipoprotein lipase. This study demonstrates that ARC can reduce key adipogenic factors by activating the AMPK in vitro and in vivo and suggests a therapeutic implication of ARC for obesity treatment. J. Cell. Biochem. 117: 2067-2077, 2016. © 2016 Wiley Periodicals, Inc.

Stem cells: insights into the secretome.

Stem cells have been considered as possible therapeutic vehicles for different health related problems such as cardiovascular and neurodegenerative diseases and cancer. Secreted molecules are key mediators in cell-cell interactions and influence the cross talk with the surrounding tissues. There is strong evidence supporting that crucial cellular functions such as proliferation, differentiation, communication and migration are strictly regulated from the cell secretome. The investigation of stem cell secretome is accumulating continuously increasing interest given the potential use of these cells in regenerative medicine. The scope of the review is to report the main findings from the investigation of stem cell secretome by the use of contemporary proteomics methods and discuss the current status of research in the field. This article is part of a Special Issue entitled: An Updated Secretome.

The potent osteo-inductive capacity of bioinspired brown seaweed-derived carbohydrate nanofibrous three-dimensional scaffolds.

This study aimed to investigate the osteo-inductive capacity of a fucoidan polysaccharide network derived from brown algae on human adipose-derived stem cells (HA-MSCs) for bone regeneration. The physiochemical properties of the scaffold including surface morphology, surface chemistry, hydrophilicity, mechanical stiffness, and porosity were thoroughly characterized. Both in vitro and in vivo measurements implied a superior cell viability, proliferation, adhesion, and osteo-inductive performance of obtained scaffolds compared to using specific osteogenic induction medium with increased irregular growth of calcium crystallites, which mimic the structure of natural bones. That scaffold was highly biocompatible and suitable for cell cultures. Various examinations, such as quantification of mineralization, alkaline phosphatase, gene expression, and immunocytochemical staining of pre-osteocyte and bone markers confirmed that HAD-MSCs differentiate into osteoblasts, even without an osteogenic induction medium. This study provides evidence for the positive relationship and synergistic effects between the physical properties of the decellularized seaweed scaffold and the chemical composition of fucoidan in promoting the osteogenic differentiation of HA-MSCs. Altogether, the natural matrices derived from brown seaweed offers a sustainable, cost-effective, non-toxic bioinspired scaffold and holds promise for future clinical applications in orthopedics.

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

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

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.

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.

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.

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.

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.

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 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.