Human Amniotic Mesenchymal Stromal Cells Research Articles

Proteomic analysis of the human amniotic mesenchymal stromal cell secretome by integrated approaches via filter-aided sample preparation

The immunomodulatory, anti-inflammatory and regenerative properties of the human amniotic mesenchymal stromal cells (hAMSCs) secretome are acknowledged but the understanding of the specific bioactive components remains incomplete. To address these limitations, the present investigation aimed to profile the proteins and peptides content of the hAMSC secretome through sample pretreatment and fractionation on 10 kDa molecular cut-off FASP (Filter Aided Sample Preparation) device and LC-MS analysis. The filter retained protein fraction underwent trypsin digestion, while the unretained was collected unchanged for intact small proteins and peptides analysis. This combined approach (C-FASP) collects in a single step two complementary fractions, advantageously saving sample volume and time of analysis. The bottom-up analysis of the C-FASP proteins fraction >10 kDa confirmed our previous findings, establishing a set of proteins consistently characterizing the hAMSC secretome. The analysis of the fraction <10 kDa, never been investigated to our knowledge, identified peptide fragments of thymosin beta 4 and beta 10, collagen alpha 1 chains I and III, alpha-enolase, and glyceraldehyde-3-phosphate dehydrogenase, involved in wound healing, anti-inflammatory response, tissue repair and regeneration, key biological activities of the secretome. C-FASP provided a comprehensive molecular profile of the hAMSC secretome offering new insights for enhanced therapeutic applications in regenerative medicine. SignificanceIn this investigation we originally present the comprehensive proteomic investigation of the human amniotic mesenchymal stromal cell secretome by combining the analysis of the proteome and of the peptidome following sample pretreatment and fractionation by Filter Aided Sample Preparation (FASP) with 10 kDa molecular cut-off in coupling with LC-MS analysis. The proteome fraction retained by FASP filter was analyzed after enzymatic digestion, while the unretained fraction, below 10 kDa molecular mass, was analyzed unchanged in its intact form. This dual approach provides novel insights, previously unexplored, into the molecular components potentially responsible for the immunomodulatory and anti-inflammatory properties of the hAMSC secretome. These findings could significantly enhance the therapeutic potential of hAMSCs in regenerative medicine.

Native characterization and QC profiling of human amniotic mesenchymal stromal cell vesicular fractions for secretome-based therapy

Human amniotic mesenchymal stromal cells (hAMSCs) have unique immunomodulatory properties making them attractive candidates for regenerative applications in inflammatory diseases. Most of their beneficial properties are mediated through their secretome. The bioactive factors concurring to its therapeutic activity are still unknown. Evidence suggests synergy between the two main components of the secretome, soluble factors and vesicular fractions, pivotal in shifting inflammation and promoting self-healing. Biological variability and the absence of quality control (QC) protocols hinder secretome-based therapy translation to clinical applications. Moreover, vesicular secretome contains a multitude of particles with varying size, cargos and functions whose complexity hinders full characterization and comprehension.This study achieved a significant advancement in secretome characterization by utilizing native, FFF-based separation and characterizing extracellular vesicles derived from hAMSCs. This was accomplished by obtaining dimensionally homogeneous fractions then characterized based on their protein content, potentially enabling the identification of subpopulations with diverse functionalities.This method proved to be successful as an independent technique for secretome profiling, with the potential to contribute to the standardization of a qualitative method. Additionally, it served as a preparative separation tool, streamlining populations before ELISA and LC-MS characterization. This approach facilitated the categorization of distinctive and recurring proteins, along with the identification of clusters associated with vesicle activity and functions. However, the presence of proteins unique to each fraction obtained through the FFF separation tool presents a challenge for further analysis of the protein content within these cargoes.

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 αvβ3 expression in MDA-MB-231 breast cancer cells. For this purpose, we employed a co-culture system using 6-well plate transwell. After 72h, 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 αvβ3 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 amniotic mesenchymal stromal cell-derived exosomes promote neuronal function by inhibiting excessive apoptosis in a hypoxia/ischemia-induced cerebral palsy model: A preclinical study

BackgroundCerebral palsy (CP) is a condition resulting from perinatal brain injury and can lead to physical disabilities. Exosomes derived from human amniotic mesenchymal stromal cells (hAMSC-Exos) hold promise as potential therapeutic options. ObjectiveThis study aimed to investigate the impact of hAMSC-Exos on neuronal cells and their role in regulating apoptosis both in vitro and in vivo. MethodshAMSC-Exos were isolated via ultracentrifugation and characterized via transmission electron microscopy, particle size analysis, and flow cytometry. In vitro, neuronal damage was induced by lipopolysaccharide (LPS). CP rat models were established via left common carotid artery ligation. Apoptosis levels in cells and CP rats were assessed using flow cytometry, quantitative reverse transcription polymerase chain reaction (RT–qPCR), Western blotting, and TUNEL analysis. ResultsThe results demonstrated successful isolation of hAMSC-Exos via ultracentrifugation, as the isolated cells were positive for CD9 (79.7%) and CD63 (80.2%). Treatment with hAMSC-Exos significantly mitigated the reduction in cell viability induced by LPS. Flow cytometry revealed that LPS-induced damage promoted apoptosis, but this effect was attenuated by treatment with hAMSC-Exos. Additionally, the expression of caspase-3 and caspase-9 and the Bcl-2/Bax ratio indicated that excessive apoptosis could be attenuated by treatment with hAMSC-Exos. Furthermore, tail vein injection of hAMSC-Exos improved the neurobehavioral function of CP rats. Histological analysis via HE and TUNEL staining showed that apoptosis-related damage was attenuated following hAMSC-Exo treatment. ConclusionsIn conclusion, hAMSC-Exos effectively promote neuronal cell survival by regulating apoptosis, indicating their potential as a promising therapeutic option for CP that merits further investigation.

The Human Placental Amniotic Membrane Mesenchymal-Stromal-Cell-Derived Conditioned Medium Inhibits Growth and Promotes Apoptosis of Human Cholangiocarcinoma Cells In Vitro and In Vivo by Suppressing IL-6/JAK2/STAT3 Signaling.

Mesenchymal stromal cells (MSCs) have recently been shown to play an important role in the growth and progression of many solid tumors, including cholangiocarcinoma (CCA). The human placental amniotic membrane (hPAM) is one of the most favorable sources of MSCs due to its availability and non-invasive harvesting procedure. However, the role of human placental amniotic membrane mesenchymal stromal cells (hPAMSCs) in the growth and progression of human CCA has not yet been determined. This study investigates the effects of conditioned medium derived from hPAMSCs (PA-CM) on the properties of three human CCA cell lines and explores possible mechanisms of action. Varying concentrations of PA-CM were used to treat CCA cells to determine their effects on the proliferation and apoptosis of CCA cells. The results showed that PA-CM inhibited the proliferation and colony-forming capacity of KKU100, KKU213A, and KKU213B cells. PA-CM also promoted the apoptosis of these CCA cells by causing the loss of mitochondrial membrane potential. Western Blotting confirmed that PA-CM induced CCA cell apoptosis by increasing the levels of the Bax/Bcl-2 ratio, cleaved caspase 3, and cleaved PARP, possibly by inhibiting the IL-6/JAK2/STAT3 signaling pathway. Moreover, our in vivo study also confirmed the suppressive effect of hPAMSCs on CCA cells by showing that PA-CM reduced tumor volume in nude mice transplanted with human CCA cells. Taken together, our results demonstrate that PA-CM has potent tumor-suppressive effects on human CCA cells and could potentially be used in combination with chemotherapy to develop a more effective treatment for CCA patients.

In Utero Cell Treatment of Hemophilia A Mice via Human Amniotic Fluid Mesenchymal Stromal Cell Engraftment.

Hemophilia is a genetic disorder linked to the sex chromosomes, resulting in impaired blood clotting due to insufficient intrinsic coagulation factors. There are approximately one million individuals worldwide with hemophilia, with hemophilia A being the most prevalent form. The current treatment for hemophilia A involves the administration of clotting factor VIII (FVIII) through regular and costly injections, which only provide temporary relief and pose inconveniences to patients. In utero transplantation (IUT) is an innovative method for addressing genetic disorders, taking advantage of the underdeveloped immune system of the fetus. This allows mesenchymal stromal cells to play a role in fetal development and potentially correct genetic abnormalities. The objective of this study was to assess the potential recovery of coagulation disorders in FVIII knockout hemophilia A mice through the administration of human amniotic fluid mesenchymal stromal cells (hAFMSCs) via IUT at the D14.5 fetal stage. The findings revealed that the transplanted human cells exhibited fusion with the recipient liver, with a ratio of approximately one human cell per 10,000 mouse cells and produced human FVIII protein in the livers of IUT-treated mice. Hemophilia A pups born to IUT recipients demonstrated substantial improvement in their coagulation issues from birth throughout the growth period of up to 12 weeks of age. Moreover, FVIII activity reached its peak at 6 weeks of age, while the levels of FVIII inhibitors remained relatively low during the 12-week testing period in mice with hemophilia. In conclusion, the results indicated that prenatal intrahepatic therapy using hAFMSCs has the potential to improve clotting issues in FVIII knockout mice, suggesting it as a potential clinical treatment for individuals with hemophilia A.

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 72h, 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.

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.

AEC and AFMSC Transplantation Preserves Fertility of Experimentally Induced Rat Varicocele by Expressing Differential Regenerative Mechanisms.

Amniotic membrane and amniotic fluid derived cells are regarded as a promising stem cell source for developing regenerative medicine techniques, although they have never been tested on male infertility diseases such as varicocele (VAR). The current study aimed to examine the effects of two distinct cell sources, human Amniotic Fluid Mesenchymal Stromal Cells (hAFMSCs) and amniotic epithelial cells (hAECs), on male fertility outcomes in a rat induced VAR model. To explain cell-dependent enhancement of reproductive outcomes in rats transplanted with hAECs and hAFMSCs, insights on testis morphology, endocannabinoid system (ECS) expression and inflammatory tissue response have been carried out alongside cell homing assessment. Both cell types survived 120 days post-transplantation by modulating the ECS main components, promoting proregenerative M2 macrophages (Mφ) recruitment and a favorable anti-inflammatory IL10 expression pattern. Of note, hAECs resulted to be more effective in restoring rat fertility rate by enhancing both structural and immunoresponse mechanisms. Moreover, immunofluorescence analysis revealed that hAECs contributed to CYP11A1 expression after transplantation, whereas hAFMSCs moved towards the expression of Sertoli cell marker, SOX9, confirming a different contribution into the mechanisms leading to testis homeostasis. These findings highlight, for the first time, a distinct role of amniotic membrane and amniotic fluid derived cells in male reproduction, thus proposing innovative targeted stem-based regenerative medicine protocols for remedying high-prevalence male infertility conditions such as VAR.

Human amniotic mesenchymal stem cellspromote endometrium regeneration in a rat model of intrauterine adhesion.

Human amniotic transplantation has been proposed to improve the therapeutic efficacy of intrauterine adhesions (IUAs). Human amniotic mesenchymal stem stromal cells (hAMSCs) can differentiate into multiple tissue types. This study aimed to investigate the mechanism by which hAMSCs transplantation promotes endometrial regeneration. The rat models with IUA were established through mechanical and infective methods, and PKH26-labeled hAMSCs were transplanted through the tail vein (combined with/without estrogen). Under three different conditions, hAMSCs differentiated into endometrium-like cells. HE and Mason staining assays, and immunohistochemistry were used to compare the changes in rat models treated with hAMSCs and/or estrogen transplantation. To define the induction of hAMSCs to endometrium-like cells in vitro, an induction medium (cytokines, estrogen) was used to investigate the differentiation of hAMSCs into endometrium-like cells. qRT-polymerase chain reaction(PCR) and western blotting were performed to detect the differentiation of hAMSCs into endometrium-like cells. A greater number of glands, fewer endometrial fibrotic areas, and stronger expression of vascular endothelial growth factorand cytokeratinin the combined group (hAMSCs transplantation combined with estrogen) than in the other treatment groups were observed. hAMSCs could be induced intoendometrium-like cells by cytokine treatment (TGF-β1, EGF, and PDGF-BB). Transplantation of hAMSCs is an effective alternative for endometrial regeneration after injury in rats. The differentiation protocol for hAMSCs will be useful for further studies on human endometrial regeneration.

The Icarus Flight of Perinatal Stem and Renal Progenitor Cells Within Immune System.

Our immune system actively fights bacteria and viruses, and it must strike a delicate balance between over- and under-reaction, just like Daedalus and Icarus in Greek mythology, who could not escape their imprisonment by flying too high or too low. Both human amniotic epithelial and mesenchymal stromal cells and the conditioned medium generated from their culture exert multiple immunosuppressive activities. They have strong immunomodulatory properties that are influenced by the types and intensity of inflammatory stimuli present in the microenvironment. Notably, very recently, the immunomodulatory activity of human adult renal stem/progenitor cells (ARPCs) has been discovered. ARPCs cause a decrease in Tregs and CD3+ CD4− CD8− (DN) T cells in the early stages of inflammation, encouraging inflammation, and an increase in the late stages of inflammation, favoring inflammation quenching. If the inflammatory trigger continues, however, ARPCs cause a further increase in DN T cells to avoid the development of a harmful inflammatory state. As in the flight of Daedalus and Icarus, who could not fly too high or too low to not destroy their wings by the heat of the sun or the humidity of the sea, in response to an inflammatory environment, stem cells seem to behave by paying attention to regulating T cells in the balance between immune tolerance and autoimmunity. Recognizing the existence of both suppressive and stimulatory properties, and the mechanisms that underpin the duality of immune reaction, will aid in the development of active immunotherapeutic approaches that manipulate the immune system to achieve therapeutic benefit.

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 inhibition of Panc1 cancer cells invasion by hAMSCs secretome through suppression of tyrosine phosphorylation of SGK223 (at Y411 site), c-Src (at Y416, Y530 sites), AKT activity, and JAK1/Stat3 signaling.

SGK223 is a scaffolding protein involving in the oncogenic tyrosine kinase signaling. SGK223 was phosphorylated at Y411 by c-Src and in response to the Epidermal growth factor receptor (EGFR). Tyrosine phosphorylated SGK223 at Y411 enables to interact with CSK resulting up regulation of c-Src activity and promotion of the cell migration. Human amniotic mesenchymal stromal cells (hAMSCs) are a population of multipotent cells that it was considered to be as a potential platform in cancer therapy. Herein, we employed a co-culture system to clarify the effects of hAMSCs secretome through tyrosine phosphorylation of c-Src, SGK223, AKT activity, and JAK1/Stat3 signaling in Panc1 pancreatic cancer cells. By using the 0.4μm pore sized transwell membranes, both cell lines were firstly co-cultured for 72h. Next, c-Src activity (tyrosine phosphorylation levels at Y530 and Y416), tyrosine phosphorylation level of SGK223 (at Y411), AKT activity, and JAK1/Stat3 signaling in Panc1 cells after treatment with hAMSCs were evaluated. Our results showed that hAMSCs have the inhibitory effects on Panc1 pancreatic cancer cells invasion and it suggests that the suppression of c-Src activity, SGK223 expression, AKT activity, and JAK1/Stat3 signaling may be as critical targets in pancreatic cancer therapy.

Human Amnion-Derived Mesenchymal Stromal Cells: A New Potential Treatment for Carbapenem-Resistant Enterobacterales in Decompensated Cirrhosis.

Background: Spontaneous bacterial peritonitis (SBP) is a severe and often fatal infection in patients with decompensated cirrhosis and ascites. The only cure for SBP is antibiotic therapy, but the emerging problem of bacterial resistance requires novel therapeutic strategies. Human amniotic mesenchymal stromal cells (hA-MSCs) possess immunomodulatory and anti-inflammatory properties that can be harnessed as a therapy in such a context. Methods: An in vitro applications of hA-MSCs in ascitic fluid (AF) of cirrhotic patients, subsequently infected with carbapenem-resistant Enterobacterales, was performed. We evaluated the effects of hA-MSCs on bacterial load, innate immunity factors, and macrophage phenotypic expression. Results: hA-MSCs added to AF significantly reduce the proliferation of both bacterial strains at 24 h and diversely affect M1 and M2 polarization, C3a complement protein, and ficolin 3 concentrations during the course of infection, in a bacterial strain-dependent fashion. Conclusion: This study shows the potential usefulness of hA-MSC in treating ascites infected with carbapenem-resistant bacteria and lays the foundation to further investigate antibacterial and anti-inflammatory roles of hA-MSC in in vivo models.

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.

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.

Noval Insights into the Role of Platelet Angiogenic Growth Factors on the Regulation of Normal Vascular Development

The role of platelets as angiogenic regulators has been the subject of intense research over the last decade, and has led to the recognition of platelets as the predominant source of key angiogenic growth factors (AGFs) in the circulation. Through the regulated delivery of these factors, platelets contribute to wound healing, maintenance of vascular integrity, and tumor angiogenesis. Prior studies have shown that thrombocytopenia in adult mice leads to hemorrhages primarily from structurally abnormal new blood vessels, both in in vivo angiogenesis assays and inside tumors. The rapid growth during fetal and neonatal life is associated with a developmental stage-specific need for accelerated angiogenesis and vascular development. However, whether and how platelets participate in normal fetal/neonatal vascular development is unknown, except for their role in blood/lymphatic separation. We hypothesized that platelets would be important for normal fetal/neonatal vascular development, and that their AGF profile would be different from that of adult platelets. As a first step, we quantified five key AGFs (VEGF-A, bFGF, ANG-1, PDGF-BB and TGF-b) by ELISA in platelet lysates and platelet-poor plasma from term cord blood samples (CB; n=12) and from the peripheral blood of healthy adult volunteers (PB; n=8). These studies demonstrated that CB platelets, just like PB platelets, are the major source of AGFs in the circulation, carrying more than 80% of the levels of these AGFs present in the blood. However, the angiogenic profiles were different in platelets of different developmental origins. Fetal/neonatal platelets contained ~70% more bFGF and VEGF-A than adult platelets (p<0.01 for both), but had significantly lower levels of ANG-1, PDGF-BB and TGF-β1 (p<0.05 vs. adult platelets). VEGF-A and bFGF are essential for the proliferation and differentiation of endothelial cells, while ANG-1, PDGFs and TGF-b concert mural cell coverage, basement membrane formation and vascular maturation, thus stabilizing and “strengthening” the vasculature. Based on these characteristics, we hypothesized that neonatal and adult platelets would have different biological effects. To test this, we first assessed the effects of CB- vs. PB-derived platelet lysates on human umbilical vascular endothelial cells (HUVEC), in a tube formation assay. In these experiments, CB platelets more potently stimulated angiogenesis, quantified as number of capillary branch points at 4, 5, and 6 hours, than PB platelets. Subsequently, we compared the proliferative effects of CB vs. PB platelet lysates on human primary CB-derived endothelial progenitor cells (EPCs) and amniotic membrane-derived mesenchymal stromal cells (MSCs). Consistent with the effects on HUVEC cells, CB platelet lysates induced approximately 50% more proliferation of EPCs, compared to PB platelet lysates (p<0.001). In contrast, PB platelet lysates were approximately twice more potent in promoting MSCs growth (p<0.001 vs. CB platelets). Since EPCs give rise to the vascular endothelium and MSCs differentiate into mural cells, these results strongly suggested that the AGF content in fetal/neonatal platelets promotes rapid angiogenesis at the expense of a stable vasculature, matching the developmental needs of rapidly growing fetuses and neonates. Finally, since birth is associated with an abrupt change in oxygen concentration (from the profoundly hypoxic intrauterine to the normoxic extrauterine environment), we assessed whether oxygen concentration would modulate the VEGF content in CB-derived cultured MKs (and thus platelets). Consistent with this hypothesis, CB MKs cultured in 5% oxygen released significantly more VEGF in the medium compared to MKs cultured in 20% oxygen (p<0.05). Our findings strongly suggest that the platelet AGF profile changes from neonatal to adult life to help regulate vascular development at different stages of life, and suggest that it might also change in response to environmental factors such as oxygen concentration. Whether a change in platelet angiogenic profile associated with the sudden increase in oxygen concentration during preterm birth contributes to diseases of prematurity characterized by disrupted vascular development (i.e. retinopathy of prematurity) requires further investigation. DisclosuresSola-Visner:Sysmex Inc.: Research Funding.

Evaluating the effect of secretome of human amniotic mesenchymal stromal cells on apoptosis induction and epithelial-mesenchymal transition inhibition in LNCaP prostate cancer cells based on 2D and 3D cell culture models.

Prostate cancer (PCa) is the second most prevalent cancer in men worldwide. Most cases of death from PCa are due to metastasis. Early stages of metastasis are mediated by epithelial-mesenchymal transition (EMT) process through which cancer cells acquire motility and invasive characteristics. Thus, more potent and novel therapeutic strategies must be designed based on the inhibition of EMT or metastasis. Herein, we employ a co-culture system to evaluate the anti-EMT effects of human amniotic mesenchymal stromal cells (hAMSCs) on LNCaP PCa cells. The RNA of treated (sample) and untreated cancer cells (control) and whole-cell lysates of related cells were prepared and analysed through quantitative real-time polymerase chain reaction (qRT-PCR) and western blot, respectively. Based on the results, the expression of vimentin, Snail and Zeb1 in LNCaP cells decreased and the expression of E-cadherin increased after treatment with hAMSCs. Furthermore, induction of the cellular apoptosis in LNCaP cells was detected. The anti-cancer activity of conditioned medium from hAMSCs was shown using hanging drop technique (a 3D cell culture model). Our findings support the idea that stem cells can be considered as a novel therapeutic approach to inhibit prostate cancer cells. SIGNIFICANCE OF THE STUDY: The anti-tumour activity of hAMSCs on LNCaP prostate cancer cells using 2D and 3D cell culture models via induction of apoptosis, suppression of EMT process and down-regulation of EGFR was shown. The results of the present study support this idea that hAMSCs may be a potent therapeutic tool to suppress tumour growth in LNCaP prostate cancer cells.

Evaluating the in vitro therapeutic effects of human amniotic mesenchymal stromal cells on MiaPaca2 pancreatic cancer cells using 2D and 3D cell culture model

Human amniotic mesenchymal stromal cells (hAMSCs) are considered as a population of multipotent cells. The molecular events associated with mesenchymal stromal cell (MSC)/tumor cell interactions should be studied to identify the role of MSCs on suppressing or inducing the key signaling pathways of tumor cells. Thus, designing therapeutic approaches is considered as important. In the present study, hAMSCs and MiaPaca2 cells were first cultured separately. In addition, both cell lines were co-cultured by using 0.4 μm pore sized transwell membranes in different times. Further, the RNA of the cells was extracted, and Bcl2, Bax, epidermal growth factor receptor (EGFR), c-Src, C-terminal Src Kinase (CSK), and SGK223 expression were analyzed through quantitative real time PCR. Furthermore, the total cell lysates of the cells were prepared and analyzed by using western blot. Based on the results, the expression of EGFR, c-Src, SGK223, and CSK in MiaPaca2 cells reduced after treating with hAMSCs. Notably, the cellular apoptosis of MiaPaca2 cells was induced in 2D cell culture system. Further, the anti-cancer activity of conditioned medium from hAMSCs was confirmed in a 3D cell culture model by using hanging drop technique. Finally, hAMSCs have inhibitory effects on pancreatic cancer cells and can be considered as a therapeutic way to suppress EGFR, c-Src, and SGK223, as the potent targets in cancer cell signaling.

Effect of Diphenyleneiodonium Chloride on Intracellular Reactive Oxygen Species Metabolism with Emphasis on NADPH Oxidase and Mitochondria in Two Therapeutically Relevant Human Cell Types.

Reactive oxygen species (ROS) have recently been recognized as important signal transducers, particularly regulating proliferation and differentiation of cells. Diphenyleneiodonium (DPI) is known as an inhibitor of the nicotinamide adenine dinucleotide phosphate oxidase (NOX) and is also affecting mitochondrial function. The aim of this study was to investigate the effect of DPI on ROS metabolism and mitochondrial function in human amniotic membrane mesenchymal stromal cells (hAMSCs), human bone marrow mesenchymal stromal cells (hBMSCs), hBMSCs induced into osteoblast-like cells, and osteosarcoma cell line MG-63. Our data suggested a combination of a membrane potential sensitive fluorescent dye, tetramethylrhodamine methyl ester (TMRM), and a ROS-sensitive dye, CM-H2DCFDA, combined with a pretreatment with mitochondria-targeted ROS scavenger MitoTEMPO as a good tool to examine effects of DPI. We observed critical differences in ROS metabolism between hAMSCs, hBMSCs, osteoblast-like cells, and MG-63 cells, which were linked to energy metabolism. In cell types using predominantly glycolysis as the energy source, such as hAMSCs, DPI predominantly interacted with NOX, and it was not toxic for the cells. In hBMSCs, the ROS turnover was influenced by NOX activity rather than by the mitochondria. In cells with aerobic metabolism, such as MG 63, the mitochondria became an additional target for DPI, and these cells were prone to the toxic effects of DPI. In summary, our data suggest that undifferentiated cells rather than differentiated parenchymal cells should be considered as potential targets for DPI.