Paracrine Effects Of Mesenchymal Stem Cells Research Articles

Some thoughts on the research of mesenchymal stem cell exosomes and wound microenvironment

Since researchers have found that the conditioned medium and exosomes of mesenchymal stem cells (MSCs) had the biological effects equivalent to those of MSCs, MSC exosomes (MSC-Exos), the representative product of MSCs' paracrine effect, have become the research focus of the "cell-free" therapy of MSCs. However, most researchers currently use conventional culture condition to culture MSCs and then isolate exosomes for the treatment of wound or other diseases. Theoretically, the paracrine effect of MSCs is directly associated with the pathological condition of the wound (disease) microenvironment or in vitro culture condition, and their paracrine components and biological effects may be altered with the changes of the wound (disease) microenvironment or in vitro culture condition. Thus, the feasibility of using traditional culture condition to culture MSCs for exosome extraction for the treatment of different diseases without considering the actual situation of the disease to be treated needs further discussion. Therefore, the author suggests that the research of MSC-Exos should consider the microenvironment of the wound (disease) to be treated. as much as possible, otherwise the extracted MSC-Exos may not be "accurate" or may not really achieve the treatment effect of MSCs. In this article, we summarized some thoughts of the author and problems related to the researches about MSC-Exos and wound microenvironment, and hoped to discuss with researchers.

Osteogenically differentiated mesenchymal stem cells promote the apoptosis of human umbilical vein endothelial cells in vitro.

The absence of blood vessels in tissue engineered bone often leads to necrosis of internal cells after implantation, ultimately affecting the process of bone repair. Herein, mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs) were cocultured to induce osteogenesis and angiogenesis. Based on the findings, the number of HUVECs in the coculture system increased in the growth medium group, but decreased in the osteogenic induction medium (OIM) group. Considering that the paracrine effects of MSCs had changed, we tested the genes expression of osteogenically differentiated MSCs. The expression of osteogenic genes in MSCs increased during osteogenesis. Further, the expression levels of pigment epithelial-derived factor (PEDF) gene and protein, an antivascular factor, were also increased. To verify whether MSCs promote HUVECs apoptosis via PEDF, PEDF was silenced via siRNA. The conditioned medium of differentiated MSCs with PEDF silencing significantly improved the proliferation and apoptosis of HUVECs. Based on further experiments, PEDF mediated the apoptosis and proliferation of HUVECs through p53, BAX/BCL-2, FAS, and c-Caspase-3. However, when PEDF was silenced with siRNA, the osteogenic potential of MSCs was affected. The results of this study provide a theoretical basis for the construction of prevascularized bone tissues in vitro.

Mesenchymal stem cell-derived exosomes: therapeutic implications for rotator cuff injury.

Rotator cuff injuries are a common clinical condition of the shoulder joint. Surgery that involves reattaching the torn tendon to its humeral head bony attachment has a somewhat lower success rate. The scar tissue formed during healing of the rotator cuff leads to poor tendon-related mechanical properties. To promote healing, a range of genetic interventions, as well as cell transplantation, and many other techniques have been explored. In recent years, the therapeutic promise of mesenchymal stem cells (MSCs) has been well documented in animal and clinical studies. Some data have suggested that MSCs can promote angiogenesis, reduce inflammation and cell proliferationand increase collagen deposition. These functions are likely paracrine effects of MSCs, particularly mediated through exosomes. Here, we review the use of MSCs-related exosomes in tissues and organs. We also discuss their potential utility for treating rotator cuff injuries, and explore the underlying mechanisms of their effects.

Biology and therapeutic potential of mesenchymal stem cell-derived exosomes.

Mesenchymal stem cells (MSC) are multipotent stromal cells with the potential to differentiate into several cell types. MSC‐based therapy has emerged as a promising strategy for various diseases. Accumulating evidence suggests that the paracrine effects of MSC are partially exerted by the secretion of soluble factors, in particular exosomes. MSC‐derived exosomes are involved in intercellular communication through transfer of proteins, RNA, DNA and bioactive lipids, which might constitute a novel intercellular communication mode. This review illustrates the current knowledge on the composition and biological functions as well as the therapeutic potential of MSC‐derived exosomes in cancer, with a focus on clinical translation opportunities.

MSC-secreted TGF-\u03b2 regulates lipopolysaccharide-stimulated macrophage M2-like polarization via the Akt/FoxO1 pathway

BackgroundAn uncontrolled inflammatory response is a critical pathophysiological feature of sepsis. Mesenchymal stem cells (MSCs) induce macrophage phenotype polarization and reduce inflammation in sepsis. MSC-secreted transforming growth factor beta (TGF-β) participated in the immune modulatory function of MSCs. However, the underlying mechanism of MSC-secreted TGF-β was not fully elucidated in regulation macrophage M2-like polarization.MethodsThe paracrine effects of MSCs on macrophage polarization were studied using a co-culture protocol with LPS-stimulated RAW264.7 cells/mouse peritoneal macrophages and MSCs. The effect of TGF-β in the co-culture system was blocked by the TGF-β receptor inhibitor. To determine the role of MSC-secreted TGF-β, we used recombinant TGF-β to culture with LPS-stimulated RAW264.7 cells. In addition, we employed antibody microarray analysis to determine the mechanisms of MSC secreted TGF-β on LPS-stimulated RAW264.7 cell/mouse peritoneal macrophage M2-like polarization. Furthermore, we used an Akt inhibitor and a FoxO1 inhibitor to inhibit the Akt/FoxO1 pathway. The nuclear translocation of FoxO1 was detected by Western blot.ResultsMSCs induced LPS-stimulated RAW264.7 cell/mouse peritoneal macrophage polarization towards the M2-like phenotype and significantly reduced pro-inflammatory cytokine levels via paracrine, which was inhibited by TGF-β receptor inhibitor. Furthermore, we found that MSC-secreted TGF-β enhanced the macrophage phagocytic ability. The antibody microarray analysis and Western blot verified that TGF-β treatment activated the Akt/FoxO1 pathway in LPS-stimulated macrophages, TGF-β-induced FoxO1 nuclear translocation and obviously expressed in the cytoplasm, the effects of TGF-β regulatory effects on LPS-stimulated macrophage were inhibited by pre-treatment with Akt inhibitor and FoxO1 inhibitor.ConclusionsTGF-β secreted by MSCs could skew LPS-stimulated macrophage polarization towards the M2-like phenotype, reduce inflammatory reactions, and improve the phagocytic ability via the Akt/FoxO1 pathway, providing potential therapeutic strategies for sepsis.

Potential and Therapeutic Efficacy of Cell-based Therapy Using Mesenchymal Stem Cells for Acute/chronic Kidney Disease.

Kidney disease can be either acute kidney injury (AKI) or chronic kidney disease (CKD) and it can lead to the development of functional organ failure. Mesenchymal stem cells (MSCs) are derived from a diverse range of human tissues. They are multipotent and have immunomodulatory effects to assist in the recovery from tissue injury and the inhibition of inflammation. Numerous studies have investigated the feasibility, safety, and efficacy of MSC-based therapies for kidney disease. Although the exact mechanism of MSC-based therapy remains uncertain, their therapeutic value in the treatment of a diverse range of kidney diseases has been studied in clinical trials. The use of MSCs is a promising therapeutic strategy for both acute and chronic kidney disease. The mechanism underlying the effects of MSCs on survival rate after transplantation and functional repair of damaged tissue is still ambiguous. The paracrine effects of MSCs on renal recovery, optimization of the microenvironment for cell survival, and control of inflammatory responses are thought to be related to their interaction with the damaged kidney environment. This review discusses recent experimental and clinical findings related to kidney disease, with a focus on the role of MSCs in kidney disease recovery, differentiation, and microenvironment. The therapeutic efficacy and current applications of MSC-based kidney disease therapies are also discussed.

Heterogenic transplantation of bone marrow-derived rhesus macaque mesenchymal stem cells ameliorates liver fibrosis induced by carbon tetrachloride in mouse.

Liver fibrosis is a disease that causes high morbidity and has become a major health problem. Liver fibrosis can lead to the end stage of liver diseases (livercirrhosisand hepatocellularcarcinoma). Currently, liver transplantation is the only effective treatment for end-stage liver disease. However, the shortage of organ donors, high cost of medical surgery, immunological rejection and transplantation complications severely hamper liver transplantation therapy. Mesenchymal stem cells (MSCs) have been regarded as promising cells for clinical applications in stem cell therapy in the treatment of liver diseases due to their unique multipotent differentiation capacity, immunoregulation and paracrine effects. Although liver fibrosis improvements by MSC transplantation in preclinical experiments as well as clinical trials have been reported, the in vivo fate of MSCs after transportation and their therapeutic mechanisms remain unclear. In this present study, we isolated MSCs from the bone marrow of rhesus macaques. The cells exhibited typical MSC markers and could differentiate into chondrocytes, osteocytes, and adipocytes, which were not affected by labeling with enhanced green fluorescent protein (EGFP). The harvested MSCs respond to interferon-γ stimulation and have the ability to inhibit lymphocyte proliferation in vitro. EGFP-labeled MSCs (1 × 106 cells) were transplanted into mice with carbon tetrachloride-induced liver fibrosis via tail vein injection. The ability of the heterogenic MSC infusion to ameliorate liver fibrosis in mice was evaluated by a blood plasma chemistry index, pathological examination and liver fibrosis-associated gene expression. Additionally, a small number of MSCs that homed and engrafted in the mouse liver tissues were evaluated by immunofluorescence analysis. Our results showed that the transplantation of heterogenic MSCs derived from monkey bone marrow can be used to treat liver fibrosis in the mouse model and that the paracrine effects of MSCs may play an important role in the improvement of liver fibrosis.

Injectable co-gels of collagen and decellularized vascular matrix improve MSC-based therapy for acute kidney injury

Transplantation of mesenchymal stem cells (MSCs) is promising for treatment of acute kidney injury (AKI), but their therapeutic effects are often limited under normal conditions. In this study, we prepared the co-gels of decellularized vascular matrix and collagen, and investigated whether the co-gels increase the therapeutic potentials of MSCs on AKI. In vitro studies indicated that the co-gels enhanced the paracrine effects of MSCs, and significantly reduced the apoptosis of MSCs under oxidative environments. When the co-gels were co-transplanted with MSCs into the kidney of model rats with ischemia-reperfusion (I/R)-induced AKI, the survival and paracrine effects of MSCs were enhanced in the injured kidney. More importantly, the co-gels increased the therapeutic effects of MSCs for AKI, as indicated by cell apoptosis, tissue damage, vascularization and renal function. Therefore, the co-gels of decellularized vascular matrix and collagen improved the therapeutic effects of MSCs, and might be promising for AKI treatment.

Mesenchymal stem cells and their conditioned medium can enhance the repair of uterine defects in a rat model

BackgroundOur aim was to examine the roles of mesenchymal stem cell (MSC) transplantation in the repair of large uterine defects. MethodsUterine defects were created in both uterine horns of female rats by a punch instrument, and bone marrow-derived MSCs, MSC-conditioned medium (MSC-CM) or vehicle were injected into the myometrium around the defect. The rate of uterine defect repair was monitored on day 2 and 4 after operation. Cytokine array of MSC-CM was performed, followed by neutralizing antibody experiments to clarify the exact cytokine participating in the MSC-CM-enhanced wound repair. ResultsTransplantation of MSCs, but not myometrial cells, significantly enhanced uterine defect repair. The transplanted MSCs were detected in the uterine horn with no signs of rejection on day 4 after transplantation, when the MSC-transplanted uterine wound was nearly healed. Moreover, uterine defect repair was also accelerated by injection of MSC-CM, indicating the paracrine effects of MSCs on uterine wound healing. Cytokine array analysis further revealed that MSC-CM contained abundant cytokines and chemokines, among which high levels of interleukin-6 (IL-6) were found. Additionally, antibodies against IL-6 were shown to block MSC-CM-enhanced uterine defect repair. ConclusionThis study demonstrated that transplantation of MSCs could enhance uterine defect repair by paracrine effects involving IL-6, which are findings that may be applied to facilitate uterine wound healing in the removal of huge intramural masses.

Paracrine effects of mesenchymal stem cells on the activation of keratocytes

AimsThe purpose of this study is to investigate the impact of mesenchymal stem cell (MSC)-derived soluble factors on the function of keratocytes, with a particular focus on the processes involved...

SDF-1 overexpression by mesenchymal stem cells enhances GAP-43-positive axonal growth following spinal cord injury.

Utilizing genetic overexpression of trophic molecules in cell populations has been a promising strategy to develop cell replacement therapies for spinal cord injury (SCI). Over-expressing the chemokine, stromal derived factor-1 (SDF-1α), which has chemotactic effects on many cells of the nervous system, offers a promising strategy to promote axonal regrowth following SCI. The purpose of this study was to explore the effects of human SDF-1α, when overexpressed by mesenchymal stem cells (MSCs), on axonal growth and motor behavior in a contusive rat model of SCI. Using a transwell migration assay, the paracrine effects of MSCs, which were engineered to secrete human SDF-1α (SDF-1-MSCs), were assessed on cultured neural stem cells (NSCs). For in vivo analyses, the SDF-1-MSCs, unaltered MSCs, or Hanks Buffered Saline Solution (vehicle) were injected into the lesion epicenter of rats at 9-days post-SCI. Behavior was analyzed for 7-weeks post-injury, using the Basso, Beattie, and Bresnahan (BBB) scale of locomotor functions. Immunohistochemistry was performed to evaluate major histopathological outcomes, including gliosis, inflammation, white matter sparing, and cavitation. New axonal outgrowth was characterized using immunohistochemistry against the neuron specific growth-associated protein-43 (GAP-43). The results of these experiments demonstrate that the overexpression of SDF-1α by MSCs can enhance the migration of NSCs in vitro. Although only modest functional improvements were observed following transplantation of SDF-1-MSCs, a significant reduction in cavitation surrounding the lesion, and an increased density of GAP-43-positive axons inside the SCI lesion/graft site were found. The results from these experiments support the potential role for utilizing SDF-1α as a treatment for enhancing growth and regeneration of axons after traumatic SCI.

The inflamed microenvironment: role on MSCs immunobiology and cancer

Inflammation and cancer are an inseparable binomial. The majority of cancers are triggered by somatic mutations and environmental factors with a common element: inflammation. Inflammation creates a microenvironment in which neoplastic cells can profit from the trophic factors secreted by inflammatory cells, useful to interfere with the anti-tumor response. Among the others, mesenchymal stem cells (MSCs) participate to microenvironment creation by a strong paracrine effect. The linkage between MSCs and inflammation is bidirectional: the inflamed microenvironment affects the complex MSCs immunobiology, but also MSCs can sustain inflammation. Here, we tried to clarify the influence of inflammation on the immunobiology of MSCs and deepen the paracrine effect of MSCs on tumor growth. MSCs were isolated from periprosthetic capsule caused by breast implant, affected by inflammation (I-MSCs). The contralateral part of the same patient, not inflamed, was used as control (C-MSCs). A panel of selected cytokines were analyzed by Real-Time PCR and ELISA. The cytokines expression was different in I-MSCs compared to C-MSCs, revealing that inflammation affects MSCs immunobiology. Then, C- and I-MSCs were indirectly co-cultured with MCF7 cells from breast adenocarcinoma. New analyses on proliferation rate and cytokines expression were performed. C- and I-MSCs gave almost the same results. The over-secretion of all the cytokines referred to the Th1 pathway and the decrease of those belonging to the Th2 pathway revealed the absence of a switch from Th1 to Th2 important to induce a chronic inflammation. The levels of TGF-β and G-CSF linked to the skill to damage the antigen-presenting cell function were decreased. In conclusion, even if MCF-7 proliferation increased after co-culture with I-MSCs, MSCs-derived paracrine effect does not sustain breast adenocarcinoma. These results absolve the breast implants from the insult to enhance adenocarcinoma onset.

Paracrine regulation in mesenchymal stem cells: the role of Rap1.

Since the first report more than a decade ago that bone marrow (BM) stem cells transplantation could promote heart regeneration following myocardial infarction (MI),1 different types of stem cells have been examined. Among those investigated to date, mesenchymal stem cells (MSCs) have attracted huge interest owing to some of their unique properties that include easy isolation, high expandability and low immunogenicity. Nonetheless despite the promising results of animal studies and clinical trials, MSC-based therapy for MI still faces some fundamental hurdles.2 One of the major challenges is the poor cell engraftment and low cell-survival rate following transplantation caused by the hostile environment of the injured heart and change to the immunologic milieu of transplanted MSCs. In addition, the mechanisms of stem cell-mediated tissue repair are not fully understood. Low cell retention and poor myocardial differentiation cannot explain the remarkable recovery of heart function following MSCs treatment. Therefore, the paracrine effects of MSCs are currently thought to have a predominant role. Although many cytokines released from MSCs contribute to heart function recovery,3 some are useless and even harmful.4 Thus, there is an urgent need to optimize MSCs before their transplantation to improve cell survival and augment their paracrine effects. Recently, Rap1, a telomeric repeat binding factor 2 interacting protein 1, has been identified as an important modulator of the nuclear factor kappa-B (NF-κB) pathway.5, 6 This pathway has been reported to regulate MSCs secretion profiling and survival.7, 8 On the basis of these findings, modulation of the NF-κB pathway to mediate MSCs therapy is feasible and important. Nonetheless total deletion of NF-κB is lethal to cells.9 Identification of an important regulator that can mediate activity of the NF-κB pathway and subsequently regulate MSC therapeutic efficacy for MI is vital. The roles of Rap1 in regulation of MSCs and the underlying mechanisms have not been classified, thus to understand how Rap1 regulates the paracrine effects and cell survival of MSC-mediated heart repair following infarction by regulation of the NF-κB pathway, therefore, is important.10

Direct intercellular communications dominate the interaction between adipose-derived MSCs and myofibroblasts against cardiac fibrosis.

The onset of cardiac fibrosis post myocardial infarction greatly impairs the function of heart. Recent advances of cell transplantation showed great benefits to restore myocardial function, among which the mesenchymal stem cells (MSCs) has gained much attention. However, the underlying cellular mechanisms of MSC therapy are still not fully understood. Although paracrine effects of MSCs on residual cardiomyocytes have been discussed, the amelioration of fibrosis was rarely studied as the hostile environment cannot support the survival of most cell populations and impairs the diffusion of soluble factors. Here in order to decipher the potential mechanism of MSC therapy for cardiac fibrosis, we investigated the interplay between MSCs and cardiac myofibroblasts (mFBs) using interactive co-culture method, with comparison to paracrine approaches, namely treatment by MSC conditioned medium and gap co-culture method. Various fibrotic features of mFBs were analyzed and the most prominent anti-fibrosis effects were always obtained using direct co-culture that allowed cell-to-cell contacts. Hepatocyte growth factor (HGF), a well-known anti-fibrosis factor, was demonstrated to be a major contributor for MSCs’ anti-fibrosis function. Moreover, physical contacts and tube-like structures between MSCs and mFBs were observed by live cell imaging and TEM which demonstrate the direct cellular interactions.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-015-0196-7) contains supplementary material, which is available to authorized users.

Mesenchymal stem cell therapy in nonhematopoietic diseases.

The extensive clinical experiments with stem cells, particularly in the treatment of oncohematological diseases, opened up the possibility of trying out stem cells with nonhematopoietic diseases. Mesenchymal stem cells (MSCs) subsequently emerged as a promising source for the regeneration and repair of various tissues in the treatment of a range of diseases due to their presence in all derived mesenchymal tissues in the adult solid organs as well as in mesoderm from embryonic tissue. This, plus their pluripotentiality and the fact that they were easily obtainable, meant that MSCs represented an important source for studies in regenerative medicine. MSCs have been shown to be capable of differentiating in various types of mesoderm derived cells as well as ectoderm cells, such as skeletal muscle cells, neurons, cardiomyocytes, osteocytes, chondrocytes, and others. MSCs in specific cultivation conditions in vitro and in host transplanted tissue, being niche dependent, arouse clinical interest in stem-cell therapy for regeneration in nonhematopoietic tissue diseases and the prospect of creating a mesenchymal stem-cell bank for research and subsequent clinical use. In the development of clinical models, always preceded by preclinical studies, there are various requirements to guarantee the safety of therapy with stem cells and their products, including the GMP procedures, cytogenetic control, identification and characterization of cells by immune cytometric analysis, and demonstration of their pluripotentiality for the prospective use of MSC for tissue regeneration in nonhematopoietic diseases [1]. In the study of MSCs, the intrinsic properties—molecular, immunocytochemistry, isolation, expansion, differentiation, and cryopreservation—have made great advances and need further discussion. The fact that MSCs do not express the antigens of histocompatibility means that they can be used in allogeneic transplantation. The development of methods for isolating the subpopulations of MSC fractions together with the perspective with this subpopulation could obtain better results than the total population in replacement therapy for correction of determined specific function [2]. Researchers and physicians are looking into the physiopathology of some diseases in light of the intrinsic cell conditions on the development of each disease and are proposing therapies based on the cells themselves or on cell-based products. Some diseases are triggered in their physiopathology by autoimmune mechanisms that could be stabilized with the paracrine effects of MSCs, such as the anti-inflammatory effect [3]; other diseases are triggered by the senescence of the cells, as in conditions of cellular apoptosis, and the MSC cells could act with the paracrine effects such as antiapoptosis and by cell replacement; there are some diseases which result from the loss of the production of certain molecules and MSCs could differentiate in cells capable of producing the molecule [4]; in other diseases, there is an interruption in signals between the tissue cells, and the MSC paracrine effects could promote the connections through the production of growth factors [5]. On the other hand, in ischemic diseases, MSCs could be differentiated in specific types of specialized cells, such as vessels and contractile cells, cardiomyocytes [6]; in genetic diseases, MSCs could ensure the delivery of genes for gene therapy [7] and could act in immunomodulation with vaccines [8]. This issue provides discussions of the points described above and takes a look into the future of cell therapy in nonhematopoetic diseases, showing that it is nearer than we expect! It is reality! Katherine Athayde Teixeira de Carvalho Gustav Steinhoff Juan Carlos Chachques

CXCR4 Receptor Overexpression in Mesenchymal Stem Cells Facilitates Treatment of Acute Lung Injury in Rats

Novel therapeutic regimens for tissue renewal incorporate mesenchymal stem cells (MSCs) as they differentiate into a variety of cell types and are a stem cell type that is easy to harvest and to expand in vitro. However, surface chemokine receptors, such as CXCR4, which are involved in the mobilization of MSCs, are expressed only on the surface of a small proportion of MSCs, and the lack of CXCR4 expression may underlie the low efficiency of homing of MSCs toward tissue damage, which results in a poor curative effect. Here, a rat CXCR4 expressing lentiviral vector was constructed and introduced into MSCs freshly prepared from rat bone marrow. The influence of CXCR4 expression on migration, proliferation, differentiation, and paracrine effects of MSCs was examined in vitro. The in vivo properties of CXCR4-MSCs were also investigated in a model of acute lung injury in rats induced by lipopolysaccharide. Expression of CXCR4 in MSCs significantly enhanced the chemotactic and paracrine characteristics of the cells in vitro but did not affect self-renewal or differentiation into alveolar and vascular endothelial cells. In vivo, CXCR4 improved MSC homing and colonization of damaged lung tissue, and furthermore, the transplanted CXCR4-MSCs suppressed the development of acute lung injury in part by modulating levels of inflammatory molecules and the neutrophil count. These results indicated that efficient mobilization of MSCs to sites of tissue injury may be due to CXCR4, and therefore, increased expression of CXCR4 may improve their therapeutic potential in the treatment of diseases where tissue damage develops.

The Potential of Umbilical Cord Derived Mesenchymal Stem Cells in Intervertebral Disc Repair

Introduction Nucleus pulposus (NP) is the center and major compartment of intervertebral disc (IVD). Mesenchymal stem cells (MSCs) are a type of stem cell source under intensive investigation for their potential to regenerate NP. MSCs have been identified from various sources with different characteristics. There are indications that fetal or close to fetal tissue sources contain cells with relatively undifferentiated phenotype with respect to MSCs from adult sources. Moreover, evidences have shown that umbilical cord-derived MSCs (CMSCs) may have better chondrogenic differentiation potential than bone marrow-derived MSCs (BMSCs).1 We hypothesize CMSCs might be a suitable stem cell source for NP regeneration. The aim of this research is to analyze the paracrine effect of MSCs on NP cells, and compare the effect of BMSCs and CMSCs in an attempt to identify a better MSC source for future clinical application. Materials and Methods Human BMSCs, CMSCs, and degenerated NP cells (three batches each) were isolated and characterized from patients undergoing spinal fusion and patients at caesarean delivery, respectively, after IRB approval was acquired. Conditioned media (CM) was collected after 48 hours exposure to MSC monolayer. Cell proliferation and cytotoxicity were assessed by MTT assay after 1, 3, and 7 days in MSC-CM. Proteoglycan content of NP cells in both types of MSC-CM were measured by DMMB assay after 14 days in culture. Gene expression of degeneration-related molecules of NP cells in MSC-CM, including CDH2, CD55, FBLN1, Sox9, KRT19, KRT18, and MGP, were determined by real-time RT-PCR. Protein expression of KRT19 in degenerated NP cells before and after MSC-CM treatment was examined by immunocytochemistry and confocal microscopy. All results were normalized to the control group in which the NP cells were cultured in basal medium. Results Human BMSCs and CMSCs that we isolated satisfied the minimum criteria of MSCs; that is, they were CD73(+), CD105(+), CD146(+), CD14(−), CD45(−), C34(−), and had tri-lineage differentiation potency. The overall metabolic activities of NP cells measured by MTT reading were significantly enhanced in MSC-CM than that in control basal medium, especially in CMSC-CM. This is accompanied by a slight increase in proteoglycan production. We demonstrated that MMP12, MGP, and KRT19 are the major differential expressed genes between scoliotic and degenerated human NP cells. We found that MGP and MMP12 were significantly downregulated, while KRT19 expression was significantly upregulated, in NP cells treated by MSC-CM, especially CMSC-CM. The increased KRT19 expression in NP cells was also confirmed at protein level by confocal microscopy. Conclusion This is the first comparative study that discussed how different sources of MSCs affect the biological activities of cultured NP cells through paracrine effect. MGP2 and KRT193 has recently been reported to be associated with IVD degeneration. In this study, MSC-CM effectively upregulated KRT19 while downregulated MMP12 and MGP, suggesting that MSC-CM may promote a recovery of NP cell phenotype. In line with this finding is that MSC-CM treatment also enhanced overall cell metabolic activities, reduced apoptosis, and enhanced proteoglycan production of NP cells in culture. In all aspects tested, CMSC-CM showed stronger effect than BMSC-CM, suggesting that CMSC is a superior source of MSCs for future clinical application for IVD regeneration. Disclosure of Interest None declared References Wang L, Tran I, Seshareddy K, Weiss ML, Detamore MS. A comparison of human bone marrow-derived mesenchymal stem cells and human umbilical cord-derived mesenchymal stromal cells for cartilage tissue engineering. Tissue Eng Part A 2009;15(8):2259–2266 Lee CR, Sakai D, Nakai T, et al. A phenotypic comparison of intervertebral disc and articular cartilage cells in the rat. Eur Spine J 2007;16(12):2174–2185 Rutges J, Creemers LB, Dhert W, et al. Variations in gene and protein expression in human nucleus pulposus in comparison with annulus fibrosus and cartilage cells: potential associations with aging and degeneration. Osteoarthritis Cartilage 2010;18(3):416–423

Hypoxia-pretreated human MSCs attenuate acute kidney injury through enhanced angiogenic and antioxidative capacities.

Hypoxia preconditioning has been confirmed as an effective strategy to enhance the therapeutic potentials of mesenchymal stem cells (MSCs), such as for myocardial ischemia. However, whether hypoxia preconditioning would produce beneficial effects on MSC-based renal repair has not been demonstrated. In the study, we aimed to determine the feasibility and efficacy of hypoxia preconditioning to enhance MSC-based therapy of acute kidney injury (AKI). MSCs were isolated from human adipose tissues. The paracrine effects of MSCs under normoxia and hypoxia were determined in vitro. Rats of AKI were induced by kidney I/R surgery and randomly divided into three groups: I/R control receiving PBS injection; MSC group receiving normal MSC injection; hypoMSC group receiving hypoxia-preconditioned MSC injection. It was demonstrated in vitro that paracrine effects of MSCs were significantly enhanced, especially angiogenic factors. Dihydroethidium (DHE) staining showed that antioxidative activities of MSCs were significantly enhanced by hypoxia stimulation. Vascularization, apoptosis, and histological injury were all significantly improved in hypoMSC injected group compared with that in control and MSC injected groups. Finally, the renal function was also significantly improved in hypoMSC injected group compared with that in the other two groups as assessed by the serum creatinine and BUN levels.

Paracrine effect of MSCs revealed by single cell gene expression assay in mouse infarcted hearts

Mesenchymal stem cells (MSCs) have recently been demonstrated as a promising stem cell type to rescue damaged myocardium after acute infarction. One of the most important mechanisms underlying their therapeutic effects is the secretion of paracrine factors. However, the expression profile of paracrine factors of MSCs in infarcted hearts, especially at single cell level, is poorly defined. We aimed to depict the transcriptional profile of paracrine factors secreted by MSCs in vivo, with particular interest in the comparison between normal and infarcted hearts. MSCs were isolated and injected into mice hearts immediately after infarction surgery. Bioluminescence imaging indicated a proportion of cells still alive even up to 12 days post surgery. Paralleled with survived cells, cardiac function was significantly improved after MSCs injection compared to that in PBS-injected mice, indicated by echocardiography and MRI. Despite increased number of vessels in MSCs-injected hearts, endothelial cells and cardiomyocytes transdifferentiation were rarely observed in infarcted hearts 5 days after infarction. Furthermore, laser capture microdissection followed by high through-put real time PCR was employed in our study, uncovering that the injected MSCs, compared to local cardiomyocytes, displayed elevated levels of secreted factors. To further investigate the regulation of those factors, we performed single cell analysis to dissect the gene expression profile of 48 MSCs in infarcted and normal hearts, respectively. Consistent with the in vivo observation, a similar regulation pattern of those factors was detected in cultured MSCs. Our study, for the first time, elucidated gene expression profiles, as well as regulation of paracrine factors, of MSCs at single cell level in vivo, indicating that paracrine factors from MSCs account for the improvement of cardiac function after infarction.

Fibrin Glue Improves the Therapeutic Effect of MSCs by Sustaining Survival and Paracrine Function

Fibrin glue has been widely investigated as a cell delivery vehicle for improving the therapeutic effects of mesenchymal stem cells (MSCs). Implanted MSCs produce their therapeutic effects by secreting paracrine factors and by replacing damaged tissues after differentiation. While the influence of fibrin glue on the differentiation potential of MSCs has been well documented, its effect on paracrine function of MSCs is largely unknown. Herein we investigated the influence of fibrin glue on the paracrine effects of MSCs. MSCs were isolated from human adipose tissue. The effects of fibrin glue on survival, migration, secretion of growth factors, and immune suppression of MSCs were investigated in vitro. MSCs in fibrin glue survived and secreted growth factors such as the vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) over 14 days. VEGF and immune modulators, including the transforming growth factor (TGF)-β1 and prostaglandin E2, secreted from MSCs in fibrin glue significantly increased under inflammatory conditions. Thus, MSCs in fibrin glue effectively suppressed immune reactions. In addition, fibrin glue protected the MSCs from oxidative stress and prevented human dermal fibroblast death induced by exposure to extreme stress. In contrast, MSCs within fibrin glue hardly migrated. These results suggest that fibrin glue may sustain survival of implanted MSCs and their paracrine function. Our results provide a mechanistic data to allow further development of MSCs with fibrin glue as a clinical treatment.