Blood-derived Mesenchymal Stromal Cells Research Articles

The effects of intraovarian injection of autologous menstrual blood-derived mesenchymal stromal cells on pregnancy outcomes in women with poor ovarian response

BackgroundAssisted reproduction faces a significant obstacle in the form of poor ovarian response (POR) to controlled ovarian stimulation. To address this challenge, mesenchymal stem cell therapy has been proposed as a potential treatment for female infertility and/or restoration of ovarian function in POR women. Our previous research has demonstrated that menstrual blood-derived-mesenchymal stromal cells (MenSCs) injected into the ovaries of women with POR can increase pregnancy rates. The objective of this study was to examine whether MenSC therapy could enhance ovarian reserve parameters and pregnancy outcomes in a larger population of individuals with POR.MethodThis study consisted of 180 infertile individuals with POR who declined oocyte donation. Participants were divided into two groups: those who received bilateral MenSCs intraovarian injection and those who received no intervention. Our primary aim was to compare the rates of spontaneous pregnancy between the two groups, followed by an investigation of any alterations in the ovarian reserve parameters, such as serum FSH, AMH, and AFC levels, as well as the ICSI/IVF outcomes, in both groups of participants.ResultsThe MenSC therapy exhibited a favourable tolerability profile and did not raise any safety concerns. Following the 2-month follow-up period, women who received MenSC treatment demonstrated a significantly higher rate of spontaneous pregnancy (P < 0.005) and an improvement in anti-Müllerian hormone (AMH) levels (P = 0.0007) and antral follicle count (AFC) (P < 0.001), whereas the control group demonstrated a considerable decline in these parameters (Both P < 0.001). The MenSC therapy led to a greater number of mature oocytes and embryos among women who underwent ICSI/IVF. Our age subgroup analysis demonstrated a significant difference in the number of spontaneous pregnancies and ICSI/IVF outcomes between the treatment and control groups only among individuals below 40 years of age.ConclusionThe results of our study indicate that MenSCs treatment may be a viable option for treating women experiencing POR. However, in order to be widely implemented in clinical practice, the clinical effectiveness of MenSCs therapy will need to be established through rigorous prospective randomized clinical trials.Trial registration: ClinicalTrials.gov Identifier: NCT05703308. Registered 01/26/2023, retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT05703308. IRCT, IRCT20180619040147N4. Registered 08/01/2020.

Bioprocess development for cord blood mesenchymal stromal cells on microcarriers in Vertical-Wheel bioreactors.

Equine mesenchymal stromal cells (MSCs) have been found to be beneficial for the treatment of many ailments, including orthopedic injuries, due to their superior differentiation potential and immunomodulating properties. Cell therapies require large cell numbers, which are not efficiently generated using conventional static expansion methods. Expansion of equine cord blood-derived MSCs (eCB-MSCs) in bioreactors, using microcarriers as an attachment surface, has the potential to generate large numbers of cells with increased reproducibility and homogeneity compared with static T-flask expansion. This study investigated the development of an expansion process using Vertical-Wheel (VW) bioreactors, a single-use bioreactor technology that incorporates a wheel instead of an impeller. Initially, microcarriers were screened at small scale to assess eCB-MSC attachment and growth and then in bioreactors to assess cell expansion and harvesting. The effect of different donors, serial passaging, and batch versus fed batch were all examined in 0.1 L VW bioreactors. The use of VW bioreactors with an appropriate microcarrier was shown to be able to produce cell densities of up to 1E6 cells/mL, while maintaining cell phenotype and functionality, thus demonstrating great potential for the use of these bioreactors to produce large cell numbers for cell therapies.

MicroRNAs as prognostic markers for chondrogenic differentiation potential of equine mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) are a promising cell source for cartilage tissue regeneration in animals and humans but with large inter-donor variation in their in vitro chondrogenic differentiation potential. Underlying molecular mechanisms responsible for culture-expanded MSC heterogeneity remains poorly understood. In this study, we sought to identify variations in miRNA signatures associated with cultured equine MSC chondrogenic differentiation potential from different donors. Neocartilage tissue generated from equine cord blood-derived MSCs (CB-MSCs) was categorized as having either high or low chondrogenic potential based on their histological appearance and quantification of glycosaminoglycan deposition. Using next-generation sequencing, we identified 30 differentially expressed miRNAs amongst undifferentiated MSC cultures that corresponded with their chondrogenic potential. Of note, MSCs with low chondrogenic potential upregulated miR-146a and miR-487b-3p, which was also observed by qRT-PCR. Our findings suggest that miRNA profiling of equine MSC cultures may have prognostic value in selecting MSC donors with regards to their chondrogenic differentiation potential.

Menstrual blood-derived mesenchymal stromal cells: impact of preconditioning on the cargo of extracellular vesicles as potential therapeutics

BackgroundMesenchymal stromal cells (MSCs) have been shown to exert their therapeutic effects through the secretion of broad spectrum of paracrine factors, including extracellular vesicles (EVs). Accordingly, EVs are being pursued as a promising alternative to cell-based therapies. Menstrual blood-derived stromal cells (MenSCs) are a type of MSC that, due to their immunomodulatory and regenerative properties, have emerged as an innovative source. Additionally, new strategies of cell priming may potentially alter the concentration and cargo of released EVs, leading to modification of their biological properties. In this study, we aimed to characterize the EVs released by MenSCs and compare their therapeutic potential under three different preconditioning conditions (proinflammatory stimuli, physioxia, and acute hypoxia).MethodsMenSCs were isolated from five healthy women. Following culturing to 80% confluence, MenSCs were exposed to different priming conditions: basal (21% O2), proinflammatory stimuli (IFNγ and TNFα, 21% O2), physioxia (1–2% O2), and acute hypoxia (< 1% O2) for 48–72 h. Conditioned media from MenSCs was collected after 48 h and EVs were isolated by a combination of ultra-filtration and differential centrifugation. An extensive characterization ranging from nano-flow cytometry (nFC) to quantitative high-throughput shotgun proteomics was performed. Bioinformatics analyses were used to derive hypotheses on their biological properties.ResultsNo differences in the morphology, size, or number of EVs released were detected between priming conditions. The proteome analysis associated with basal MenSC-EVs prominently revealed their immunomodulatory and regenerative capabilities. Furthermore, quantitative proteomic analysis of differentially produced MenSC-EVs provided sufficient evidence for the utility of the differential preconditioning in purpose-tailoring EVs for their therapeutic application: proinflammatory priming enhanced the anti-inflammatory, regenerative and immunomodulatory capacity in the innate response of EVs, physioxia priming also improves tissue regeneration, angiogenesis and their immunomodulatory capacity targeting on the adaptive response, while acute hypoxia priming, increased hemostasis and apoptotic processes regulation in MenSC-EVs, also by stimulating immunomodulation mainly through the adaptive response.ConclusionsPriming of MenSCs under proinflammatory and hypoxic conditions affected the cargo proteome of EVs released, resulting in different therapeutic potential, and thus warrants experimental exploration with the aim to generate better-defined MSC-derived bioproducts.

First clinical application of cord blood mesenchymal stromal cells in children with multi-drug resistant nephrotic syndrome

Background and objectivesChildren with multi-drug resistant idiopathic nephrotic syndrome (MDR-INS) usually progress to end-stage kidney disease with a consistent risk of disease recurrence after transplantation. New therapeutic options are needed for these patients. Mesenchymal stromal cells (MSCs) are multipotential non-hematopoietic cells with several immunomodulatory properties and growing clinical applications. Cord blood-derived MSC have peculiar anti-inflammatory and immunosuppressive properties. We aimed at assessing safety and efficacy of cord-blood-derived MSCs (CB-MSCs) in children with MDR-INS.Design, setting, participantsProspective, open-label, single arm phase I–II pilot study. Pediatric patients with MDR-INS, resistant to at least two lines of therapy, were enrolled. Allogenic CB-MSCs were administered intravenously on days 0, 14, and 21 at a dose of 1.5 × 106 cells/kg. Patients were followed for at least 12 months. The primary outcomes were safety and toxicity. The secondary outcome was remission at 12 months evaluated by urinary protein/urinary creatinine ratio (uPr/uCr). Circulating regulatory T cells (Tregs) were monitored.ResultsEleven pediatric patients with MDR-INS (10 females, median age 13 years) resistant to a median of 3 previous lines of therapy were enrolled. All patients completed the CB-MSC infusion schedule. No patient experienced any infusion-related adverse event or toxicity. Nine patients were assessable for efficacy. At the 12 months follow-up after the treatment, the median uPr/uCr did not change significantly from baseline (8.13 vs. 9.07; p = 0.98), while 3 patients were in partial or complete remission. A lower baseline uPr/uCr was a predictor of remission (2.55 vs. 8.74; p = 0.0238). Tregs count was not associated with CB-MSCs therapy.ConclusionsCB-MSCs are safe and may have a role in the immunosuppressive therapy of pediatric patients with MDR-INS. This preliminary experience paves the way toward further phase II studies addressing MSC efficacy in immune-mediated kidney diseases.

Possible Effect of the use of Mesenchymal Stromal Cells in the Treatment of Autism Spectrum Disorders: A Review.

Autism spectrum disorder (ASD) represents a set of heterogeneous neurodevelopmental conditions defined by impaired social interactions and repetitive behaviors. The number of reported cases has increased over the past decades, and ASD is now a major public health burden. So far, only treatments to alleviate symptoms are available, with still unmet need for an effective disease treatment to reduce ASD core symptoms. Genetic predisposition alone can only explain a small fraction of the ASD cases. It has been reported that environmental factors interacting with specific inter-individual genetic background may induce immune dysfunctions and contribute to the incidence of ASD. Such dysfunctions can be observed at the central level, with increased microglial cells and activation in ASD brains or in the peripheral blood, as reflected by high circulating levels of pro-inflammatory cytokines, abnormal activation of T-cell subsets, presence of auto-antibodies and of dysregulated microbiota profiles. Altogether, the dysfunction of immune processes may result from immunogenetically-determined inefficient immune responses against a given challenge followed by chronic inflammation and autoimmunity. In this context, immunomodulatory therapies might offer a valid therapeutic option. Mesenchymal stromal cells (MSC) immunoregulatory and immunosuppressive properties constitute a strong rationale for their use to improve ASD clinical symptoms. In vitro studies and pre-clinical models have shown that MSC can induce synapse formation and enhance synaptic function with consequent improvement of ASD-like symptoms in mice. In addition, two preliminary human trials based on the infusion of cord blood-derived MSC showed the safety and tolerability of the procedure in children with ASD and reported promising clinical improvement of core symptoms. We review herein the immune dysfunctions associated with ASD provided, the rationale for using MSC to treat patients with ASD and summarize the current available studies addressing this subject.

Effects of intravenous administration of peripheral blood-derived mesenchymal stromal cells after infusion of lipopolysaccharide in horses.

BackgroundA systemic and dysregulated immune response to infection contributes to morbidity and mortality associated with sepsis. Peripheral blood‐derived mesenchymal stromal cells (PB‐MSC) mitigate inflammation in animal models of sepsis. Allogeneic PB‐MSC administered IV to horses is well‐tolerated but therapeutic benefits are unknown.HypothesisAfter IV lipopolysaccharide (LPS) infusion, horses treated with PB‐MSC would have less severe clinical signs, clinicopathological abnormalities, inflammatory cytokine gene expression, and oxidative stress compared to controls administered a placebo.AnimalsSixteen horses were included in this study.MethodsA randomized placebo‐controlled experimental trial was performed. Sixteen healthy horses were assigned to 1 of 2 treatment groups (1 × 109 PB‐MSC or saline placebo). Treatments were administered 30 minutes after completion of LPS infusion of approximately 30 ng/kg. Clinical signs, clinicopathological variables, inflammatory cytokine gene expression, and oxidative stress markers were assessed at various time points over a 24‐hour period.ResultsA predictable response to IV LPS infusion was observed in all horses. At the dose administered, there was no significant effect of PB‐MSC on clinical signs, clinicopathological variables, or inflammatory cytokine gene expression at any time point. Antioxidant potential was not different between treatment groups, but intracellular ROS increased over time in the placebo group. Other variables that changed over time were likely due to effects of IV LPS infusion.Conclusions and Clinical ImportanceAdministration of allogeneic PB‐MSC did not cause clinically detectable adverse effects in healthy horses. The dose of PB‐MSC used here is unlikely to exert a beneficial effect in endotoxemic horses.

The Proliferation and Stemness of Peripheral Blood-Derived Mesenchymal Stromal Cells Were Enhanced by Hypoxia.

This study aimed to address the dilemma of low peripheral blood-derived mesenchymal stromal cell (PBMSC) activity and reduced phenotype in bone or cartilage tissue engineering. Rat PBMSCs (rPBMSCs) were obtained by density gradient centrifugation, and stromal cell characteristics were confirmed by flow cytometry (FCM) and multi-differentiation potential induction experiments. Cell growth curve, viability experiments, and clone formation experiments were performed by [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (MTS) and cell counting, and the cell cycle was confirmed by cell FCM. The proliferation signal pathway and stemness-related proteins were detected by molecular methods including Western blot and real-time polymerase chain reaction. CD73, CD90, and CD105 were highly expressed, and CD14, CD19, CD34, CD45, and HLA-DR were barely expressed in rPBMSCs. rPBMSCs possessed the potential to differentiate into chondrocytes, adipocytes, and osteoblasts under their respective induction conditions. Cell growth curve and viability experiments were performed under hypoxic conditions: 19% O2, 5% O2, and 1% O2. Specifically, 5% O2 accelerated the proliferation and expression of the stemness of PBMSCs. Cycle experiments proved that hypoxia promoted the cell transition from the G1 phase to the S phase. Molecular experiments confirmed that 5% O2 hypoxia significantly elevated the expressions of hypoxia-inducible factor 1α and β-catenin and simultaneously the expressions of cycle-related genes including CyclinE/CDK2 and stemness-related genes including Nanog and SOX2. The appropriate concentration of hypoxia (i.e., 5% O2) enhanced the proliferation and stemness of rPBMSCs and increased the multidirectional differentiation potential of stromal cells. The proposed culture method could improve the viability and maintain the phenotype of rPBMSCs in cartilage or bone tissue engineering.

Intrapericardial Administration of Secretomes from Menstrual Blood-Derived Mesenchymal Stromal Cells: Effects on Immune-Related Genes in a Porcine Model of Myocardial Infarction

Acute myocardial infarction (AMI) is a manifestation of ischemic heart disease where the immune system plays an important role in the re-establishment of homeostasis. We hypothesize that the anti-inflammatory activity of secretomes from menstrual blood-derived mesenchymal stromal cells (S-MenSCs) and IFNγ/TNFα-primed MenSCs (S-MenSCs*) may be considered a therapeutic option for the treatment of AMI. To assess this hypothesis, we have evaluated the effect of S-MenSCs and S-MenSCs* on cardiac function parameters and the involvement of immune-related genes using a porcine model of AMI. Twelve pigs were randomly divided into three biogroups: AMI/Placebo, AMI/S-MenSCs, and AMI/S-MenSCs*. AMI models were generated using a closed chest coronary occlusion-reperfusion procedure and, after 72 h, the different treatments were intrapericardially administered. Cardiac function parameters were monitored by magnetic resonance imaging before and 7 days post-therapy. Transcriptomic analyses in the infarcted tissue identified 571 transcripts associated with the Gene Ontology term Immune response, of which 57 were differentially expressed when different biogroups were compared. Moreover, a prediction of the interactions between differentially expressed genes (DEGs) and miRNAs from secretomes revealed that some DEGs in the infarction area, such as STAT3, IGFR1, or BCL6 could be targeted by previously identified miRNAs in secretomes from MenSCs. In conclusion, the intrapericardial administration of secretome early after infarction has a significant impact on the expression of immune-related genes in the infarcted myocardium. This confirms the immunomodulatory potential of intrapericardially delivered secretomes and opens new therapeutic perspectives in myocardial infarction treatment.

3D bioassembly of cell-instructive chondrogenic and osteogenic hydrogel microspheres containing allogeneic stem cells for hybrid biofabrication of osteochondral constructs

Recently developed modular bioassembly techniques hold tremendous potential in tissue engineering and regenerative medicine, due to their ability to recreate the complex microarchitecture of native tissue. Here, we developed a novel approach to fabricate hybrid tissue-engineered constructs adopting high-throughput microfluidic and 3D bioassembly strategies. Osteochondral tissue fabrication was adopted as an example in this study, because of the challenges in fabricating load bearing osteochondral tissue constructs with phenotypically distinct zonal architecture. By developing cell-instructive chondrogenic and osteogenic bioink microsphere modules in high-throughput, together with precise manipulation of the 3D bioassembly process, we successfully fabricated hybrid engineered osteochondral tissue in vitro with integrated but distinct cartilage and bone layers. Furthermore, by encapsulating allogeneic umbilical cord blood-derived mesenchymal stromal cells, and demonstrating chondrogenic and osteogenic differentiation, the hybrid biofabrication of hydrogel microspheres in this 3D bioassembly model offers potential for an off-the-shelf, single-surgery strategy for osteochondral tissue repair.

Menstrual blood-derived mesenchymal stromal cells efficiently ameliorate experimental autoimmune encephalomyelitis by inhibiting T cell activation in mice

BackgroundImmunosuppressive properties grant mesenchymal stromal cells (MSCs) promising potential for treating autoimmune diseases. As autologous MSCs suffer from limited availability, the readily available allogeneic MSCs isolated from menstrual blood (MB-MSCs) donated by young, healthy individuals offer great potential. Here, we evaluate the therapeutic potential of MB-MSCs as ready-to-use allo-MSCs in multiple sclerosis, an autoimmune disease developed by the activation of myelin sheath-reactive Th1 and Th17 cells, by application in its animal model experimental autoimmune encephalomyelitis (EAE).MethodsWe assessed the therapeutic effect of MB-MSCs transplanted via either intravenous (i.v.) or intraperitoneal (i.p.) route in EAE in comparison with umbilical cord-derived MSCs (UC-MSCs). We used histology to assess myelin sheath integrity and infiltrated immune cells in CNS and flow cytometry to evaluate EAE-associated inflammatory T cells and antigen-presenting cells in lymphoid organs.ResultsWe observed disease-ameliorating effects of MB-MSCs when transplanted at various stages of EAE (day − 1, 6, 10, and 19), via either i.v. or i.p. route, with a potency comparable to UC-MSCs. We observed reduced Th1 and Th17 cell responses in mice that had received MB-MSCs via either i.v. or i.p. injection. The repressed Th1 and Th17 cell responses were associated with a reduced frequency of plasmacytoid dendritic cells (pDCs) and a suppressed co-stimulatory capacity of pDCs, cDCs, and B cells.ConclusionsOur data demonstrate that the readily available MB-MSCs significantly reduced the disease severity of EAE upon transplantation. Thus, they have the potential to be developed as ready-to-use allo-MSCs in MS-related inflammation.Graphical abstract

Menstrual Blood-Derived Mesenchymal Stromal Cells as a Resource for Regenerative Medicine

Menstrual Blood-Derived Mesenchymal Stromal Cells as a Resource for Regenerative Medicine

A Fibrin Coating Method of Polypropylene Meshes Enables the Adhesion of Menstrual Blood-Derived Mesenchymal Stromal Cells: A New Delivery Strategy for Stem Cell-Based Therapies.

Polypropylene (PP) mesh is well-known as a gold standard of all prosthetic materials of choice for the reinforcement of soft tissues in case of hernia, organ prolapse, and urinary incontinence. The adverse effects that follow surgical mesh implantation remain an unmet medical challenge. Herein, it is outlined a new approach to allow viability and adhesion of human menstrual blood-derived mesenchymal stromal cells (MenSCs) on PP surgical meshes. A multilayered fibrin coating, based on fibrinogen and thrombin from a commercial fibrin sealant, was optimized to guarantee a homogeneous and stratified film on PP mesh. MenSCs were seeded on the optimized fibrin-coated meshes and their adhesion, viability, phenotype, gene expression, and immunomodulatory capacity were fully evaluated. This coating guaranteed MenSC viability, adhesion and did not trigger any change in their stemness and inflammatory profile. Additionally, MenSCs seeded on fibrin-coated meshes significantly decreased CD4+ and CD8+ T cell proliferation, compared to in vitro stimulated lymphocytes (p < 0.0001). Hence, the proposed fibrin coating for PP surgical meshes may allow the local administration of stromal cells and the reduction of the exacerbated inflammatory response following mesh implantation surgery. Reproducible and easy to adapt to other cell types, this method undoubtedly requires a multidisciplinary and translational approach to be improved for future clinical uses.

Latent Tri-lineage Potential of Human Menstrual Blood-Derived Mesenchymal Stromal Cells Revealed by Specific In Vitro Culture Conditions.

Human menstrual blood-derived mesenchymal stromal cells (MenSCs) have become not only an important source of stromal cells for cell therapy but also a cellular source for neurologic disorders in vitro modeling. By using culture protocols originally developed in our laboratory, we show that MenSCs can be converted into floating neurospheres (NSs) using the Fast-N-Spheres medium for 24-72h and can be transdifferentiated into functional dopaminergic-like (DALNs, ~ 26% TH + /DAT + flow cytometry) and cholinergic-like neurons (ChLNs, ~ 46% ChAT + /VAChT flow cytometry) which responded to dopamine- and acetylcholine-triggered neuronal Ca2+ inward stimuli when cultured with the NeuroForsk and the Cholinergic-N-Run medium, respectively in a timely fashion (i.e., 4-7days). Here, we also report a direct transdifferentiation method to induce MenSCs into functional astrocyte-like cells (ALCs) by incubation of MenSCs in commercial Gibco® Astrocyte medium in 7days. The MSC-derived ALCs (~ 59% GFAP + /S100β +) were found to respond to glutamate-induced Ca2+ inward stimuli. Altogether, these results show that MenSCs are a reliable source to obtain functional neurogenic cells to further investigate the neurobiology of neurologic disorders.

A Chemically Defined, Xeno- and Blood-Free Culture Medium Sustains Increased Production of Small Extracellular Vesicles From Mesenchymal Stem Cells.

Cell therapy is witnessing a notable shift toward cell-free treatments based on paracrine factors, in particular, towards small extracellular vesicles (sEV), that mimic the functional effect of the parental cells. While numerous sEV-based applications are currently in advanced preclinical stages, their promised translation depends on overcoming the manufacturing hurdles posed by the large-scale production of purified sEV. Unquestionably, the culture medium used with the parental cells plays a key role in the sEV’s secretion rate and content. An essential requisite is the use of a serum-, xeno-, and blood-free medium to meet the regulatory entity requirements of clinical-grade sEV’s production. Here, we evaluated OxiumTMEXO, a regulatory complying medium, with respect to production capacity and conservation of the EV’s characteristics and functionality and the parental cell’s phenotype and viability. A comparative study was established with standard DMEM and a commercially available culture medium developed specifically for sEV production. Under similar conditions, OxiumTMEXO displayed a three-fold increase of sEV secretion, with an enrichment of particles ranging between 51 and 200 nm. These results were obtained through direct quantification from the conditioned medium to avoid the isolation method’s interference and variability and were compared to the two culture media under evaluation. The higher yield obtained was consistent with several harvest time points (2, 4, and 6 days) and different cell sources, incluiding umbilical cord-, menstrual blood-derived mesenchymal stromal cells and fibroblasts. Additionally, the stem cell phenotype and viability of the parental cell remained unchanged. Furthermore, OxiumTMEXO-sEV showed a similar expression pattern of the vesicular markers CD63, CD9, and CD81, with respect to sEV derived from the other conditions. The in vitro internalization assays in different target cell types and the pharmacokinetic profile of intraperitoneally administered sEV in vivo indicated that the higher EV production rate did not affect the uptake kinetics or the systemic biodistribution in healthy mice. In conclusion, the OxiumTMEXO medium sustains an efficient and robust production of large quantities of sEV, conserving the classic functional properties of internalization into acceptor target cells and biodistribution in vivo, supplying the amount and quality of EVs for the development of cell-free therapies.

Reduction of Graft-versus-Host-Disease in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) Mice by Cotransplantation of Syngeneic Human Umbilical Cord-Derived Mesenchymal Stromal Cells

Graft-versus-host disease (GVHD) is one of the major complications following hematopoietic stem cell transplantation, which remains the sole curative therapy for many malignant diseases of the hematopoietic system. The immunomodulatory potential of mesenchymal stromal cells (MSCs) to treat GVHD is currently being tested in various preclinical and clinical trials. Because the results of the preclinical and clinical trials on the use of MSCs to treat GVHD have not been consistent, we analyzed the potential beneficial effects of syngeneic versus allogenic treatment, culture expansion of MSCs, and various MSC cell doses and time points of MSC transplantation in a murine GVHD model. We established the murine GVHD model based on the transplantation of umbilical cord blood-derived hematopoietic stem cells (UC-HSCs) and used this model to assess the therapeutic potential of umbilical cord blood-derived MSCs (UC-MSCs). The use of HSC and MSC populations derived from the same donor allowed us to exclude third-party cells and test the UC-HSCs and UC-MSCs in a matched setting. Moreover, we were able to compare various doses, transplantation time points, and the influence of culture expansion of MSCs on the impact of treatment. This resulted in 16 different treatment groups. The most efficient setting for treatment of UC-HSC-induced GVHD reactions was based on the simultaneous administration of 1×106 culture-expanded, syngeneically matched UC-MSCs. This therapy effectively reduced the number of CD8+ T cells in the blood, protected the mice from weight loss, and prolonged their survival until the end of observation period. Taken together, our data show beneficial effects of (1) syngeneic over allogeneic UC-HSCs and UC-MSCs, (2) culture-expanded cells over freshly isolated primary cells, (3) simultaneous over sequential administration, and (4) high doses of UC-MSCs. The animal model of GVHD established here is now available for more detailed studies, including a comparative analysis of the efficacy of MSCs derived from alternative sources, such as adipose tissue and bone marrow.

Intrinsic Angiogenic Potential and Migration Capacity of Human Mesenchymal Stromal Cells Derived from Menstrual Blood and Bone Marrow.

Several therapies are being developed to increase blood circulation in ischemic tissues. Despite bone marrow-derived mesenchymal stromal cells (bmMSC) are still the most studied, an interesting and less invasive MSC source is the menstrual blood, which has shown great angiogenic capabilities. Therefore, the aim of this study was to evaluate the angiogenic properties of menstrual blood-derived mesenchymal stromal cells (mbMSC) in vitro and in vivo and compared to bmMSC. MSC’s intrinsic angiogenic capacity was assessed by sprouting and migration assays. mbMSC presented higher invasion and longer sprouts in 3D culture. Additionally, both MSC-spheroids showed cells expressing CD31. mbMSC and bmMSC were able to migrate after scratch wound in vitro, nonetheless, only mbMSC demonstrated ability to engraft in the chick embryo, migrating to perivascular, perineural, and chondrogenic regions. In order to study the paracrine effects, mbMSC and bmMSC conditioned mediums were capable of stimulating HUVEC’s tube-like formation and migration. Both cells expressed VEGF-A and FGF2. Meanwhile, PDGF-B was expressed exclusively in mbMSC. Our results indicated that mbMSC and bmMSC presented a promising angiogenic potential. However, mbMSC seems to have additional advantages since it can be obtained by non-invasive procedure and expresses PDGF-B, an important molecule for vascular formation and remodeling.

De novo Vessel Formation Through Cross-Talk of Blood-Derived Cells and Mesenchymal Stromal Cells in the Absence of Pre-existing Vascular Structures.

BackgroundThe generation of functional blood vessels remains a key challenge for regenerative medicine. Optimized in vitro culture set-ups mimicking the in vivo perivascular niche environment during tissue repair may provide information about the biological function and contribution of progenitor cells to postnatal vasculogenesis, thereby enhancing their therapeutic potential.AimWe established a fibrin-based xeno-free human 3D in vitro vascular niche model to study the interaction of mesenchymal stromal cells (MSC) with peripheral blood mononuclear cells (PBMC) including circulating progenitor cells in the absence of endothelial cells (EC), and to investigate the contribution of this cross-talk to neo-vessel formation.Materials and MethodsBone marrow-derived MSC were co-cultured with whole PBMC, enriched monocytes (Mo), enriched T cells, and Mo together with T cells, respectively, obtained from leukocyte reduction chambers generated during the process of single-donor platelet apheresis. Cells were embedded in 3D fibrin matrices, using exclusively human-derived culture components without external growth factors. Cytokine secretion was analyzed in supernatants of 3D cultures by cytokine array, vascular endothelial growth factor (VEGF) secretion was quantified by ELISA. Cellular and structural re-arrangements were characterized by immunofluorescence and confocal laser-scanning microscopy of topographically intact 3D fibrin gels.Results3D co-cultures of MSC with PBMC, and enriched Mo together with enriched T cells, respectively, generated, within 2 weeks, complex CD31+/CD34+ vascular structures, surrounded by basement membrane collagen type-IV+ cells and matrix, in association with increased VEGF secretion. PBMC contained CD31+CD34+CD45dimCD14– progenitor-type cells, and EC of neo-vessels were PBMC-derived. Vascular structures showed intraluminal CD45+ cells that underwent apoptosis thereby creating a lumen. Cross-talk of MSC with enriched Mo provided a pro-angiogenic paracrine environment. MSC co-cultured with enriched T cells formed “cell-in-cell” structures generated through internalization of T cells by CD31+CD45dim⁣/– cells. No vascular structures were detected in co-cultures of MSC with either Mo or T cells.ConclusionOur xeno-free 3D in vitro vascular niche model demonstrates that a complex synergistic network of cellular, extracellular and paracrine cross-talk can contribute to de novo vascular development through self-organization via co-operation of immune cells with blood-derived progenitor cells and MSC, and thereby may open a new perspective for advanced vascular tissue engineering in regenerative medicine.

Human Adipose Tissue-Derived Stromal Cells Suppress Human, but Not Murine Lymphocyte Proliferation, via Indoleamine 2,3-Dioxygenase Activity.

Over recent years, mesenchymal stromal cells (MSC) have gained immense attraction in immunotherapy, regenerative medicine and tissue engineering. MSC microenvironment modulation occurs through synergy of direct cell–cell contact, and secreted soluble factors and extracellular vesicles (EV). MSC-derived EV have been suggested as cell-free immunomodulatory alternative to MSC; however, previous findings have challenged this. Furthermore, recent data suggest that evaluating the mechanism of action of human MSC (hMSC) in animal models might promote adverse immune reactions or lack of functionality due to xeno-incompatibilities. In this study, we first assessed the immunomodulatory strength of different human MSC sources on in vitro stimulated T cells and compared this to interferon-gamma (IFNγ) primed MSC conditioned medium (CM) and EV. Second, we addressed the main molecular mechanisms, and third, we assessed the MSC in vitro immunosuppressive effect across interspecies barriers. We identified human adipose tissue-derived stromal cells (ASC) with strongest immunomodulatory strength, followed by bone marrow (BM) and cord blood-derived MSC (CB). Whilst CM from primed ASC managed to exert analogous effects as their cellular counterpart, EV derived thereof did not, reproducing previous findings. IFNγ-induced indoleamine 2,3-dioxygenase (IDO) activity was identified as key mechanism to suppress human lymphocyte proliferation, as in the presence of the IDO inhibitor epacadostat (Epac) a stimulation of proliferation was seen. In addition, we revealed MSC immunosuppressive effects to be species-specific, because human cells failed to suppress murine lymphocyte proliferation. In summary, ASC were the strongest immunomodulators with the IDO-kynurenine pathway being key within the human system. Importantly, the in vitro lack of interspecies immunomodulatory strength suggests that preclinical data need to be carefully interpreted especially when considering a possible translation to clinical field.

Umbilical cord blood-derived mesenchymal stromal cells promote myeloid-derived suppressor cell proliferation by secreting HLA-G to reduce acute graft-versus-host disease after hematopoietic stem cell transplantation

Background aimsMesenchymal stem cells (MSCs) use multiple mechanisms to constrain both innate and adaptive immune responses to prevent graft-versus-host disease (GVHD). Myeloid-derived suppressor cells (MDSCs), as a heterogeneous population of early myeloid progenitor cells originating from bone marrow, are a naturally occurring immune regulatory population associated with inhibition of ongoing inflammatory responses, indicating their potential for GVHD therapy. There is accumulating evidence that MSCs and MDSCs do not act independently, but rather establish crosstalk. However, the role of MSCs in MDSC expansion and activation in GVHD remains unexplored. MethodsIn vitro experiments included 2 groups: peripheral blood mononuclear cells (PBMCs) after mobilization and human umbilical cord blood-derived MSCs (UCB-MSCs) co-cultured with PBMCs. The number and functional difference of MDSCs in PBMCs were determined by flow cytometry. The culture supernatants of co-cultured cells were analyzed to identify cytokines involved in MDSC proliferation. The relationship between MSCs and MDSCs was clarified in GVHD and graft-versus-leukemia (GVL) animal models. ResultsIn vitro experiments confirmed that UCB-MSCs secreted HLA-G protein to promote and maintain the proliferation of MDSCs in peripheral blood after granulocyte colony-stimulating factor mobilization, and UCB-MSCs mediated the function of MDSCs to inhibit the proliferation of T cells and promote the proliferation of regulatory T cells. UCB-MSCs overexpressing HLA-G induced MDSC production in recipient mice, improved the ability of MDSCs to suppress T cells and further reduced acute GVHD (aGVHD) symptoms and survival time without influencing GVL effects. ConclusionsUCB-MSCs expanded MDSCs via HLA-G/Ig-like transcript 4, reducing the severity of aGVHD without affecting GVL. The immunosuppressive potential of MSCs for the treatment of aGVHD significantly affects the development of MDSCs, thereby consolidating the position of MSCs in the prevention and treatment of aGVHD.