Secretome Of Human Mesenchymal Stem Cells Research Articles

Human Umbilical Cord-Mesenchymal Stem Cells Promote Extracellular Matrix Remodeling in Microglia.

Human mesenchymal stem cells modulate the immune response and are good candidates for cell therapy in neuroinflammatory brain disorders affecting both adult and premature infants. Recent evidence indicates that through their secretome, mesenchymal stem cells direct microglia, brain-resident immune cells, toward pro-regenerative functions, but the mechanisms underlying microglial phenotypic transition are still under investigation. Using an in vitro coculture approach combined with transcriptomic analysis, we identified the extracellular matrix as the most relevant pathway altered by the human mesenchymal stem cell secretome in the response of microglia to inflammatory cytokines. We confirmed extracellular matrix remodeling in microglia exposed to the mesenchymal stem cell secretome via immunofluorescence analysis of the matrix component fibronectin and the extracellular crosslinking enzyme transglutaminase-2. Furthermore, an analysis of hallmark microglial functions revealed that changes in the extracellular matrix enhance ruffle formation by microglia and cell motility. These findings point to extracellular matrix changes, associated plasma membrane remodeling, and enhanced microglial migration as novel mechanisms by which mesenchymal stem cells contribute to the pro-regenerative microglial transition.

Human mesenchymal stem cell secretome lowers caspase-3 levels and apoptosis in hepatocytes of cholestatic rats

Liver cirrhosis is ranked 15th as a cause of morbidity and is ranked 11th as a leading cause of mortality in the world. Definitive therapy is liver transplantation, but it is constrained by difficulties in finding a transplant donor Hu MSCs cell-based therapy can help liver regeneration directly, through hepatogenic differentiation, or indirectly through the paracrine secretome. This study aims to determine the effect of Human Mesenchymal Stem Cell Secretome administration on caspase 3 levels and apoptotic hepatic cell count in a rat model with cholestasis after choledochal duct ligation receiving standard UDCA (Urso Deoxy Cholid Acid) therapy. Twenty-four male Wistar rats were subjected to a choledochal duct ligation and were included in this randomized experimental study. After surgical intervention, all rats were randomly assigned into 4 group; control, administration of UDCA, Hu MSC-S, and a combination of UDCA and Human Mesenchymal Stem Cell Secretome for 4 weeks. Caspase-3 levels were assessed from the blood sample, while the apoptosis of hepatocyte evaluated using histopathologic examination of the liver. Caspase-3 levels was significantly lower in the UDCA and Human Mesenchymal Stem Cell Secretome treated groups (6.23 ± 0,30), compared to the UDCA (8.77 ± 0.55) and Human Mesenchymal Stem Cell Secretome alone (7.30 ± 0.42) (ANOVA p < 0,05). The apoptosis of hepatocyte was significantly lower in the combination group (8,88 ± 5,46) compared to the UDCA (29,00 ± 4,52) and Human Mesenchymal Stem Cell Secretome alone (20,83 ± 4,14) (ANOVA p < 0,05). In conclusion, addition of Human Mesenchymal Stem Cell Secretome to UDCA lowered caspase-3 levels and apoptotic cell count in rats with hepatic cholestasis after choledochal duct ligation and better compared to each alone. Keywords: Mesenchymal Stem Cell Secretome, Capsase-3, Apoptotic Cell, Liver Regeneration, Cholestasis

Human mesenchymal stem cell secretome increases hepatocyte growth factor expression and promotes liver regeneration in Wistar rats with cholestasis

Globally, the annual mortality rate due to liver cirrhosis is 2.2%, which ranks 12th as the cause of death in the United States. Human mesenchymal stem cell secretome (Hu-MSC-S) is known to reduce liver injury and inflammation. Thus, the present study aims to determine the effect of Hu-MSC-S on hepatocyte growth factor (HGF) expression and liver regeneration in cholestatic rats after choledochal duct ligation receiving standard ursodeoxycholic acid (UDCA) therapy. A randomized experimental study was conducted on 24 male Wistar rats. All rats were randomly assigned into four groups such as control, UDCA, Hu-MSC-S, and UDCA+Hu-MSC-S. The expression of HGF and mitotic index as markers of liver regeneration were evaluated using histopathologic examination. Both HGF expression and the mitotic index were significantly higher in the UDCA and Hu-MSC-S groups (P < 0.05) compared to the control group. The combined administration of UDCA and Hu-MSC-S resulted in significantly higher HGF expression and mitotic index than the individual intervention (P < 0.05). The additional administration of Hu-MSC-S increased HGF expression and mitotic index in rat model of hepatic cholestasis. In conclusion, HuMSC-S administration could be considered as a therapy in patients who received standard UDCA therapy for liver regeneration.

Effect of human mesenchymal stem cell secretome administration on morphine self-administration and relapse in two animal models of opioid dependence

The present study investigates the possible therapeutic effects of human mesenchymal stem cell-derived secretome on morphine dependence and relapse. This was studied in a new model of chronic voluntary morphine intake in Wistar rats which shows classic signs of morphine intoxication and a severe naloxone-induced withdrawal syndrome. A single intranasal-systemic administration of MSCs secretome fully inhibited (>95%; p < 0.001) voluntary morphine intake and reduced the post-deprivation relapse intake by 50% (p < 0.02). Since several studies suggest a significant genetic contribution to the chronic use of many addictive drugs, the effect of MSCs secretome on morphine self-administration was further studied in rats bred as high alcohol consumers (UChB rats). Sub-chronic intraperitoneal administration of morphine before access to increasing concentrations of morphine solutions and water were available to the animals, led UChB rats to prefer ingesting morphine solutions over water, attaining levels of oral morphine intake in the range of those in the Wistar model. Intranasally administered MSCs secretome to UChB rats dose-dependently inhibited morphine self-administration by 72% (p < 0.001); while a single intranasal dose of MSC-secretome administered during a morphine deprivation period imposed on chronic morphine consumer UChB rats inhibited re-access morphine relapse intake by 80 to 85% (p < 0.0001). Both in the Wistar and the UChB rat models, MSCs-secretome administration reversed the morphine-induced increases in brain oxidative stress and neuroinflammation, considered as key engines perpetuating drug relapse. Overall, present preclinical studies suggest that products secreted by human mesenchymal stem cells may be of value in the treatment of opioid addiction.

Preclinical Assessment of Mesenchymal-Stem-Cell-Based Therapies in Spinocerebellar Ataxia Type 3.

The low regeneration potential of the central nervous system (CNS) represents a challenge for the development of new therapeutic strategies for neurodegenerative diseases, including spinocerebellar ataxias. Spinocerebellar ataxia type 3 (SCA3)—or Machado–Joseph disease (MJD)—is the most common dominant ataxia, being mainly characterized by motor deficits; however, SCA3/MJD has a complex and heterogeneous pathophysiology, involving many CNS brain regions, contributing to the lack of effective therapies. Mesenchymal stem cells (MSCs) have been proposed as a potential therapeutic tool for CNS disorders. Beyond their differentiation potential, MSCs secrete a broad range of neuroregulatory factors that can promote relevant neuroprotective and immunomodulatory actions in different pathophysiological contexts. The objective of this work was to study the effects of (1) human MSC transplantation and (2) human MSC secretome (CM) administration on disease progression in vivo, using the CMVMJD135 mouse model of SCA3/MJD. Our results showed that a single CM administration was more beneficial than MSC transplantation—particularly in the cerebellum and basal ganglia—while no motor improvement was observed when these cell-based therapeutic approaches were applied in the spinal cord. However, the effects observed were mild and transient, suggesting that continuous or repeated administration would be needed, which should be further tested.

Antibacterial and antibiofilm activity of bone marrow-derived human mesenchymal stem cells secretome against Vibrio cholerae

The ability of V. cholerae to survive and spread in the aquatic environment combined with the scarcity of effective antimicrobial agents, especially those effective against multidrug-resistant strains highlights the need for alternative non-antibiotic approaches for the treatment of V. cholerae infections. The aim of this study was to specifically examine the potential direct effect of unstimulated MSC secretome on V. cholerae killing and biofilm formation as a representative of non-invasive enteric bacterial pathogen. The bmMSCs were characterized by the presence of CD44 and CD73 and the absence of CD45 and CD34 molecular markers. Moreover, self-regeneration and differentiation capacity of MSCs into adipocytes and osteogenic lineages was assessed by immunohistology (IHC) method. The antibacterial activity of unstimulated MSCs supernatant against V. cholerae in broth microdilution assay decreased the bacterial suspension from 108 CFU/ml to 107 CFU/ml and showed a significant antimicrobial activity in a dose-dependent manner at dilutions of 1:8 to 1:128 (P < 0.05). The role of MSC secretome without preconditioning in the prevention of biofilm formation was assessed through plate-crystal violet assay and showed high antibiofilm activity against V. cholerae also in dose-dependent manner. As antibacterial mechanisms of MSC secretome are different from conventional antibiotics, together with its antibiofilm activity, proposes its application as a novel therapeutic approach combatting multi‐drug resistant pathogens with no fear of developing antimicrobial resistance.

Characterizing the impact of 2D and 3D culture conditions on the therapeutic effects of human mesenchymal stem cell secretome on corneal wound healing in vitro and ex vivo

The therapeutic effects of secreted factors (secretome) produced by bone marrow-derived human mesenchymal stem cells (MSCs) were evaluated as a function of their growth in 2D culture conditions and on 3D electrospun fiber scaffolds.Electrospun fiber scaffolds composed of polycaprolactone and gelatin were fabricated to provide a 3D microenvironment for MSCs, and their mechanical properties were optimized to be similar to corneal tissue. The secretome produced by the MSCs cultured on 3D fiber matrices versus 2D culture dishes were analyzed using a Luminex immunoassay, and the secretome of MSCs cultured on the 3D versus 2D substrates showed substantial compositional differences. Concentrations of factors such as HGF and ICAM-1 were increased over 5 times in 3D cultures compared to 2D cultures. In vitro proliferation and scratch-based wound healing assays were performed to compare the effects of the secretome on corneal fibroblast cells (CFCs) when delivered synchronously from co-cultured MSCs through a trans-well co-culture system versus asynchronously after harvesting the factors separately and adding them to the media. Cell viability of CFCs was sustained for 6 days when co-cultured with MSCs seeded on the fibers but decreased with time under other conditions. Scratch assays showed 95% closure at 48 h when CFCs were co-cultured with MSCs seeded on fibers, while the control group only exhibited 50% closure at 48 h. Electrospun fibers seeded with MSCs were then applied to a rabbit corneal organ culture system, and MSCs seeded on fibers promoted faster epithelialization and less scarring. Corneas were fixed and stained for alpha smooth muscle actin (α-SMA), and then analyzed by confocal microscopy. Immunostaining showed that expression of α-SMA was lower in corneas treated with MSCs seeded on fibers, suggesting suppression of myofibroblastic transformation.MSCs cultured on electrospun fibers facilitate wound healing in CFCs and on explanted corneas through differential secretome profiles compared to MSCs cultured on 2D substrates. Future work is merited to further understand the nature and basis of these differences and their effects in animal models. Statement of significancePrevious studies have shown that the secretome of bone marrow-derived mesenchymal stem cells (MSC) is promotes corneal wound healing by facilitating improved wound closure rates and reduction of scarring and neovascularization. The present research is significant because it provides evidence for the modulation of the secretome as a function of the MSC culture environment. This leads to differential expression of therapeutic factors secreted, which can impact corneal epithelial and stromal healing after severe injury.In addition, this article shows that co-continuous delivery of the MSC secretome improves cell migration and proliferation over aliquoted delivery, and that MSCs grown on three-dimensional electrospun fiber constructs may provide a favorable microenvironment for cultured MSCs and as a carrier to deliver their secreted factors to the ocular surface.

Towards cell therapy of polycystic ovary syndrome (PCOS): human mesenchymal stem cells secretome inhibits androgen production by pcos theca cells

Background & Aim Background Polycystic Ovarian Syndrome (PCOS) is a metabolic disorder characterized by inflammation, infertility and excess ovarian androgen production by theca cells. Though its etiology is not fully understood. Women with PCOS exhibit increased expression of CYP17A1, and CYP11A1 genes involved in androgen production. Due to the robust anti-inflammatory ability of human bone marrow Mesenchymal stem cells (hMSCs), we evaluated its utility as a novel cell-therapy by injecting hMSCs directly into the ovaries of PCOS mouse model. It showed a significant improvement in hyperandrogenemia. hMSCs also secrete various factors in media, known as secretome. The literature shows that the secretome of hMSCs has the same regenerative and reparative function likes hMSCs. Hypothesis We hypothesize that hMSCs secretome contains the factors that are able to inhibit the androgen biosynthesis in PCOS theca cells. Methods, Results & Conclusion Material and Methods Theca cells, collected from normal cycling and PCOS patient underwent ovariectomy, were seeded on culture dishes pre-coated with the extracellular matrix at a density of 6 × 104 cells/ml and cultured for 24 hours. Cells were then treated with either 3T3 conditioned media as a control (C) or Mesenchymal stem cells conditioned media (CM). The C and CM were prepared through collecting the serum-free supernatant of 3T3 and hMSCs at an optimal confluence, 48 hours later. The mRNA and protein expression of CYP17A1, CYP11A1, and DENND1A.V2 were quantified by RT-PCR and Western blot, while testosterone level in media was measured by RIA assay. Results Human PCOS theca cells treated with hMSCs CM for 48 hours secreted significantly lower level of testosterone (146 ± 13.6 ng/dl) compared to control C (215 ± 11.3 ng/dl) (P Conclusion The therapeutic capabilities of hMSCs in PCOS are partially mediated by its secretome which contains various growth factors and anti-inflammatory cytokines both free and within extracellular vesicles. Stem cell therapy, either by cell implantation or via secretome injection might potentially be a novel approach for effective treatment of PCOS patients.

Human Mesenchymal Stem Cell Secretome from Bone Marrow or Adipose-Derived Tissue Sources for Treatment of Hypoxia-Induced Pulmonary Epithelial Injury

Alveolar epithelial dysfunction induced by hypoxic stress plays a significant role in the pathological process of lung ischemia-reperfusion injury (IRI). Mesenchymal stem cell (MSC) therapies have demonstrated efficacy in exerting protective immunomodulatory effects, thereby reducing airway inflammation in several pulmonary diseases. Aim: This study assesses the protective effects of MSC secretome from different cell sources, human bone marrow (BMSC) and adipose tissue (ADSC), in attenuating hypoxia-induced cellular stress and inflammation in pulmonary epithelial cells. Methods: Pulmonary epithelial cells, primary rat alveolar epithelial cells (AEC) and A549 cell line were pre-treated with BMSC, or ADSC conditioned medium (CM) and subjected to hypoxia for 24 h. Results: Both MSC-CM improved cell viability, reduced secretion of pro-inflammatory mediators and enhanced IL-10 anti-inflammatory cytokine production in hypoxic injured primary rat AECs. ADSC-CM reduced hypoxic cellular injury by mechanisms which include: inhibition of p38 MAPK phosphorylation and nuclear translocation of subunits in primary AECs. Both MSC-CM enhanced translocation of Bcl-2 to the nucleus, expression of cytoprotective glucose-regulated proteins (GRP) and restored matrix metalloproteinases (MMP) function, thereby promoting repair and cellular homeostasis, whereas inhibition of GRP chaperones was detrimental to cell survival. Conclusions: Elucidation of the protective mechanisms exerted by the MSC secretome is an essential step for maximizing the therapeutic effects, in addition to developing therapeutic targets-specific strategies for various pulmonary syndromes.

Anti-inflammatory effects of human embryonic stem cell-derived mesenchymal stem cells secretome preconditioned with diazoxide, trimetazidine and MG-132 on LPS-induced systemic inflammation mouse model

Systemic inflammatory response syndrome is a complex pathophysiologic and immunologic response to an insult. Sepsis is a life-threatening condition happening when the body’s response to infection causes injury to its own tissues and organs. Stem cell therapy is a new approach to modulate immune responses. Mesenchymal stem cells (MSCs) establish a regenerative niche by secreting secretome and modulating immune responses. MSC secretome can be leveraged for therapeutic applications if production of secretary molecules were optimized. Pharmacological preconditioning using small molecules can increase survival of MSCs after transplantation. The aim of this study was to investigate the effect of secretome of human embryonic-derived mesenchymal stem cells (hESC-MSCs) preconditioned with MG-132,Trimetazidine (TMZ) and Diazoxide (DZ) on immunomodulatory efficiency of these cells in Lipo polysaccharide (LPS) challenged mice models. Mice were injected intraperitoneally with LPS and groups of animals were intraperitoneally given 1 ml 30× secretome 6 h after LPS injection. Serum levels of biochemical parameters were then measured by an auto analyser and serum inflammatory cytokine levels were analysed using commercially available RayBio Mouse Inflammation Antibody Array. Ultimately, histopathology and survival studies were conducted. The results showed that TMZ and DZ-conditioned medium significantly increasing the survival and improvement of histopathological score. We found that MG-132-conditioned medium failed to show significant outcomes. This study demonstrated that human MSC secretome has the potential to control inflammation.

Joshua Mayourian: Rising to the Challenge.

Joshua Mayourian: Rising to the Challenge.

Mesenchymal stem cell secretome reduces pain and subchondral bone alterations in a mouse osteoarthritis model

Purpose: Mesenchymal stem cells (MSCs) represent a promising biological therapeutic option as an OA modifying treatment and have shown clinical improvement. Next to their differentiation potential into several lineages, they have the ability to influence their environment by secreting trophic mediators. These secreted factors could possibly counteract inflammatory and catabolic processes and attract endogenous repair cells. By using the MSC secretome as an OA treatment instead of the MSCs themselves, the possibilities to provide a more standardized and affordable therapeutic option would be largely increased. Together with a decrease of legal and safety issues, this would greatly enhance the clinical applicability of cell-based treatment as a true disease modifying anti-osteoarthritic drug. Aim of our study was to explore the anti-osteoarthritic effects of MSC secretome on pain, subchondral bone changes, cartilage damage, synovial thickness and synovial macrophage subtypes in a mouse osteoarthritis model. Methods: For the production of MSC Conditioned Medium (MSC-CM), human bone marrow derived MSCs from 3 donors were stimulated with IFNγ and TNFα (50 ng/mL each). After 24 hours of stimulation, medium was replaced by αMem without Phenol Red containing 0.05% BSA. This medium was collected after 24 hours, concentrated using 3k cut-off filters, frozen and designated MSC-CM. OA was induced unilaterally in 54 male C57/Bl6 mice by two intra-articular collagenase injections (collagenase-induced osteoarthritis model). After 7 days, mice were randomly assigned to 3 consecutive injections with 20.000 MSCs, MSC-CM from 20.000 MSCs or medium only (control). Weight distribution over the left and right hind limbs was evaluated as an indicator of pain using an incapitance tester. Other outcome measures were evaluated by histology at 5 and 21 days after treatment. Results: Seven days after induction of OA (day 0), 41.2 6.3% of weight was distributed on the affected limb, indicating pain when compared to before OA (Fig. 1, P<0.0001). We observed an early normalization in weight bearing in MSC-CM injected animals at day 7 compared to day 0, indicating early pain reduction (P = 0.001). Moreover, mice that received MSC-CM displayed significantly less pain compared to the medium control at day 7 (P = 0.001). OA induction lead to loss of subchondral bone tissue in the medial tibia plateau, 5 days after the first collagenase injection. (Fig. 2, P = 0.003). The percentage of subchondral bone in the MSC-CM treated group as well as the MSC treated group was not statistically different from the healthy control group, indicating amelioration of OA related decrease of subchondral bone volume. No treatment effects were seen on cartilage damage, synovial inflammation or the presence of iNOS, CD206, or CD163 positive macrophages (Fig. 3A–C). Conclusions: In this study, we found that injection of human MSC secretome resulted in an early pain reduction and amelioration of early subchondral bone changes, in a comparable manner to injection of MSCs themselves. Possibly, these two aspects are related, since subchondral bone marrow edema or bone marrow lesions have clinically been shown to be correlated with patients perceived degree of pain. Synovitis is another OA feature which has clinically been related to pain. We did not find a treatment effect on synovial thickness or macrophage phenotype in our study. Additional studies are warranted to further unravel the mechanisms behind the therapeutic effects of MSCs and their secretome. This way, we can evolve this biological approach towards a true disease modifying anti-osteoarthritic drug, in which the use of secretome would greatly enhance the standardization, affordability and clinical translatability.

Proteomic analysis of the secretome of human bone marrow-derived mesenchymal stem cells primed by pro-inflammatory cytokines

Mesenchymal stem cells (MSC) represent an impressive opportunity in term of regenerative medicine and immunosuppressive therapy. Although it is clear that upon transplantation MSC exert most of their therapeutic effects through the secretion of bioactive molecules, the effects of a pro-inflammatory recipient environment on MSC secretome have not been characterized. In this study, we used a label free mass spectrometry based quantitative proteomic approach to analyze how pro-inflammatory cytokines modulate the composition of the human MSC secretome. We found that pro-inflammatory cytokines have a strong impact on the secretome of human bone marrow-derived MSC and that the large majority of cytokine-induced proteins are involved in inflammation and/or angiogenesis. Comparative analyses with results recently obtained on mouse MSC secretome stimulated under the same conditions reveals both analogies and differences in the effect of pro-inflammatory cytokines on MSC secretome in the two organisms. In particular, functional analyses confirmed that tissue inhibitor of metalloproteinase-1 (TIMP1) is a key effector molecule responsible for the anti-angiogenic properties of both human and mouse MSC within an inflammatory microenvironment. Mass spectrometry data are available via ProteomeXchange with identifier PXD005746 SignificanceThe secretion of a broad range of bioactive molecules is believed to be the main mechanism by which MSC exert specific therapeutic effects. MSC are very versatile and respond to specific environments by producing and releasing a variety of effector molecules. To the best of our knowledge this is the first study aimed at describing the secretome of human MSC primed using a mixture of cytokines, to mimic pro-inflammatory conditions encountered in vivo, by a quantitative high-resolution mass spectrometry based approach. The main output of the study concerns the identification of a list of specific proteins involved in inflammation and angiogenesis which are overrepresented in stimulated MSC secretome. The data complement a previous study on the secretome of mouse MSC stimulated under the same conditions. Comparative analyses reveal analogies and differences in the biological processes affected by overrepresented proteins in the two organisms. In particular, the key role of TIMP-1 for the anti-angiogenic properties of stimulated MSC secretome already observed in mouse is confirmed in human. Overall, these studies represent key steps necessary to characterize the different biology of MSC in the two organisms and design successful pre-clinical experiments as well as clinical trials.

Impact of the Secretome of Human Mesenchymal Stem Cells on Brain Structure and Animal Behavior in a Rat Model of Parkinson's Disease.

Research in the last decade strongly suggests that mesenchymal stem cell (MSC)‐mediated therapeutic benefits are mainly due to their secretome, which has been proposed as a possible therapeutic tool for the treatment of Parkinson's disease (PD). Indeed, it has been shown that the MSC secretome increases neurogenesis and cell survival, and has numerous neuroprotective actions under different conditions. Additionally, using dynamic culturing conditions (through computer‐controlled bioreactors) can further modulate the MSC secretome, thereby generating a more potent neurotrophic factor cocktail (i.e., conditioned medium). In this study, we have characterized the MSC secretome by proteomic‐based analysis, investigating its therapeutic effects on the physiological recovery of a 6‐hydroxidopamine (6‐OHDA) PD rat model. For this purpose, we injected MSC secretome into the substantia nigra (SNc) and striatum (STR), characterizing the behavioral performance and determining histological parameters for injected animals versus untreated groups. We observed that the secretome potentiated the increase of dopaminergic neurons (i.e., tyrosine hydroxylase‐positive cells) and neuronal terminals in the SNc and STR, respectively, thereby supporting the recovery observed in the Parkinsonian rats’ motor performance outcomes (assessed by rotarod and staircase tests). Finally, proteomic characterization of the MSC secretome (through combined mass spectrometry analysis and Bioplex assays) revealed the presence of important neuroregulatory molecules, namely cystatin C, glia‐derived nexin, galectin‐1, pigment epithelium‐derived factor, vascular endothelial growth factor, brain‐derived neurotrophic factor, interleukin‐6, and glial cell line‐derived neurotrophic factor. Overall, we concluded that the use of human MSC secretome alone was able to partially revert the motor phenotype and the neuronal structure of 6‐OHDA PD animals. This indicates that the human MSC secretome could represent a novel therapeutic for the treatment of PD. Stem Cells Translational Medicine 2017;6:634–646

Microenvironment Regulation of Pluripotent Stem Cell-Derived Neural Progenitor Aggregates by Human Mesenchymal Stem Cell Secretome

Neural progenitor cells (NPCs) derived from pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced PSCs, are promising cell source for the treatment of various neurological diseases. NPC derivation from PSCs is regulated by microenvironment factors that influence cell fate via paracrine and autocrine effects. In this study, ESC-derived NPC aggregates were replated in the secretomes of bone marrow-derived human mesenchymal stem cells (hMSCs) generated under hypoxia or normoxia to investigate the effects of hMSC secretome on NPC cellular behaviors. The results demonstrated that hMSC secretomes stimulated endogenous secretion of extracellular matrices from NPC aggregates and enhanced cell adhesion and proliferation. NPC functional differentiation measured by migration length, neurite extension, and the yield of neural and glial cells were also increased by threefold to fourfold. Inhibition of fibroblast growth factor-2, transforming growth factor-β1, and brain-derived neurotrophic factor signaling differentially reduced the adherent cell number, migration length, and neurite extension, suggesting the regulatory effects of a broad spectrum of hMSC-derived factors. In summary, ESC-derived NPC aggregates in hypoxic hMSC secretomes may represent a suitable combination to promote the engraftment and neurogenesis in vivo.

Characterization of human mesenchymal stem cell secretome at early steps of adipocyte and osteoblast differentiation

BackgroundIt is well established that adipose tissue plays a key role in energy storage and release but is also a secretory organ and a source of stem cells. Among different lineages, stem cells are able to differentiate into adipocytes and osteoblasts. As secreted proteins could regulate the balance between both lineages, we aimed at characterizing the secretome of human multipotent adipose-derived stem cell (hMADS) at an early step of commitment to adipocytes and osteoblasts.ResultsA proteomic approach, using mono-dimensional electrophoresis and tandem mass spectrometry, allowed us to identify a total of 73 proteins at day 0 and day 3 of adipocyte and osteoblast differentiation. Analysis of identified proteins showed that 52 % corresponded to classical secreted proteins characterized by a signal peptide, that 37 % previously described in the extracellular compartment were devoid of signal peptide and that 11 % neither exhibited a signal peptide nor had been previously described extracellularly. These proteins were classified into 8 clusters according to their function. Quantitative analysis has been performed for 8 candidates: PAI-1, PEDF, BIGH3, PTX3, SPARC, ENO1, GRP78 and MMP2. Among them, PAI-1 was detected at day 0 and day 3 of osteoblast differentiation but never in adipocyte secretome. Furthermore we showed that PAI-1 mRNA was down-regulated in the bone of ovariectomized mice.ConclusionGiven its regulation during the early events of hMADS cell differentiation and its status in ovariectomized mice, PAI-1 could play a role in the adipocyte/osteoblast balance and thus in bone diseases such as osteoporosis.