Harnessing mesenchymal stem cell secretome: Effect of extracellular matrices on proangiogenic signaling

The low engraftment and retention rate of mesenchymal stem cells (MSCs) at the target site indicates that the potential benefits of MSC-based therapies can be attributed to their paracrine signaling. In this study, the extracellular matrices (ECMs) deposited by bone marrow-derived human MSCs in the presence and absence of ascorbic acid was characterized. MSCs were seeded on top of decellularized ECM (dECM) and the concentrations of proangiogenic and antiangiogenic molecules released in culture (conditioned) media was compared. Effects of ECM derived from MSCs with different passage numbers on MSC secretome was also investigated. Our study revealed that the expression of proangiogenesis-related factors were upregulated when MSCs were harvested on dECMs, irrespective of media supplementation, as compared with those cultured on tissue culture plates. In addition, dECM generated in the presence of ascorbic acid promoted the expression of proangiogenic molecules as compared with dECM-derived in absence of media supplementation. Further, it was observed that the effectiveness of dECM to stimulate proangiogenic signaling of MSCs was reduced as cell passage number was increased from P3 to P5. The proliferation as well as capillary morphogenesis of human umbilical vein endothelial cells (HUVECs) in the presence of conditioned media were enhanced compared with the normal HUVECs culture media. These data indicate that the secretory signatures of MSCs and consequently, the therapeutic efficacy of MSCs can be regulated by presentation of dECM composition and variation of its composition.     

Proteomic techniques for characterisation of mesenchymal stem cell secretome

Mesenchymal stem cells (MSCs) are multipotent cells with a substantial potential in human regenerative medicine due to their ability to migrate to sites of injury, capability to suppress immune response and accessibility in large amount from patient's own bone marrow or fat tissue. It has been increasingly observed that the transplanted MSCs did not necessarily engraft and differentiate at the site of injury but might exert their therapeutic effects through secreted trophic signals. The MSCs secrete a variety of autocrine/paracrine factors, called secretome, that support regenerative processes in the damaged tissue, induce angiogenesis, protect cells from apoptotic cell death and modulate immune system. The cell culture medium conditioned by MSCs or osteogenic, chondrogenic as well as adipogenic precursors derived from MSCs has become a subject of intensive proteomic profiling in the search for and identification of released factors and microvesicles that might be applicable in regenerative medicine. Jointly with the methods for MSC isolation, expansion and differentiation, proteomic analysis of MSC secretome was enabled recently mainly due to the extensive development in protein separation techniques, mass spectrometry, immunological methods and bioinformatics. This review describes proteomic techniques currently applied or prospectively applicable in MSC secretomics, with a particular focus on preparation of the secretome sample, protein/peptide separation, mass spectrometry and protein quantification techniques, analysis of posttranslational modifications, immunological techniques, isolation and characterisation of secreted vesicles and exosomes, analysis of cytokine-encoding mRNAs and bioinformatics.     

PEDF from mouse mesenchymal stem cell secretome attracts fibroblasts

Conditioned medium (secretome) derived from an enriched stem cell culture stimulates chemotaxis of human fibroblasts. These cells are classified as multipotent murine mesenchymal stromal cells (mMSC) by immunochemical analysis of marker proteins. Proteomic analysis of mMSC secretome identifies nineteen secreted proteins, including extracellular matrix structural proteins, collagen processing enzymes, pigment epithelium-derived factor (PEDF) and cystatin C. Immunodepletion and reconstitution experiments show that PEDF is the predominant fibroblast chemoattractant in the conditioned medium, and immunofluorescence microscopy shows strong staining for PEDF in the cytoplasm, at the cell surface, and in intercellular space between mMSCs. This stimulatory effect of PEDF on fibroblast chemotaxis is in contrast to the PEDF-mediated inhibition of endothelial cell migration, reported previously. These differential functional effects of PEDF toward fibroblasts and endothelial cells may serve to program an ordered temporal sequence of scaffold building followed by angiogenesis during wound healing.     

Harnessing and honing mesenchymal stem/stromal cells for the amelioration of graft-versus-host disease

Allogeneic hematopoietic stem cell transplantation is a deterministic curative procedure for various hematologic disorders and congenital immunodeficiency. Despite its increased use, the mortality rate for patients undergoing this procedure remains high, mainly due to the perceived risk of exacerbating graft-versus-host disease (GVHD). However, even with immunosuppressive agents, some patients still develop GVHD. Advanced mesenchymal stem/stromal cell (MSC) strategies have been proposed to achieve better therapeutic outcomes, given their immunosuppressive potential. However, the efficacy and trial designs have varied among the studies, and some research findings appear contradictory due to the challenges in characterizing the in vivo effects of MSCs. This review aims to provide real insights into this clinical entity, emphasizing diagnostic, and therapeutic considerations and generating pathophysiology hypotheses to identify research avenues. The indications and timing for the clinical application of MSCs are still subject to debate.     

Modulation of bone marrow mesenchymal stem cell secretome by ECM-like hydrogels

It has been demonstrated that bone marrow mesenchymal stem cell (BM-MSCs) transplantation has beneficial effects on several central nervous system (CNS) debilitating conditions. Growing evidence indicate that trophic factors secreted by these cells are the key mechanism by which they are acting. These cells are frequently used in combination with 3D artificial matrices, for instance hydrogels, in tissue engineering-based approaches. However, so far, no study has been reported on the influence of such matrices, namely the presence or absence of extracellular matrix motifs, on BM-MSCs secretome and its effects in neuronal cell populations. In this sense, we herein studied the impact of a hydrogel, gellan gum, on the behavior and secretome of BM-MSCs, both in its commercial available form (commonly used in tissue engineering) and in a fibronectin peptide-modified form. The results showed that in the presence of a peptide in the gellan gum hydrogel, BM-MSCs presented higher proliferation and metabolic activity than in the regular hydrogel. Moreover, the typical spindle shape morphology of BM-MSCs was only observed in the modified hydrogel. The effects of the secretome of BM-MSCs were also affected by the chemical nature of the extracellular matrix. BM-MSCs cultured in the modified hydrogel were able to secrete factors that induced higher metabolic viabilities and neuronal cell densities, when compared to those of the unmodified hydrogel. Thus adding a peptide sequence to the gellan gum had a significant effect on the morphology, activity, proliferation and secretome of BM-MSCs. These results highlight the importance of mimicking the extracellular matrix when BM-MSCs are cultured in hydrogels for CNS applications.     

Type 2 diabetes alters mesenchymal stem cell secretome composition and angiogenic properties

This study aimed at characterizing the impact of type 2 diabetes mellitus (T2DM) on the bone marrow mesenchymal stem cell (BMMSC) secretome and angiogenic properties. BMMSCs from Zucker diabetic fatty rats (ZDF) (a T2DM model) and Zucker LEAN littermates (control) were cultured. The supernatant conditioned media (CM) from BMMSCs of diabetic and control rats were collected and analysed. Compared to results obtained using CM from LEAN‐BMMSCs, the bioactive content of ZDF‐BMMSC CM (i) differently affects endothelial cell (HUVEC) functions in vitro by inducing increased (3.5‐fold; P < 0.01) formation of tubule‐like structures and migration of these cells (3‐fold; P < 0.001), as well as promotes improved vascular formation in vivo, and (ii) contains different levels of angiogenic factors (e.g. IGF1) and mediators, such as OSTP, CATD, FMOD LTBP1 and LTBP2, which are involved in angiogenesis and/or extracellular matrix composition. Addition of neutralizing antibodies against IGF‐1, LTBP1 or LTBP2 in the CM of BMMSCs from diabetic rats decreased its stimulatory effect on HUVEC migration by approximately 60%, 40% or 40%, respectively. These results demonstrate that BMMSCs from T2DM rats have a unique secretome with distinct angiogenic properties and provide new insights into the role of BMMSCs in aberrant angiogenesis in the diabetic milieu.     

Disagreements in the therapeutic use of mesenchymal stem cell-derived secretome

In a recent article, the authors provide a detailed summary of the characteristics and biological functions of mesenchymal stem cells (MSCs), as well as a discussion on the potential mechanisms of action of MSC-based therapies. They describe the morphology, biogenesis, and current isolation techniques of exosomes, one of the most important fractions of the MSC-derived secretome. They also summarize the characteristics of MSC-derived exosomes and highlight their functions and therapeutic potential for tissue/organ regeneration and for kidney, liver, cardiovascular, neurological, and musculoskeletal diseases, as well as cutaneous wound healing. Despite the fact that MSCs are regarded as an important pillar of regenerative medicine, their regenerative potential has been demonstrated to be limited in a number of pathological conditions. The negative effects of MSC-based cell therapy have heightened interest in the therapeutic use of MSC-derived secretome. On the other hand, MSC-derived exosomes and microvesicles possess the potential to have a significant impact on disease development, including cancer. MSCs can interact with tumor cells and promote mutual exchange and induction of cellular markers by exchanging secretome. Furthermore, enzymes secreted into and activated within exosomes can result in tumor cells acquiring new properties. As a result, therapeutic applications of MSC-derived secretomes must be approached with extreme caution.     

Potential application for mesenchymal stem cells in the treatment of cardiovascular diseases

Stem cells isolated from various sources have been shown to vary in their differentiation capacity or pluripotentiality. Two groups of stem cells, embryonic and adult stem cells, may be capable of differentiating into any desired tissue or cell type, which offers hope for the development of therapeutic applications for a large number of disorders. However, major limitations with the use of embryonic stem cells for human disease have led researchers to focus on adult stem cells as therapeutic agents. Investigators have begun to examine postnatal sources of pluripotent stem cells, such as bone marrow stroma or adipose tissue, as sources of mesenchymal stem cells. The following review focuses on recent research on the use of stem cells for the treatment of cardiovascular and pulmonary diseases and the future application of mesenchymal stem cells for the treatment of a variety of cardiovascular disorders.     

Intranasal delivery of mesenchymal stem cell secretome repairs the brain of Alzheimer’s mice

The multiplicity of systems affected in Alzheimer’s disease (AD) brains calls for multi-target therapies. Although mesenchymal stem cells (MSC) are promising candidates, their clinical application is limited because of risks related to their direct implantation in the host. This could be overcome by exploiting their paracrine action. We herein demonstrate that in vivo systemic administration of secretome collected from MSC exposed in vitro to AD mouse brain homogenates (MSC-CS), fully replicates the cell-mediated neuroreparative effects in APP/PS1 AD mice. We found a complete but transient memory recovery by 7 days, which vanished by 14 days, after a single MSC-CS intravenous administration in 12-month or 22–24-month-old mice. Treatment significantly reduced plaque load, microglia activation, and expression of cytokines in astrocytes in younger, but not aged, mice at 7 days. To optimize efficacy, we established a sustained treatment protocol in aged mice through intranasal route. Once-weekly intranasal administration of MSC-CS induced persistent memory recovery, with dramatic reduction of plaques surrounded by a lower density of β-amyloid oligomers. Gliosis and the phagocytic marker CD68 were decreased. We found a higher neuronal density in cortex and hippocampus, associated with a reduction in hippocampal shrinkage and a longer lifespan indicating healthier conditions of MSC-CS-treated compared to vehicle-treated APP/PS1 mice. Our data prove that MSC-CS displays a great multi-level therapeutic potential, and lay the foundation for identifying the therapeutic secretome bioreactors leading to the development of an efficacious multi-reparative cocktail drug, towards abrogating the need for MSC implantation and risks related to their direct use.Subject terms: Neural ageing, Neurological disorders, Inflammation     

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

Background  

It 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.     

Review of the adipose derived stem cell secretome

Recent advances in protein detection and analysis have lead to multiple in depth studies that analyze the adipose-derived stem cell (ASC) secretome. These studies differ significantly in their methods of secretome preparation and analysis. Most of them use a pro-differentiation or pro-inflammatory stimulus to observe differential expression of secreted proteins. In spite of the variance in methodologies used, 68 proteins are reported to be commonly expressed in a majority of the studies and may serve as potential candidates for conserved secretome proteins.     

The effect of Trimetazidine and Diazoxide on immunomodulatory activity of human embryonic stem cell-derived mesenchymal stem cell secretome

Comprehensive proteome profiling of the factors secreted by mesenchymal stem cells (MSCs), referred to as secretome, revealed that it consists of cytokines, chemokines, growth factors, extracellular matrix proteins, and components of regeneration, vascularization, and hematopoiesis pathways. Harnessing this MSC secretome for therapeutic applications requires the optimization of production of secretary molecules. A variety of preconditioning methods have been introduced, which subject cells to stimulatory molecules to create the preferred response and stimulate persistent effects. Pharmacological preconditioning uses small molecules and drugs to increase survival of MSCs after transplantation or prolong release of effective secretary factors such as cytokines that improve immune system responses. In this study, we investigated the effect of secretome of human embryonic-derived mesenchymal stem cells (hESC-MSCs) preconditioned with Trimetazidine (TMZ) and Diazoxide (DZ) on immunomodulatory efficiency of these cells in LPS-induced peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from human peripheral blood and treated with concentrated hESC-MSC-derived conditioned medium and then, the secreted levels of IL-10, TNFα and IL-1β were assessed by ELISA after induction with LPS. The results showed that TMZ and DZ-conditioned medium significantly enhanced immunomodulatory potential of hESC-MSCs by increasing the secretion of IL-10, TNFα and IL-1β from LPS- induced PBMCs. We also found that hESC-MSCs did not secrete mentioned cytokines prior to or after the preconditioning with TMZ and DZ. In conclusion, our results implied that TMZ and DZ can be used to promote the immunomodulatory effects of hESC-MSC secretome. It is obvious that for applying of these findings in clinical demands, the potency of different pre-conditioned MSCs secretome on immune response needs to be more clarified.     

Advances in mesenchymal stem cell transplantation for the treatment of osteoporosis

Osteoporosis is a systemic metabolic bone disease with characteristics of bone loss and microstructural degeneration. The personal and societal costs of osteoporosis are increasing year by year as the ageing of population, posing challenges to public health care. Homing disorders, impaired capability of osteogenic differentiation, senescence of mesenchymal stem cells (MSCs), an imbalanced microenvironment, and disordered immunoregulation play important roles during the pathogenesis of osteoporosis. The MSC transplantation promises to increase osteoblast differentiation and block osteoclast activation, and to rebalance bone formation and resorption. Preclinical investigations on MSC transplantation in the osteoporosis treatment provide evidences of enhancing osteogenic differentiation, increasing bone mineral density, and halting the deterioration of osteoporosis. Meanwhile, the latest techniques, such as gene modification, targeted modification and co‐transplantation, are promising approaches to enhance the therapeutic effect and efficacy of MSCs. In addition, clinical trials of MSC therapy to treat osteoporosis are underway, which will fill the gap of clinical data. Although MSCs tend to be effective to treat osteoporosis, the urgent issues of safety, transplant efficiency and standardization of the manufacturing process have to be settled. Moreover, a comprehensive evaluation of clinical trials, including safety and efficacy, is still needed as an important basis for clinical translation.     

The role of stem cells for treatment of cardiovascular disease

Cardiovascular disease is a global cause of mortality and morbidity. Current treatments fail to address the underlying scarring and cell loss, which are the causes of ischaemic heart failure. Cellular transplantation can overcome these problems and new impetus has been injected into this field following the isolation of human embryonic and adult stem cells. These cells have shown remarkable ability to produce cardiomyocytes and vascular cells in vitro and in vivo. Initial transplantation studies have demonstrated functional benefits and it is hoped further randomised clinical trials will concur with initial findings. Much basic science remains to be unearthed, such as the signals for homing, differentiation and engraftment of transplanted cells. Further matters of concern are the role of cell fusion and the mechanisms by which transplanted cells improve cardiac function. In spite of initial progress made in stem cell therapy there is still much to be done and we are some way off from achieving the goal of effective cellular regeneration.     

The mesenchymal stem cell secretome: A new paradigm towards cell-free therapeutic mode in regenerative medicine

Mesenchymal Stem Cells (MSCs) have been shown to be a promising candidate for cell-based therapy. The therapeutic potential of MSCs, towards tissue repair and wound healing is essentially based on their paracrine effects. Numerous pre-clinical and clinical studies of MSCs have yielded encouraging results. Further, these cells have been shown to be relatively safe for clinical applications. MSCs harvested from numerous anatomical locations including the bone marrow, adipose tissue, Wharton’s jelly of the umbilical cord etc., display similar immunophenotypic profiles. However, there is a large body of evidence showing that MSCs secrete a variety of biologically active molecules such as growth factors, chemokines, and cytokines. Despite the similarity in their immunophenotype, the secretome of MSCs appears to vary significantly, depending on the age of the host and niches where the cells reside. Thus, by implication, proteomics-based profiling suggests that the therapeutic potential of the different MSC populations must also be different. Analysis of the secretome points to its influence on varied biological processes such as angiogenesis, neurogenesis, tissue repair, immunomodulation, wound healing, anti-fibrotic and anti-tumour for tissue maintenance and regeneration. Though MSC based therapy has been shown to be relatively safe, from a clinical standpoint, the use of cell-free infusions can altogether circumvent the administration of viable cells for therapy. Understanding the secretome of in vitro cultured MSC populations, by the analysis of the corresponding conditioned medium, will enable us to evaluate its utility as a new therapeutic option. This review will focus on the accumulating evidence that points to the therapeutic potential of the conditioned medium, both from pre-clinical and clinical studies. Finally, this review will emphasize the importance of profiling the conditioned medium for assessing its potential for cell-free therapy therapy.     

The secretome of mesenchymal stem cells: Potential implications for neuroregeneration

Mesenchymal stem cells have shown regenerative properties in many tissues. This feature had originally been ascribed to their multipotency and thus their ability to differentiate into tissue-specific cells. However, many researchers consider the secretome of mesenchymal stem cells the most important player in the observed reparative effects of these cells. In this review, we specifically focus on the potential neuroregenerative effect of mesenchymal stem cells, summarize several possible mechanisms of neuroregeneration and list key factors mediating this effect. We illustrate examples of mesenchymal stem cell treatment in central nervous system disorders including stroke, neurodegenerative disorders (such as Parkinson's disease, Huntington's disease, multiple system atrophy and cerebellar ataxia) and inflammatory disease (such as multiple sclerosis). We specifically highlight studies where mesenchymal stem cells have entered clinical trials.     

间充质干细胞外泌体在阿尔兹海默症治疗中的研究进展

阿尔兹海默症（AD）是一种病理机制复杂,以进行性认知功能障碍为主的中枢神经系统疾病,目前仍缺乏有效的治疗方法。多项研究结果显示,间充质干细胞（MSCs）外泌体能够促进抗炎、调节免疫功能、加强Aβ降解、促进神经细胞轴突生长等,能很好地针对AD的核心病理机制发挥效果从而达到治疗效果。本文主要介绍MSCs外泌体在各项AD病理机制治疗中的研究进展。     

Assessing the effect of human dental pulp mesenchymal stem cell secretome on human oral,breast, and melanoma cancer cell lines

BackgroundThe secretome of the dental pulp mesenchymal stem cells (DPMSCS-S) have an array of regenerative potential and could aid in the rehabilitation of cancer patients post-therapeutic interventions, although caution is required as DPMSC-S have shown to augment prostate cancer cells. Thus, it is vital to assess if these pro-carcinogenic effects extend to other cancer types.ObjectiveTo assess if DPMSC-S has any pro-carcinogenic effect on oral cancer, breast cancer, and melanoma cell lines.Materials and methodsConditioned media obtained from the isolated and characterized DPMSC (DPMSC-CM) were profiled using bead-based multiplex assay. AW13515 (oral cancer), MDA-MB-231 (breast cancer), and A-375 (melanoma) cell lines were exposed to 20%, 50%, and 100% DPMSC-CM for 24, 48, and 72 h. DPMSC-CM effect on the cancer cell properties and secretome were assessed.ResultsDPMSC-CM augmented invasion, adhesion, multi-drug resistance, DNA repair, and mitochondrial repair in AW13516 through upregulation of growth factors Ang-2, EGF, M−CSF, PDGF-AA, PDGF-BB, pro-inflammatory cytokines TNF-α, IL-2, downregulation of anti-inflammatory cytokine TGF-β1, and pro-inflammatory cytokine IL-4. In MDA-MB-231, invasion, and multi-drug resistance were augmented through upregulation of growth factors EGF, EPO, G-CSF, HGF, M−CSF, PDGF-AA, and pro-inflammatory cytokine TNF-α, CXCL10, IL-12p70. EMT, invasion, migration, and adhesion were augmented in A-375 through upregulation of growth factors Ang-2, EGF, PDGF-BB, TGF-α, pro-inflammatory cytokines TNF-α, and IL-17A.ConclusionDPMSC-CM can augment the carcinogenic properties of oral cancer, breast cancer, and melanoma cells, further animal model studies are required to validate our in-vitro findings.     

间充质干细胞在肝脏疾病应用的研究现况

间充质干细胞（MSC）属于成体干细胞的一种,是一类具有自我更新和多向分化能力的多能干细胞。其来源丰富,免疫原性低,目前体内外实验均发现MSC可促进损伤肝脏修复,改善症状,提高存活率。通过调节肝脏局部和全身炎症反应和免疫紊乱发挥治疗作用。本文就MSC治疗肝脏疾病的研究现况进行综述。     

间充质干细胞外泌体治疗肺部疾病的研究进展

肺部疾病作为一种呼吸系统常见病,与重工业快速发展、环境污染等密切相关,死亡率极高。近来研究发现间充质干细胞(mesenchymal stem cells,MSCs)能够自主归巢至疾病损伤部位、修复受损组织并参与调节全身炎症和免疫反应,在临床上有较好的应用前景。外泌体是一种细胞外膜囊泡,可能通过调节细胞间通讯参与生物系统中的各种活动,分泌至细胞外环境的外泌体也会影响宿主免疫系统。而MSCs外泌体作为MSCs旁分泌过程中的一种介质,携带有蛋白质、脂质、m RNA和microRNA等生物活性物质,比基于细胞的治疗有更高的安全性,其潜在的医学价值受到了研究者的广泛关注。本文综述了近年来研究中MSCs外泌体在常见肺部疾病治疗中的作用机制,将为其在未来的临床应用方面提供依据。