Human Mesenchymal Stem/stromal Cells Research Articles

Changes in the Transcriptome Profiles of Human Amnion-Derived Mesenchymal Stromal/Stem Cells Induced by Three-Dimensional Culture: A Potential Priming Strategy to Improve Their Properties.

Mesenchymal stromal/stem cells (MSCs) are believed to function in vivo as a homeostatic tool that shows therapeutic properties for tissue repair/regeneration. Conventionally, these cells are expanded in two-dimensional (2D) cultures, and, in that case, MSCs undergo genotypic/phenotypic changes resulting in a loss of their therapeutic capabilities. Moreover, several clinical trials using MSCs have shown controversial results with moderate/insufficient therapeutic responses. Different priming methods were tested to improve MSC effects, and three-dimensional (3D) culturing techniques were also examined. MSC spheroids display increased therapeutic properties, and, in this context, it is crucial to understand molecular changes underlying spheroid generation. To address these limitations, we performed RNA-seq on human amnion-derived MSCs (hAMSCs) cultured in both 2D and 3D conditions and examined the transcriptome changes associated with hAMSC spheroid formation. We found a large number of 3D culture-sensitive genes and identified selected genes related to 3D hAMSC therapeutic effects. In particular, we observed that these genes can regulate proliferation/differentiation, as well as immunomodulatory and angiogenic processes. We validated RNA-seq results by qRT-PCR and methylome analysis and investigation of secreted factors. Overall, our results showed that hAMSC spheroid culture represents a promising approach to cell-based therapy that could significantly impact hAMSC application in the field of regenerative medicine.

Therapeutic Efficacy of Human Mesenchymal Stem Cells With Different Delivery Route and Dosages in Rat Models of Spinal Cord Injury

Recent studies have shown that the use of mesenchymal stem/stromal cells (MSCs) may be a promising strategy for treating spinal cord injury (SCI). This study aimed to explore the effectiveness of human umbilical cord-derived MSCs (hUC-MSCs) with different administration routes and dosages on SCI rats. Following T10-spinal cord contusion in Sprague-Dawley rats (N = 60), three different dosages of hUC-MSCs were intrathecally injected into rats (SCI-ITH) after 24 h. Intravenous injection of hUC-MSCs (SCI-i.v.) and methylprednisolone reagent (SCI-PC) were used as positive controls (N = 10/group). A SCI control group without treatment and a sham operation group were injected with Multiple Electrolyte Injection solution. The locomotor function was assessed by Basso Beattie Bresnahan (BBB) rating score, magnetic resonance imaging (MRI), histopathology, and immunofluorescence. ELISA was conducted to further analyze the nerve injury and inflammation in the rat SCI model. Following SCI, BBB scores were significantly lower in the SCI groups compared with the sham operation group, but all the treated groups showed the recovery of hind-limb motor function, and rats receiving the high-dose intrathecal injection of hUC-MSCs (SCI-ITH-H) showed improved outcomes compared with rats in hUC-MSCs i.v. and positive control groups. Magnetic resonance imaging revealed significant edema and spinal cord lesion in the SCI groups, and significant recovery was observed in the medium and high-dose hUC-MSCs ITH groups. Histopathological staining showed that the necrotic area in spinal cord tissue was significantly reduced in the hUC-MSCs ITH-H group, and the immunofluorescence staining confirmed the neuroprotection effect of hUC-MSCs infused on SCI rats. The increase of inflammatory cytokines was repressed in hUC-MSCs ITH-H group. Our results confirmed that hUC-MSC administered via intrathecal injection has dose-dependent neuroprotection effect in SCI rats.

Amnion-Derived Mesenchymal Stromal/Stem Cell Paracrine Signals Potentiate Human Liver Organoid Differentiation: Translational Implications for Liver Regeneration.

The prevalence of end-stage liver diseases has reached very high levels globally. The election treatment for affected patients is orthotopic liver transplantation, which is a very complex procedure, and due to the limited number of suitable organ donors, considerable research is being done on alternative therapeutic options. For instance, the use of cell therapy, such as the transplantation of hepatocytes to promote liver repair/regeneration, has been explored, but standardized protocols to produce suitable human hepatocytes are still limited. On the other hand, liver progenitor and multipotent stem cells offer potential cell sources that could be used clinically. Different studies have reported regarding the therapeutic effects of transplanted mesenchymal stromal/stem cells (MSCs) on end-stage liver diseases. Moreover, it has been shown that delivery of MSC-derived conditioned medium (MSC-CM) can reduce cell death and enhance liver proliferation in fulminant hepatic failure. Therefore, it is believed that MSC-CM contains many factors that probably support liver regeneration. In our work, we used an in vitro model of human liver organoids to study if the paracrine components secreted by human amnion-derived MSCs (hAMSCs) affected liver stem/progenitor cell differentiation. In particular, we differentiated liver organoids derived from bipotent EpCAM+ human liver cells and tested the effects of hAMSC secretome, derived from both two-dimensional (2D) and three-dimensional (3D) hAMSC cultures, on that model. Our analysis showed that conditioned medium (CM) produced by 3D hAMSCs was able to induce an over-expression of mature hepatocyte markers, such as ALB, NTCP, and CYP3A4, compared with both 2D hAMSC cultures and the conventional differentiation medium (DM). These data were confirmed by the over-production of ALB protein and over-activity of CYP3A4 observed in organoids grown in 3D hAMSC-CM. Liver repair dysfunction plays a role in the development of liver diseases, and effective repair likely requires the normal functioning of liver stem/progenitor cells. Herein, we showed that hAMSC-CM produced mainly by 3D cultures had the potential to increase hepatic stem/progenitor cell differentiation, demonstrating that soluble factors secreted by those cells are potentially responsible for the reaction. This work shows a potential approach to improve liver repair/regeneration also in a transplantation setting.

Pluripotent-derived Mesenchymal Stem/stromal Cells: an Overview of the Derivation Protocol Efficacies and the Differences Among the Derived Cells.

Mesenchymal stem/stromal cells (MSCs) are remarkable tools for regenerative medicine. Therapeutic approaches using these cells can promote increased activity and viability in several cell types through diverse mechanisms such as paracrine and immunomodulatory activities, contributing substantially to tissue regeneration and functional recovery. However, biological samples of human MSCs, usually obtained from adult tissues, often exhibit variable behavior during in vitro culture, especially with respect to cell population heterogeneity, replicative senescence, and consequent loss of functionality. Accordingly, it is necessary to establish standard protocols to generate high-quality, stable cell cultures, for example, by using pluripotent stem cells (PSCs) in derivation protocols of MSC-like cells since PSCs maintain their characteristics consistently during culture. However, the available protocols seem to generate distinct populations of PSC-derivedMSCs (PSC-MSCs) with peculiar attributes, which do not always resemble bona fide primary MSCs. The present review addresses the developmental basis behind some of these derivation protocols, exposing the differences among them and discussing the functional properties of PSC-MSCs, shedding light on elements that may help determine standard characterizations and criteria to evaluate and define these cells.

The Chondrogenic Potential of First-Trimester and Term Placental Mesenchymal Stem/Stromal Cells.

Mesenchymal stem/stromal cells (MSCs) are a well-established cell source for cartilage engineering, but challenges remain as differentiation often results in chondrocyte hypertrophy. Chondrogenic potential also varies with MSC source and donor age. We assessed the chondrogenic potential of first-trimester and term placental MSCs and compared their response to commonly used bone marrow MSCs (BM-MSCs). MSCs were isolated from first-trimester and term placentae. BM-MSCs were commercially obtained. Chondrogenesis was induced by micromass culture in commercial chondrogenic media for 7, 14, or 21 days. Pellets were assessed for glycosaminoglycan (GAG) content, and types I, II, and X collagen. Gene expression was profiled using Qiagen RT2 human MSC arrays. At day 0, first-trimester and term MSCs expression levels of many chondrogenic genes to BM-MSC after 21 days of culture. Only first trimester MSCs showed significant changes in chondrogenic gene expression during induction compared to day 0 undifferentiated MSCs (greater BMP4, KAT2B, and reduced GDF6 expression). Additionally, first-trimester MSCs showed significantly greater expression of ABCB1 (at days 14 and 21) and BMP4 (at days 7, 14, 21) compared with term MSCs. Both first-trimester and term pellets showed increased GAG content over time and term MSCs had significantly GAG greater compared with BM-MSCs at days 7 and 14. Type II collagen was present in all pellets but unlike BM-MSCs, type I collagen was not observed in first-trimester or term MSC pellets. These data highlight differences in BM-MSC and placental MSC chondrogenesis and demonstrate that placental MSCs may be an alternative cell source.

Reprogrammed Synovial Fluid-Derived Mesenchymal Stem/Stromal Cells Acquire Enhanced Therapeutic Potential for Articular Cartilage Repair.

Functions of mesenchymal stem/stromal cells (MSCs) are affected by patient-dependent factors such as age and health condition. To tackle this problem, we used the cellular reprogramming technique to epigenetically alter human MSCs derived from the synovial fluid of joints with osteoarthritis (OA) to explore the potential of reprogrammed MSCs for repairing articular cartilage. MSCs isolated from the synovial fluid of three patients' OA knees (Pa-MSCs) were reprogrammed through overexpression of pluripotency factors and then induced for differentiation to establish reprogrammed MSC (Re-MSC) lines. We compared the in vitro growth characteristics, chondrogenesis for articular cartilage chondrocytes, and immunomodulatory capacity. We also evaluated the capability of Re-MSCs to repair articular cartilage damage in an animal model with spontaneous OA. Our results showed that Re-MSCs increased the in vitro proliferative capacity and improved chondrogenic differentiation toward articular cartilage-like chondrocyte phenotypes with increased THBS4 and SIX1 and decreased ALPL and COL10A1, compared to Pa-MSCs. In addition, Re-MSC-derived chondrocytes expressing elevated COL2A and COL2B were more mature than parental cell-derived ones. The enhancement in chondrogenesis of Re-MSC involves the upregulation of sonic hedgehog signaling. Moreover, Re-MSCs improved the repair of articular cartilage in an animal model of spontaneous OA. Epigenetic reprogramming promotes MSCs harvested from OA patients to increase phenotypic characteristics and gain robust functions. In addition, Re-MSCs acquire an enhanced potential for articular cartilage repair. Our study here demonstrates that the reprogramming strategy provides a potential solution to the challenge of variation in MSC quality.

Harnessing TRAIL-Induced Apoptosis Pathway for Cancer Immunotherapy and Associated Challenges.

The immune cytokine tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has attracted rapidly evolving attention as a cancer treatment modality because of its competence to selectively eliminate tumor cells without instigating toxicity in vivo. TRAIL has revealed encouraging promise in preclinical reports in animal models as a cancer treatment option; however, the foremost constraint of the TRAIL therapy is the advancement of TRAIL resistance through a myriad of mechanisms in tumor cells. Investigations have documented that improvement of the expression of anti-apoptotic proteins and survival or proliferation involved signaling pathways concurrently suppressing the expression of pro-apoptotic proteins along with down-regulation of expression of TRAILR1 and TRAILR2, also known as death receptor 4 and 5 (DR4/5) are reliable for tumor cells resistance to TRAIL. Therefore, it seems that the development of a therapeutic approach for overcoming TRAIL resistance is of paramount importance. Studies currently have shown that combined treatment with anti-tumor agents, ranging from synthetic agents to natural products, and TRAIL could result in induction of apoptosis in TRAIL-resistant cells. Also, human mesenchymal stem/stromal cells (MSCs) engineered to generate and deliver TRAIL can provide both targeted and continued delivery of this apoptosis-inducing cytokine. Similarly, nanoparticle (NPs)-based TRAIL delivery offers novel platforms to defeat barricades to TRAIL therapeutic delivery. In the current review, we will focus on underlying mechanisms contributed to inducing resistance to TRAIL in tumor cells, and also discuss recent findings concerning the therapeutic efficacy of combined treatment of TRAIL with other antitumor compounds, and also TRAIL-delivery using human MSCs and NPs to overcome tumor cells resistance to TRAIL.

Human meniscus allograft augmentation by allogeneic mesenchymal stromal/stem cell injections.

Meniscus allograft transplantations (MATs) represent established surgical procedures with proven outcomes. Yet, storage as frozen specimens and limited cellular repopulation may impair graft viability. This proof‐of‐concept study tests the feasibility of injecting allogeneic mesenchymal stromal/stem cells (MSCs) in meniscus allograft tissue. We investigated the injectable cell quantity, survival rate, migration, and proliferation ability of MSCs up to 28 days of incubation. In this controlled laboratory study, seven fresh‐frozen human allografts were injected with human allogeneic MSCs. Cells were labeled and histological characteristics were microscopically imaged up to 28 days. Mock‐injected menisci were included as negative controls in each experiment. Toluidine blue staining demonstrated that a 100‐µl volume can be injected while retracting and rotating the inserted needle. Immediately after injection, labeled MSCs were distributed throughout the injection channel and eventually migrated into the surrounding tissues. Histological assessment revealed that MSCs cluster in disc‐like shapes, parallel to the intrinsic lamination of the meniscus and around the vascular network. Quantification showed that more than 60% of cells were present in horizontally injected grafts and more than 30% were observed in vertically injected samples. On Day 14, cells adopted a spindle‐shaped morphology and exhibited proliferative and migratory behaviors. On Day 28, live/dead ratio assessment revealed an approximately 80% cell survival. The study demonstrated the feasibility of injecting doses of MSCs (>0.1 million) in meniscus allograft tissue with active cell proliferation, migration, and robust cell survival.

IL-1β-pre-conditioned mesenchymal stem/stromal cells' secretome modulates the inflammatory response and aggrecan deposition in intervertebral disc.

Mesenchymal stem/stromal cells (MSCs) have been increasingly used in clinical trials for low-back pain (LBP) and intervertebral disc (IVD) degeneration with promising results. Their action mechanisms are not fully understood, but they reduce IVD pro-inflammatory markers in a pro-inflammatory/degenerative IVD microenvironment. In this study the therapeutic potential of the MSC secretome, as an alternative cell-free approach for treating degenerated IVDs, was examined. Human bone marrow-derived MSC secretome (MSCsec) was collected after 48 h of preconditioning in IL-1β (10 ng/mL) and low oxygen (6 % O2), mimicking the degenerative IVD. IL-1β-pre-conditioning of MSCs increased secretion of pro-inflammatory markers hIL-6, hIL-8, hMCP-1, etc. The therapeutic effect of MSCsec was tested in a pro-inflammatory/degenerative IVD ex vivo model. MSCsec down-regulated IVD gene expression of pro-inflammatory cytokines (bIL-6, bIL-8) and matrix degrading enzyme bMMP1, while bMMP3 and bTIMP2 were up-regulated, at 48 h. After 14 d, MSCsec-treated IVDs revealed increased aggrecan deposition, although no differences in other ECM components were observed. Protein analysis of the MSCsec-treated IVD supernatant revealed a significant increase of CXCL1, MCP-1, MIP-3α, IL-6, IL-8 and GRO α/β/γ (related to TNF, NOD-like receptor and neutrophil chemotaxis signalling), and a decrease of IFN-γ, IL-10, IL-4, IL-5 and TNF-α (associated with T-cell receptor signalling). MSCsec-treated IVD supernatants did not promote angiogenesis and neurogenesis in vitro. Overall, MSCsec can be a safe therapeutic approach, presenting a strong immunomodulatory role in degenerated IVD while potentiating aggrecan deposition, which can open new perspectives on the use of MSCsec as a cell-based/ cell-free therapeutic approach to LBP.

Fibroblastic differentiation of mesenchymal stem/stromal cells (MSCs) is enhanced by hypoxia in 3D cultures treated with bone morphogenetic protein 6 (BMP6) and growth and differentiation factor 5 (GDF5).

Culture conditions and differentiation cocktails may facilitate cell maturation and extracellular matrix (ECM) secretion and support the production of engineered fibroblastic tissues with applications in ligament regeneration. The objective of this study is to investigate the potential of two connective tissue-related ligands (i.e., BMP6 and GDF5) to mediate collagenous ECM synthesis and tissue maturation in vitro under normoxic and hypoxic conditions based on the hypothesis that BMP6 and GDF5 are components of normal paracrine signalling events that support connective tissue homeostasis. Human adipose-derived MSCs were seeded on 3D-printed medical-grade polycaprolactone (PCL) scaffolds using a bioreactor and incubated in media containing GDF5 and/or BMP6 for 21days in either normoxic (5% oxygen) or hypoxic (2% oxygen) conditions. Constructs were harvested on Day 3 and 21 for cell viability analysis by live/dead staining, structural analysis by scanning electron microscopy, mRNA levels by RTqPCR analysis, and in situ deposition of proteins by immunofluorescence microscopy. Pro-fibroblastic gene expression is enhanced by hypoxic culture conditions compared to normoxic conditions. Hypoxia renders cells more responsive to treatment with BMP6 as reflected by increased expression of ECM mRNA levels on Day 3 with sustained expression until Day 21. GDF5 was not particularly effective either in the absence or presence of BMP6. Fibroblastic differentiation of MSCs is selectively enhanced by BMP6 and not GDF5. Environmental factors (i.e., hypoxia) also influenced the responsiveness of cells to this morphogen.

Diabetic Kidney Disease Alters the Transcriptome and Function of Human Adipose-Derived Mesenchymal Stromal Cells but Maintains Immunomodulatory and Paracrine Activities Important for Renal Repair.

Mesenchymal stem/stromal cells (MSCs) facilitate repair in experimental diabetic kidney disease (DKD). However, the hyperglycemic and uremic milieu may diminish regenerative capacity of patient-derived therapy. We hypothesized that DKD reduces human MSC paracrine function. Adipose-derived MSC from 38 participants with DKD and 16 control subjects were assessed for cell surface markers, trilineage differentiation, RNA sequencing (RNA-seq), in vitro function (coculture or conditioned medium experiments with T cells and human kidney cells [HK-2]), secretome profile, and cellular senescence abundance. The direction of association between MSC function and patient characteristics were also tested. RNA-seq analysis identified 353 differentially expressed genes and downregulation of several immunomodulatory genes/pathways in DKD-MSC versus Control-MSC. DKD-MSC phenotype, differentiation, and tube formation capacity were preserved, but migration was reduced. DKD-MSC with and without interferon-γ priming inhibited T-cell proliferation greater than Control-MSC. DKD-MSC medium contained higher levels of anti-inflammatory cytokines (indoleamine 2,3-deoxygenase 1 and prostaglandin-E2) and prorepair factors (hepatocyte growth factor and stromal cell-derived factor 1) but lower IL-6 versus control-MSC medium. DKD-MSC medium protected high glucose plus transforming growth factor-β-exposed HK-2 cells by reducing apoptotic, fibrotic, and inflammatory marker expression. Few DKD-MSC functions were affected by patient characteristics, including age, sex, BMI, hemoglobin A1c, kidney function, and urine albumin excretion. However, senescence-associated β-galactosidase activity was lower in DKD-MSC from participants on metformin therapy. Therefore, while DKD altered the transcriptome and migratory function of culture-expanded MSCs, DKD-MSC functionality, trophic factor secretion, and immunomodulatory activities contributing to repair remained intact. These observations support testing of patient-derived MSC therapy and may inform preconditioning regimens in DKD clinical trials.

Mesenchymal stem cell derived extracellular vesicles as treatment for osteoarthritis in a rat high fat diet groove model

Purpose: Mesenchymal stromal/stem cells (MSC) hold a great promise to control the imbalance between anabolic and catabolic processes in an OA joint due to their immunomodulatory and regenerative capacities. Recently, intra-articular injections with human MSC entered the clinical trials as a novel, low invasive and relatively cheap therapy for OA. Although, the results of this novel treatment are promising, it remains unclear how MSC exert their beneficial effects in inflammation and tissue (cartilage) repair. Increasing evidence suggests that the therapeutic efficacy of MSC depends on paracrine signaling. Recently we have shown that MSC-derived extracellular vesicles (MSC-EVs) are an important component of this signaling and have a major contribution to cartilage regeneration and inhibition of TNF-alpha stimulated inflammatory responses in chondrocytes in vitro. Extracellular vesicles (EVs) are secreted by all cell types and range in size from 40 - 100 nm (exosomes) and from 100 -1000 nm (microvesicles). They increasingly appear to play an important role in intercellular communication by transportation of important molecules (peptides, mRNA, miRNA, enzymes). MSC-derived EVs have a great potential to serve as a safer cell free therapy for OA. The purpose of this study was to investigate if MSC-derived EVs can inhibit inflammation and promote cartilage repair to a similar extent as MSCs in a rat high fat diet groove model of OA. Methods: 36 male Wistar-han (Crl:WI(Han) rats were fed a high fat diet for 24 weeks, starting at an age of 12 weeks old. In week 12 of the study the rats received unilateral surgery where local cartilage damage was induced at the right knee joint by making five longitudinal grooves on the femoral condyles. In week 13 the rats were randomized into 3 groups and given an intraarticular injection with either PBS, 2 million human bone marrow derived MSCs or EVs derived from 2 million MSCs. The extracellular vesicles group received 5 of these doses with 5 days interval. Before, during and after the injections pain behavior was monitored by dynamic weight bearing and via measuring the left and right hind paw withdrawal responses to the application of von Frey hairs. uCT scans were made at week 0 and week 24, and used to determine the number of osteophytes and cartilage mineralization in the tibia. Additionally, tibial subchondral plate thickness, trabecular bone thickness, and trabecular bone volume fraction (total volume/trabecular bone volume) were measured using ImageJ 1.47v. At endpoint animals were sacrificed and the knee joints were harvested. Joint tissue is currently under preparation for histological staining to determine the OARSI histopathological score for rats. Data is represented as mean +/- standard deviation (SD) and the difference between the means in the three groups was statistically tested using one way ANOVA. Results: Results from the von Frey tests showed a clear decrease in 50% withdrawal threshold in the right hind paw, which was expected as the right side was the grooved limb. However, no difference between the three treatment groups in either left or right paw was seen. The mean osteophyte number in the PBS group was 4.7 (+/- 0.65) versus 3.6 (+/-2.15) in the MSC group and 4.3 (+/-1.8) in the MSC-EV group (figure 1A). In the MSC and MSC-EV treatment groups the variation in osteophyte number and total osteophyte diameter between the animals was large compared to the PBS group. Cartilage mineralization were observed in 9 of 12 animals in the PBS and MSC group and in 5 of 12 animals in the MSC-EV group. Both the number and total diameter was lowest in the MSC-EV group, but there was no significant difference as compared with control groups (Figure 1B). Other parameters measured on uCT like bone volume fraction, subchondral plate and trabecular bone thickness were similar between the 3 groups. Results from the OARSI histopathological score for rats is currently under preparation. Conclusions: Preliminary results from this in vivo study show no significant differences between PBS, MSC or MSC-EV treatment on uCT data or von Frey test. However, we do observe a large spread in osteophyte score and total diameter in the MSC and MSC-EV treated groups, which could be a sign that individual animals are responding differently to the treatments. Data from the OARSI scoring will help us determine if the response really is variable for MSC and MSC-EV treatment groups. This would be in accordance with observations made in humans, as many clinical trials that test MSC-based therapies report a substantial nonresponder group. In summary, although osteophytes are one of the hallmarks of osteoarthritis, we feel a complete picture of the joint damage including cartilage and synovium is needed to draw conclusions on the effect of the treatments in this study.

Intrapancreatic MSC transplantation facilitates pancreatic islet regeneration

BackgroundType 1 diabetes mellitus (T1D) is characterized by the autoimmune destruction of the pancreatic β cells. The transplantation of mesenchymal stromal/stem cells (MSC) was reported to rescue the damaged pancreatic niche. However, there is an ongoing discussion on whether direct physical contact between MSC and pancreatic islets results in a superior outcome as opposed to indirect effects of soluble factors released from the MSC entrapped in the lung microvasculature after systemic administration. Hence, MSC were studied in direct contact (DC) and indirect contact (IDC) with murine pancreatic β cell line MIN6-cells damaged by nitrosourea derivative streptozotocin (STZ) in vitro. Further, the protective and antidiabetic outcome of MSC transplantation was evaluated through the intrapancreatic route (IPR) and intravenous route (IVR) in STZ-induced diabetic NMRI nude mice.MethodsMSC were investigated in culture with STZ-damaged MIN6-cells, either under direct contact (DC) or separated through a semi-permeable membrane (IDC). Moreover, multiple low doses of STZ were administered to NMRI nude mice for the induction of hyperglycemia. 0.5 × 106 adipose-derived mesenchymal stem cells (ADMSC) were transferred through direct injection into the pancreas (IPR) or the tail vein (IVR), respectively. Bromodeoxyuridine (BrdU) was injected for the detection of proliferating islet cells in vivo, and real-time polymerase chain reaction (RT-PCR) was employed for the measurement of the expression of growth factor and immunomodulatory genes in the murine pancreas and human MSC. Phosphorylation of AKT and ERK was analyzed with Western blotting.ResultsThe administration of MSC through IPR ameliorated hyperglycemia in contrast to IVR, STZ, and non-diabetic control in a 30-day window. IPR resulted in a higher number of replicating islet cells, number of islets, islet area, growth factor (EGF), and balancing of the Th1/Th2 response in vivo. Physical contact also provided a superior protection to MIN6-cells from STZ through the AKT and ERK pathway in vitro in comparison with IDC.ConclusionOur study suggests that the physical contact between MSC and pancreatic islet cells is required to fully unfold their protective potential.

Impact of 2Gy γ-irradiation on the hallmark characteristics of human bone marrow-derived MSCs.

Two gray γ-irradiation is a widely employed basic module for total body irradiation (TBI) in allogeneic hematopoietic cell transplantation (HCT). The effects of γ-irradiation on hematopoietic and immune cells have been well investigated, but its effects on the bone marrow microenvironment (BMM) are unknown. Given the crucial contribution of mesenchymal/stromal stem cells (MSCs) in the BMM to hematopoiesis and osteogenesis, we investigated whether γ-irradiation affects the hallmark characteristics of human bone marrow-derived MSCs (BM-MSCs). Expansion of 2Gy γ-irradiated BM-MSCs was delayed but eventually recovered. Colony formation and osteogenic, adipogenic, and chondrogenic differentiation capabilities of these cells were extensively suppressed. Irradiation of BM-MSCs did not affect the expansion of CD34 + hematopoietic stem and progenitor cells or production of CD11b + mature myeloid cells in co-cultures. However, it reduced production of CD19 + B-cells, as well as expression of CXCL12 and interleukin-7, which are essential for B-cell lymphopoiesis, in 2Gy γ-irradiated BM-MSCs. Collectively, colony formation, osteogenic differentiation, and B-cell lymphopoiesis-supportive capabilities of γ-irradiated BM-MSCs were reduced. These effects may predispose survivors receiving HCT with TBI to defective bone formation and a perturbed humoral immune response.

The ATP synthase inhibition induces an AMPK-dependent glycolytic switch of mesenchymal stem cells that enhances their immunotherapeutic potential.

Objectives: Mesenchymal Stem/Stromal Cells (MSC) are promising therapeutic tools for inflammatory diseases due to their potent immunoregulatory capacities. Their suppressive activity mainly depends on inflammatory cues that have been recently associated with changes in MSC bioenergetic status towards a glycolytic metabolism. However, the molecular mechanisms behind this metabolic reprogramming and its impact on MSC therapeutic properties have not been investigated.Methods: Human and murine-derived MSC were metabolically reprogramed using pro-inflammatory cytokines, an inhibitor of ATP synthase (oligomycin), or 2-deoxy-D-glucose (2DG). The immunosuppressive activity of these cells was tested in vitro using co-culture experiments with pro-inflammatory T cells and in vivo with the Delayed-Type Hypersensitivity (DTH) and the Graph versus Host Disease (GVHD) murine models.Results: We found that the oligomycin-mediated pro-glycolytic switch of MSC significantly enhanced their immunosuppressive properties in vitro. Conversely, glycolysis inhibition using 2DG significantly reduced MSC immunoregulatory effects. Moreover, in vivo, MSC glycolytic reprogramming significantly increased their therapeutic benefit in the DTH and GVHD mouse models. Finally, we demonstrated that the MSC glycolytic switch effect partly depends on the activation of the AMPK signaling pathway.Conclusion: Altogether, our findings show that AMPK-dependent glycolytic reprogramming of MSC using an ATP synthase inhibitor contributes to their immunosuppressive and therapeutic functions, and suggest that pro-glycolytic drugs might be used to improve MSC-based therapy.

Structural and Functional Mapping of Mesenchymal Bodies.

The formation of spheroids with mesenchymal stem/stromal cells (MSCs), mesenchymal bodies (MBs), is usually performed using bioreactors or conventional well plates. While these methods promote the formation of a large number of spheroids, they provide limited control over their structure or over the regulation of their environment. It has therefore been hard to elucidate the mechanisms orchestrating the structural organization and the induction of the trophic functions of MBs until now. We have recently demonstrated an integrated droplet-based microfluidic platform for the high-density formation and culture of MBs, as well as for the quantitative characterization of the structural and functional organization of cells within them. The protocol starts with a suspension of a few hundred MSCs encapsulated within microfluidic droplets held in capillary traps. After droplet immobilization, MSCs start clustering and form densely packed spherical aggregates that display a tight size distribution. Quantitative imaging is used to provide a robust demonstration that human MSCs self-organize in a hierarchical manner, by taking advantage of the good fit between the microfluidic chip and conventional microscopy techniques. Moreover, the structural organization within the MBs is found to correlate with the induction of osteo-endocrine functions (i.e., COX-2 and VEGF-A expression). Therefore, the present platform provides a unique method to link the structural organization in MBs to their functional properties. Graphic abstract: Droplet microfluidic platform for integrated formation, culture, and characterization of mesenchymal bodies (MBs). The device is equipped with a droplet production area (flow focusing) and a culture chamber that enables the culture of 270 MBs in parallel. A layer-by-layer analysis revealed a hierarchical developmental organization within MBs.

Electrospun PCL/PLA Scaffolds Are More Suitable Carriers of Placental Mesenchymal Stromal Cells Than Collagen/Elastin Scaffolds and Prevent Wound Contraction in a Mouse Model of Wound Healing.

Mesenchymal stem/stromal cells (MSCs) exert beneficial effects during wound healing, and cell-seeded scaffolds are a promising method of application. Here, we compared the suitability of a clinically used collagen/elastin scaffold (Matriderm) with an electrospun Poly(ε-caprolactone)/poly(l-lactide) (PCL/PLA) scaffold as carriers for human amnion-derived MSCs (hAMSCs). We created an epidermal-like PCL/PLA scaffold and evaluated its microstructural, mechanical, and functional properties. Sequential spinning of different PCL/PLA concentrations resulted in a wide-meshed layer designed for cell-seeding and a dense-meshed layer for apical protection. The Matriderm and PCL/PLA scaffolds then were seeded with hAMSCs, with or without Matrigel coating. The quantity and quality of the adherent cells were evaluated in vitro. The results showed that hAMSCs adhered to and infiltrated both scaffold types but on day 3, more cells were observed on PCL/PLA than on Matriderm. Apoptosis and proliferation rates were similar for all carriers except the coated Matriderm, where apoptotic cells were significantly enhanced. On day 8, the number of cells decreased on all carrier types except the coated Matriderm, which had consistently low cell numbers. Uncoated Matriderm had the highest percentage of proliferative cells and lowest apoptosis rate of all carrier types. Each carrier also was topically applied to skin wound sites in a mouse model and analyzed in vivo over 14 days via optical imaging and histological methods, which showed detectable hAMSCs on all carrier types on day 8. On day 14, all wounds exhibited newly formed epidermis, and all carriers were well-integrated into the underlying dermis and showing signs of degradation. However, only wounds treated with uncoated PCL/PLA maintained a round appearance with minimal contraction. Overall, the results support a 3-day in vitro culture of scaffolds with hAMSCs before wound application. The PCL/PLA scaffold showed higher cell adherence than Matriderm, and the effect of the Matrigel coating was negligible, as all carrier types maintained sufficient numbers of transplanted cells in the wound area. The anti-contractive effects of the PCL/PLA scaffold offer potential new therapeutic approaches to wound care.

A Shaking-Culture Method for Generating Bone Marrow Derived Mesenchymal Stromal/Stem Cell-Spheroids With Enhanced Multipotency in vitro.

Mesenchymal stromal/stem cells (MSCs), which generally expand into adherent monolayers, readily lose their proliferative and multilineage potential following repeated passages. Floating culture systems can be used to generate MSC spheroids, which are expected to overcome limitations associated with conventional adherent cultures while facilitating scaffold-free cell transplantation. However, the phenotypic characteristics of spheroids after long-term culture are unknown. In addition, regenerative therapies require new culture systems to maintain their undifferentiated state. In this study, we established a novel culture method employing three-dimensional (3D) “shaking” to generate MSC spheroids using bone marrow derived MSCs. Floating 3D cultures of mouse or human MSCs formed spheroids after shaking (85–95 rpm), within 1 month. These spheroids maintained their osteogenic-, adipogenic-, and chondrogenic-differentiation capacity. The adipogenic-differentiation capacity of adherent cultured mouse and human MSCs, which is lost following several passages, was remarkedly restored by shaking-culture. Notably, human MSC spheroids exhibited a renewable “undifferentiated MSC-pool” property, wherein undifferentiated MSCs grew from spheroids seeded repeatedly on a plastic culture dish. These data suggest that the shaking-culture method maintains and restores multipotency that is lost following monolayer expansion and thereby shows potential as a promising strategy for regenerative therapies with mesenchymal tissues.

Lysosomal protein surface expression discriminates fat- from bone-forming human mesenchymal precursor cells.

Tissue resident mesenchymal stem/stromal cells (MSCs) occupy perivascular spaces. Profiling human adipose perivascular mesenchyme with antibody arrays identified 16 novel surface antigens, including endolysosomal protein CD107a. Surface CD107a expression segregates MSCs into functionally distinct subsets. In culture, CD107alow cells demonstrate high colony formation, osteoprogenitor cell frequency, and osteogenic potential. Conversely, CD107ahigh cells include almost exclusively adipocyte progenitor cells. Accordingly, human CD107alow cells drove dramatic bone formation after intramuscular transplantation in mice, and induced spine fusion in rats, whereas CD107ahigh cells did not. CD107a protein trafficking to the cell surface is associated with exocytosis during early adipogenic differentiation. RNA sequencing also suggested that CD107alow cells are precursors of CD107ahigh cells. These results document the molecular and functional diversity of perivascular regenerative cells, and show that relocation to cell surface of a lysosomal protein marks the transition from osteo- to adipogenic potential in native human MSCs, a population of substantial therapeutic interest.

TNF-α and IL-1β sensitize human MSC for IFN-γ signaling and enhance neutrophil recruitment.

During inflammatory processes, tissue environmental cues are influencing the immunoregulatory properties of tissue-resident mesenchymal stem/stromal cells (MSC). In this study, we elucidated one of the molecular and cellular responses of human MSC exposed to combinations of inflammatory cytokines. We showed that during multi-cytokine priming by TNF-α, IL-1β, and IFN-γ, IL-1β further augmented the well-established immunoregulatory activity induced by TNF-α/IFN-γ. On the molecular level, TNF-α and IL-1β enhanced the expression of IFN-γ receptor (IFN-γR) via NF 'kappa-light-chain-enhancer' of activated B-cells (NF-κΒ) signaling. In turn, enhanced responsiveness to IFN-γ stimulation activated STAT5 and p38-MAPK signaling. This molecular feedback resulted in an increased IL-8 release and augmented recruitment of polymorphonuclear granulocytes (PMN). Our study suggests the possibility that responses of MSC to multi-cytokine priming regimens may be exploited therapeutically to fine-tune inflammatory activity in tissues. This study elucidates molecular mechanisms underlying the immunological priming of mesenchymal stromal cells (MSC) and their interaction with neutrophils.