Effects of Dietary Supplementation with a Ferulic Acid-Rich Bioactive Component of Wheat Bran in a Murine Model of Graft-Versus-Host Disease.

Graft-versus-host disease (GvHD) is a common and severe complication following allogeneic hematopoietic stem cell transplantation (HSCT). Its prevention and treatment is a major challenge. Ferulic acid (FA) has anti-inflammatory and antioxidant properties that could be attractive in this setting. Our aim was to evaluate a bioactive ingredient derived from wheat bran (WB), selected for its high concentration of FA, in a murine model of GvHD. The ingredient was obtained via a bioprocess involving hydrolysis and spray-drying. GvHD was induced via HSCT between MHC-mismatched mouse strains. FA treatment was administered orally. Survival and disease scores (weight loss, hunching, activity, fur texture, and skin integrity, each scored between 0 and 2 depending on disease severity) were recorded daily, histological evaluation was performed at the end of the experiment, and serum inflammatory cytokines were analyzed on days 9 and 28. Treatment with FA did not protect GvHD mice from death, nor did it diminish GvHD scores. However, histological analysis showed that ulcers with large areas of inflammatory cells, vessels, and keratin were less common in skin samples from FA-treated mice. Areas of intense inflammatory response were also seen in fewer small intestine samples from treated mice. In addition, a slight decrease in INF-γ and TNF-α expression was observed in the serum of treated mice on day 28. The results showed some local effect of the ingredient intervention, but that the dose used may not be sufficient to control or reduce the inflammatory response at the systemic level in mice with GvHD. Higher dosages of FA may have an impact when evaluating the immunomodulatory capabilities of the hydrolyzed WB ingredient. Thus, further experiments and the use of technological strategies that enrich the ingredients in soluble ferulic acid to improve its efficacy in this setting are warranted.

Therapeutic potential of mesenchymal stromal/stem cells in critical-care patients with systemic inflammatory response syndrome.

BACKGROUND: Despite notable advances in the support and treatment of patients admitted to the intensive care unit (ICU), the management of those who develop a systemic inflammatory response syndrome (SIRS) still constitutes an unmet medical need. MAIN BODY: Both the initial injury (trauma, pancreatitis, infections) and the derived uncontrolled response promote a hyperinflammatory status that leads to systemic hypotension, tissue hypoperfusion and multiple organ failure. Mesenchymal stromal/stem cells (MSCs) are emerging as a potential therapy for severe ICU patients due to their potent immunomodulatory, anti-inflammatory, regenerative and systemic homeostasis-regulating properties. MSCs have demonstrated clinical benefits in several inflammatory-based diseases, but their role in SIRS needs to be further explored. CONCLUSION: In the current review, after briefly overviewing SIRS physiopathology, we explore the potential mechanisms why MSC therapy could aid in the recovery of this condition and the pre-clinical and early clinical evidence generated to date.

Uncovering perturbations in human hematopoiesis associated with healthy aging and myeloid malignancies at single-cell resolution.

Early hematopoiesis is a continuous process in which hematopoietic stem and progenitor cells (HSPCs) gradually differentiate toward specific lineages. Aging and myeloid malignant transformation are characterized by changes in the composition and regulation of HSPCs. In this study, we used single-cell RNA sequencing (scRNA-seq) to characterize an enriched population of human HSPCs obtained from young and elderly healthy individuals.Based on their transcriptional profile, we identified changes in the proportions of progenitor compartments during aging, and differences in their functionality, as evidenced by gene set enrichment analysis. Trajectory inference revealed that altered gene expression dynamics accompanied cell differentiation, which could explain aging-associated changes in hematopoiesis. Next, we focused on key regulators of transcription by constructing gene regulatory networks (GRNs) and detected regulons that were specifically active in elderly individuals. Using previous findings in healthy cells as a reference, we analyzed scRNA-seq data obtained from patients with myelodysplastic syndrome (MDS) and detected specific alterations of the expression dynamics of genes involved in erythroid differentiation in all patients with MDS such as TRIB2. In addition, the comparison between transcriptional programs and GRNs regulating normal HSPCs and MDS HSPCs allowed identification of regulons that were specifically active in MDS cases such as SMAD1, HOXA6, POU2F2, and RUNX1 suggesting a role of these transcription factors (TFs) in the pathogenesis of the disease.In summary, we demonstrate that the combination of single-cell technologies with computational analysis tools enable the study of a variety of cellular mechanisms involved in complex biological systems such as early hematopoiesis and can be used to dissect perturbed differentiation trajectories associated with perturbations such as aging and malignant transformation. Furthermore, the identification of abnormal regulatory mechanisms associated with myeloid malignancies could be exploited for personalized therapeutic approaches in individual patients.

Our blood contains many different types of cells; red blood cells carry oxygen through the body, platelets help to stop bleeding and a variety of white blood cells fight infections. All of these critical components come from a pool of immature cells in bone marrow, which can develop and specialise into any of these. However, as we get older, these immature cells can accumulate damage, including mutations in specific genes. This increases the risk of diseases such as myelodysplastic syndromes (MDS), a type of cancer in which the cells cannot develop and the patient does not have enough healthy mature blood cells. The changes in gene activity in the immature cells have previously been studied using samples from young and elderly people, as well as individuals with MDS. These studies examined large numbers of cells together, revealing differences between young and elderly people, and individuals with MDS. However, this does not describe how the different types alter their behaviour. To address this, Ainciburu, Ezponda et al. used a technique called single-cell RNA sequencing to study the gene activity in individual immature blood cells. This revealed changes associated with maturation that may account for the different combinations of cell populations in younger and older people. The results confirmed findings from previous studies and suggested new genes involved in ageing or MDS. Ainciburu, Ezponda et al. used these results to create an analytical system that highlights gene activity differences in individual MDS patients that are independent of age-related changes. These results provide new insights that could help further research into the development of MDS and the ageing process. In addition, scientists could study other diseases using this approach of analysing individual patients' gene activity. In future, this could help to personalise clinical decisions on diagnosis and treatment.

The transcription factor DDIT3 is a potential driver of dyserythropoiesis in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are hematopoietic stem cell (HSC) malignancies characterized by ineffective hematopoiesis, with increased incidence in older individuals. Here we analyze the transcriptome of human HSCs purified from young and older healthy adults, as well as MDS patients, identifying transcriptional alterations following different patterns of expression. While aging-associated lesions seem to predispose HSCs to myeloid transformation, disease-specific alterations may trigger MDS development. Among MDS-specific lesions, we detect the upregulation of the transcription factor DNA Damage Inducible Transcript 3 (DDIT3). Overexpression of DDIT3 in human healthy HSCs induces an MDS-like transcriptional state, and dyserythropoiesis, an effect associated with a failure in the activation of transcriptional programs required for normal erythroid differentiation. Moreover, DDIT3 knockdown in CD34+ cells from MDS patients with anemia is able to restore erythropoiesis. These results identify DDIT3 as a driver of dyserythropoiesis, and a potential therapeutic target to restore the inefficient erythroid differentiation characterizing MDS patients.

Mesenchymal Stromal Cells Combined With Elastin-Like Recombinamers Increase Angiogenesis In Vivo After Hindlimb Ischemia.

Hindlimb ischemia is an unmet medical need, especially for those patients unable to undergo vascular surgery. Cellular therapy, mainly through mesenchymal stromal cell (MSC) administration, may be a potentially attractive approach in this setting. In the current work, we aimed to assess the potential of the combination of MSCs with a proangiogenic elastin-like recombinamer (ELR)-based hydrogel in a hindlimb ischemia murine model. Human bone marrow MSCs were isolated from four healthy donors, while ELR biomaterials were genetically engineered. Hindlimb ischemia was induced through ligation of the right femoral artery, and mice were intramuscularly injected with ELR biomaterial, 0.5 × 106 MSCs or the combination, and also compared to untreated animals. Tissue perfusion was monitored using laser Doppler perfusion imaging. Histological analysis of hindlimbs was performed after hematoxylin and eosin staining. Immunofluorescence with anti-human mitochondria antibody was used for human MSC detection, and the biomaterial was detected by elastin staining. To analyze the capillary density, immunostaining with an anti-CD31 antibody was performed. Our results show that the injection of MSCs significantly improves tissue reperfusion from day 7 (p = 0.0044) to day 21 (p = 0.0216), similar to the infusion of MSC + ELR (p = 0.0038, p = 0.0014), without significant differences between both groups. After histological evaluation, ELR hydrogels induced minimal inflammation in the injection sites, showing biocompatibility. MSCs persisted with the biomaterial after 21 days, both in vitro and in vivo. Finally, we observed a higher blood vessel density when mice were treated with MSCs compared to control (p<0.0001), but this effect was maximized and significantly different to the remaining experimental conditions when mice were treated with the combination of MSCs and the ELR biomaterial (p < 0.0001). In summary, the combination of an ELR-based hydrogel with MSCs may improve the angiogenic effects of both strategies on revascularization of ischemic tissues.

Eltrombopag increases the hematopoietic supporting ability of mesenchymal stem/stromal cells.

Background: Eltrombopag (EP) is a small molecule that acts directly on hematopoietic stem cells (HSCs) and megakaryocytes to stimulate the hematopoietic process. Mesenchymal stem/stromal cells (MSCs) are key hematopoietic niche regulators. Objectives: We aimed to determine whether EP has any effect on MSC function and properties (especially on their hematopoietic-supporting ability) and if so, what changes (e.g. genome-wide transcriptomic alterations) are induced in MSC after EP treatment. Design/Methods: MSCs were isolated from 12 healthy donors and treated with 15 µM and 50 µM of EP for 24 h. The toxicity of the drug on MSCs and their differentiation ability were analyzed, as well as the transcriptomic profile, reactive oxygen species (ROS) and DNA damage and the changes induced in the clonogenic capacity of HSCs. Results: The results show that EP also modifies MSC functions, decreasing their adipogenic differentiation, increasing the expression of genes involved in hypoxia and other pathways related to oxygen homeostasis, and enhancing their ability to support hematopoiesis in vitro. Conclusion: Our findings support the use of EP in cases where hematopoiesis is defective, despite its well-known direct effects on hematopoietic cells. Our findings suggest that further studies on the effects of EP on MSCs from patients with aplastic anemia are warranted.

Co-administration of human MSC overexpressing HIF-1α increases human CD34+ cell engraftment in vivo.

BACKGROUND: Poor graft function or graft failure after allogeneic stem cell transplantation is an unmet medical need, in which mesenchymal stromal cells (MSC) constitute an attractive potential therapeutic approach. Hypoxia-inducible factor-1α (HIF-1α) overexpression in MSC (HIF-MSC) potentiates the angiogenic and immunomodulatory properties of these cells, so we hypothesized that co-transplantation of MSC-HIF with CD34+ human cord blood cells would also enhance hematopoietic stem cell engraftment and function both in vitro and in vivo. METHODS: Human MSC were obtained from dental pulp. Lentiviral overexpression of HIF-1α was performed transducing cells with pWPI-green fluorescent protein (GFP) (MSC WT) or pWPI-HIF-1α-GFP (HIF-MSC) expression vectors. Human cord blood CD34+ cells were co-cultured with MSC WT or HIF-MSC (4:1) for 72 h. Then, viability (Annexin V and 7-AAD), cell cycle, ROS expression and immunophenotyping of key molecules involved in engraftment (CXCR4, CD34, ITGA4, c-KIT) were evaluated by flow cytometry in CD34+ cells. In addition, CD34+ cells clonal expansion was analyzed by clonogenic assays. Finally, in vivo engraftment was measured by flow cytometry 4-weeks after CD34+ cell transplantation with or without intrabone MSC WT or HIF-MSC in NOD/SCID mice. RESULTS: We did not observe significant differences in viability, cell cycle and ROS expression between CD34+ cells co-cultured with MSC WT or HIF-MSC. Nevertheless, a significant increase in CD34, CXCR4 and ITGA4 expression (p = 0.009; p = 0.001; p = 0.013, respectively) was observed in CD34+ cells co-cultured with HIF-MSC compared to MSC WT. In addition, CD34+ cells cultured with HIF-MSC displayed a higher CFU-GM clonogenic potential than those cultured with MSC WT (p = 0.048). We also observed a significant increase in CD34+ cells engraftment ability when they were co-transplanted with HIF-MSC compared to CD34+ co-transplanted with MSC WT (p = 0.016) or alone (p = 0.015) in both the injected and contralateral femurs (p = 0.024, p = 0.008 respectively). CONCLUSIONS: Co-transplantation of human CD34+ cells with HIF-MSC enhances cell engraftment in vivo. This is probably due to the ability of HIF-MSC to increase clonogenic capacity of hematopoietic cells and to induce the expression of adhesion molecules involved in graft survival in the hematopoietic niche.

MSCs from polytrauma patients: preliminary comparative study with MSCs from elective-surgery patients.

BACKGROUND: Polytrauma is a major clinical problem due to its impact on morbidity and mortality, especially among the younger population. Its pathophysiology is not completely elucidated, and the study of the involvement of certain cell populations with therapeutic potential, such as mesenchymal stromal cells (MSCs), is an area of growing interest, as mesenchymal cells have anti-inflammatory, immunoregulatory, and osteogenic potential. METHODS: In the present preliminary work, we have evaluated the characteristics of MSCs in terms of proliferation, immunophenotype, cell cycle, clonogenic capacity, and multilineage differentiation ability in a series of 18 patients with polytrauma and compared them to those from otherwise healthy patients undergoing elective spinal surgery. RESULTS: MSCs from polytrauma patients displayed higher proliferative potential with significantly higher cumulative population doublings, increased expression of some important cell adhesion molecules (CD105, CD166), and an early pre-osteogenic differentiation ability compared to those of the control group. CONCLUSIONS: MSCs could potentially be of help in the repair process of polytrauma patients contribute to both cell-tissue repair and anti-inflammatory response. This potential should be further explored in larger studies.

CRISPR-Cas9 Technology as a Tool to Target Gene Drivers in Cancer: Proof of Concept and New Opportunities to Treat Chronic Myeloid Leukemia.

Chronic myeloid leukemia (CML) is a hematopoietic malignancy produced by a unique oncogenic event involving the constitutively active tyrosine-kinase (TK) BCR/ABL1. TK inhibitors (TKI) changed its prognosis and natural history. Unfortunately, ABL1 remains unaffected by TKIs. Leukemic stem cells (LSCs) remain, and resistant mutations arise during treatment. To address this problem, we have designed a therapeutic CRISPR-Cas9 deletion system targeting BCR/ABL1. The system was efficiently electroporated to cell lines, LSCs from a CML murine model, and LSCs from CML patients at diagnosis, generating a specific ABL1 null mutation at high efficiency and allowing the edited leukemic cells to be detected and tracked. The CRISPR-Cas9 deletion system triggered cell proliferation arrest and apoptosis in murine and human CML cell lines. Patient and murine-derived xenografts with CRISPR-edited LSCs in NOD SCID gamma niches revealed that normal multipotency and repopulation ability of CRISPR edited LSCs were fully restored. Normal hematopoiesis was restored, avoiding myeloid bias. To the best of our knowledge, we show for the first time how a CRISPR-Cas9 deletion system efficiently interrupts BCR/ABL1 oncogene in primary LSCs to bestow a therapeutic benefit. This study is a proof of concept for genome editing in all those diseases, like CML, sustained by a single oncogenic event, opening up new therapeutic opportunities.

Targeting aberrant DNA methylation in mesenchymal stromal cells as a treatment for myeloma bone disease.

Multiple myeloma (MM) progression and myeloma-associated bone disease (MBD) are highly dependent on bone marrow mesenchymal stromal cells (MSCs). MM-MSCs exhibit abnormal transcriptomes, suggesting the involvement of epigenetic mechanisms governing their tumor-promoting functions and prolonged osteoblast suppression. Here, we identify widespread DNA methylation alterations of bone marrow-isolated MSCs from distinct MM stages, particularly in Homeobox genes involved in osteogenic differentiation that associate with their aberrant expression. Moreover, these DNA methylation changes are recapitulated in vitro by exposing MSCs from healthy individuals to MM cells. Pharmacological targeting of DNMTs and G9a with dual inhibitor CM-272 reverts the expression of hypermethylated osteogenic regulators and promotes osteoblast differentiation of myeloma MSCs. Most importantly, CM-272 treatment prevents tumor-associated bone loss and reduces tumor burden in a murine myeloma model. Our results demonstrate that epigenetic aberrancies mediate the impairment of bone formation in MM, and its targeting by CM-272 is able to reverse MBD.

Improving hematopoietic engraftment: Potential role of mesenchymal stromal cell-derived extracellular vesicles.

The therapeutic effects of mesenchymal stromal cells (MSCs) in graft failure or poor graft function after allogenic hematopoietic stem cell transplantation (HSCT) are currently undergoing clinical evaluation. MSCs exert their functions, at least partially, through the secretion of extracellular vesicles (MSC-EVs). The available information on the biological potential of MSC-EVs to improve hematopoietic function, both in in vitro studies and in reported preclinical models, focusing on the possible mechanisms of these effects are summarized in the current review. The potential advantages of EVs over MSCs are also discussed, as well as the limitations and uncertainties in terms of isolation, characterization, mechanism of action in this setting, and industrial scalability that should be addressed for their potential clinical application.

Deferasirox reduces oxidative DNA damage in bone marrow cells from myelodysplastic patients and improves their differentiation capacity.

Patients with low-risk myelodysplastic syndromes (MDS) usually develop iron overload. This leads to a high level of oxidative stress in the bone marrow (BM) and increases haematopoietic cell dysfunction. Our objective was to analyse whether chelation with deferasirox (DFX) alleviates the consequences of oxidative stress and improves BM cell functionality. We analysed 13 iron-overloaded MDS patients' samples before and 4-10 months after treatment with DFX. Using multiparametric flow cytometry analysis, we measured intracellular reactive oxygen species (ROS), DNA oxidation and double strand breaks. Haematopoietic differentiation capacity was analysed by colony-forming unit (CFU) assays. Compared to healthy donors, MDS showed a higher level of intracellular ROS and DNA oxidative damage in BM cells. DNA oxidative damage decreased following DFX treatment. Furthermore, the clonogenic assays carried out before treatment suggest an impaired haematopoietic differentiation. DFX seems to improve this capacity, as illustrated by a decreased cluster/CFU ratio, which reached values similar to controls. We conclude that BM cells from MDS are subject to higher oxidative stress conditions and show an impaired haematopoietic differentiation. These adverse features seem to be partially rectified after DFX treatment.

The Incorporation of Extracellular Vesicles from Mesenchymal Stromal Cells Into CD34+ Cells Increases Their Clonogenic Capacity and Bone Marrow Lodging Ability.

Mesenchymal stromal cells (MSC) may exert their functions by the release of extracellular vesicles (EV). Our aim was to analyze changes induced in CD34+ cells after the incorporation of MSC-EV. MSC-EV were characterized by flow cytometry (FC), Western blot, electron microscopy, and nanoparticle tracking analysis. EV incorporation into CD34+ cells was confirmed by FC and confocal microscopy, and then reverse transcription polymerase chain reaction and arrays were performed in modified CD34+ cells. Apoptosis and cell cycle were also evaluated by FC, phosphorylation of signal activator of transcription 5 (STAT5) by WES Simple, and clonal growth by clonogenic assays. Human engraftment was analyzed 4 weeks after CD34+ cell transplantation in nonobese diabetic/severe combined immunodeficient mice. Our results showed that MSC-EV incorporation induced a downregulation of proapoptotic genes, an overexpression of genes involved in colony formation, and an activation of the Janus kinase (JAK)-STAT pathway in CD34+ cells. A significant decrease in apoptosis and an increased CD44 expression were confirmed by FC, and increased levels of phospho-STAT5 were confirmed by WES Simple in CD34+ cells with MSC-EV. In addition, these cells displayed a higher colony-forming unit granulocyte/macrophage clonogenic potential. Finally, the in vivo bone marrow lodging ability of human CD34+ cells with MSC-EV was significantly increased in the injected femurs. In summary, the incorporation of MSC-EV induces genomic and functional changes in CD34+ cells, increasing their clonogenic capacity and their bone marrow lodging ability. Stem Cells 2019;37:1357-1368.

Autologous mesenchymal stromal cells embedded in tricalcium phosphate for posterolateral spinal fusion: results of a prospective phase I/II clinical trial with long-term follow-up.

BACKGROUND: Posterolateral spinal fusion with autologous bone graft is considered the "gold standard" for lumbar degenerative disc disease (DDD) when surgical treatment is indicated. The potential role of mesenchymal stromal cells (MSCs) to replace the bone graft in this setting has not been fully addressed. OBJECTIVE: To analyze the safety, feasibility and potential clinical efficacy of the implantation of autologous MSCs embedded with tricalcium phosphate as a therapeutic alternative to bone graft in patients with DDD during posterolateral spine fusion. STUDY DESIGN: Phase I/II single-arm prospective clinical trial. METHODS: Eleven patients with monosegmental DDD at L4-L5 or L5-S1 level were included. Autologous bone marrow-derived MSC were expanded in our Good Manufacturing Practice (GMP) Facility and implanted during spinal surgery embedded in a tricalcium phosphate carrier. Monitoring of patients included a postoperative period of 12 months with four visits (after the 1st, 3rd, 6th, and 12th month), with clinical and radiological assessment that included the visual analog scale (VAS), the Oswestry disability index (ODI), the Short-Form Health Survey (SF-36), the vertebral fusion grade observed through a simple Rx, and the evaluation of possible complications or adverse reactions. In addition, all patients were further followed up to 5 years for outcome. RESULTS: Median age of patients included was 44 years (range 30-58 years), and male/female ratio was (6/5) L4-L5 and L5-S1 DDD was present five and six patients, respectively. Autologous MSCs were expanded in all cases. There were no adverse effects related to cell implantation. Regarding efficacy, both VAS and ODI scores improved after surgery. Radiologically, 80% of patients achieved lumbar fusion at the end of the follow-up. No adverse effects related to the procedure were recorded. CONCLUSIONS: The use of autologous MSCs for spine fusion in patients with monosegmental degenerative disc disease is feasible, safe, and potentially effective. TRIAL REGISTRATION: no. EudraCT: 2010-018335-17 ; code Identifier: NCT01513694 ( clinicaltrials.gov ).

Human Bone Marrow Mesenchymal Stromal Cells Promote Bone Regeneration in a Xenogeneic Rabbit Model: A Preclinical Study.

Significant research efforts have been undertaken during the last decades to treat musculoskeletal disorders and improve patient's mobility and quality of life. The goal is the return of function as quickly and completely as possible. Cellular therapy has been increasingly employed in this setting. The design of this study was focused on cell-based alternatives. The present study aimed at investigating the bone regeneration capacity of xenogeneic human bone marrow-derived mesenchymal stromal cell (hMSC) implantation with tricalcium phosphate (TCP) granules in an immunocompetent rabbit model of critical-size bone defects at the femoral condyles. Two experimental groups, TCP and hMSC + TCP, were compared. Combination of TCP and hMSC did not affect cell viability or osteogenic differentiation. We also observed significantly higher bone regeneration in vivo in the hMSC + TCP group, which also displayed better TCP osteointegration. Also, evidence of hMSC contribution to a better TCP osteointegration was noticed. Finally, no inflammatory reaction was detected, besides the xenotransplantation of human cells into an immunocompetent recipient. In summary, hMSC combined with TCP granules is a potential combination for bone regeneration purposes that provides better preclinical results compared to TCP alone.

Biocompatibility of two model elastin-like recombinamer-based hydrogels formed through physical or chemical cross-linking for various applications in tissue engineering and regenerative medicine.

Biocompatibility studies, especially innate immunity induction, in vitro and in vivo cytotoxicity, and fibrosis, are often lacking for many novel biomaterials including recombinant protein-based ones, such as elastin-like recombinamers (ELRs), and has not been extensively explored in the scientific literature, in contrast to traditional biomaterials. Herein, we present the results from a set of experiments designed to elucidate the preliminary biocompatibility of 2 types of ELRs that are able to form extracellular matrix-like hydrogels through either physical or chemical cross-linking both of which are intended for different applications in tissue engineering and regenerative medicine. Initially, we present in vitro cytocompatibility results obtained upon culturing human umbilical vein endothelial cells on ELR substrates, showing optimal proliferation up to 9 days. Regarding in vivo cytocompatibility, luciferase-expressing hMSCs were viable for at least 4 weeks in terms of bioluminescence emission when embedded in ELR hydrogels and injected subcutaneously into immunosuppressed mice. Furthermore, both types of ELR-based hydrogels were injected subcutaneously in immunocompetent mice and serum TNFα, IL-1ß, IL-4, IL-6, and IL-10 concentrations were measured by enzyme-linked immunosorbent assay, confirming the lack of inflammatory response, as also observed upon macroscopic and histological evaluation. All these findings suggest that both types of ELRs possess broad biocompatibility, thus making them very promising for tissue engineering and regenerative medicine-related applications.

Mesenchymal Stromal Cell Irradiation Interferes with the Adipogenic/Osteogenic Differentiation Balance and Improves Their Hematopoietic-Supporting Ability.

Bone marrow mesenchymal stromal cells (MSCs) are precursors of adipocytes and osteoblasts and key regulators of hematopoiesis. Irradiation is widely used in conditioning regimens. Although MSCs are radio-resistant, the effects of low-dose irradiation on their behavior have not been extensively explored. Our aim was to evaluate the effect of 2.5 Gy on MSCs. Cells from 25 healthy donors were either irradiated or not (the latter were used as controls). Cells were characterized following International Society for Cellular Therapy criteria, including in vitro differentiation assays. Apoptosis was evaluated by annexin V/7-amino-actinomycin staining. Gene expression profiling and reverse transcriptase (RT)-PCR of relevant genes was also performed. Finally, long-term bone marrow cultures were performed to test the hematopoietic-supporting ability. Our results showed that immunophenotypic characterization and viability of irradiated cells was comparable with that of control cells. Gene expression profiling showed 50 genes differentially expressed. By RT-PCR, SDF-1 and ANGPT were overexpressed, whereas COL1A1 was downregulated in irradiated cells (P = .015, P = .007, and P = .031, respectively). Interestingly, differentiation of irradiated cells was skewed toward osteogenesis, whereas adipogenesis was impaired. Higher expression of genes involved in osteogenesis as SPP1 (P = .039) and lower of genes involved in adipogenesis, CEBPA and PPARG (P = .003 and P = .019), together with an increase in the mineralization capacity (Alizarin Red) was observed in irradiated cells. After differentiation, adipocyte counts were decreased in irradiated cells at days 7, 14, and 21 (P = .018 P = .046, and P = .018, respectively). Also, colony-forming unit granulocyte macrophage number in long-term bone marrow cultures was significantly higher in irradiated cells after 4 and 5 weeks (P = .046 and P = .007). In summary, the irradiation of MSCs with 2.5 Gy improves their hematopoietic-supporting ability by increasing osteogenic differentiation and decreasing adipogenesis.

Mesenchymal stromal cells (MSC) from JAK2+ myeloproliferative neoplasms differ from normal MSC and contribute to the maintenance of neoplastic hematopoiesis.

There is evidence of continuous bidirectional cross-talk between malignant cells and bone marrow-derived mesenchymal stromal cells (BM-MSC), which favors the emergence and progression of myeloproliferative neoplastic (MPN) diseases. In the current work we have compared the function and gene expression profile of BM-MSC from healthy donors (HD-MSC) and patients with MPN (JAK2V617F), showing no differences in the morphology, proliferation and differentiation capacity between both groups. However, BM-MSC from MPN expressed higher mean fluorescence intensity (MIF) of CD73, CD44 and CD90, whereas CD105 was lower when compared to controls. Gene expression profile of BM-MSC showed a total of 169 genes that were differentially expressed in BM-MSC from MPN patients compared to HD-MSC. In addition, we studied the ability of BM-MSC to support the growth and survival of hematopoietic stem/progenitor cells (HSPC), showing a significant increase in the number of CFU-GM colonies when MPN-HSPC were co-cultured with MPN-MSC. Furthermore, MPN-MSC showed alteration in the expression of genes associated to the maintenance of hematopoiesis, with an overexpression of SPP1 and NF-kB, and a downregulation of ANGPT1 and THPO. Our results suggest that BM-MSC from JAK2+ patients differ from their normal counterparts and favor the maintenance of malignant clonal hematopoietic cells.

Regeneration of hyaline cartilage promoted by xenogeneic mesenchymal stromal cells embedded within elastin-like recombinamer-based bioactive hydrogels.

Over the last decades, novel therapeutic tools for osteochondral regeneration have arisen from the combination of mesenchymal stromal cells (MSCs) and highly specialized smart biomaterials, such as hydrogel-forming elastin-like recombinamers (ELRs), which could serve as cell-carriers. Herein, we evaluate the delivery of xenogeneic human MSCs (hMSCs) within an injectable ELR-based hydrogel carrier for osteochondral regeneration in rabbits. First, a critical-size osteochondral defect was created in the femora of the animals and subsequently filled with the ELR-based hydrogel alone or with embedded hMSCs. Regeneration outcomes were evaluated after three months by gross assessment, magnetic resonance imaging and computed tomography, showing complete filling of the defect and the de novo formation of hyaline-like cartilage and subchondral bone in the hMSC-treated knees. Furthermore, histological sectioning and staining of every sample confirmed regeneration of the full cartilage thickness and early subchondral bone repair, which was more similar to the native cartilage in the case of the cell-loaded ELR-based hydrogel. Overall histological differences between the two groups were assessed semi-quantitatively using the Wakitani scale and found to be statistically significant (p < 0.05). Immunofluorescence against a human mitochondrial antibody three months post-implantation showed that the hMSCs were integrated into the de novo formed tissue, thus suggesting their ability to overcome the interspecies barrier. Hence, we conclude that the use of xenogeneic MSCs embedded in an ELR-based hydrogel leads to the successful regeneration of hyaline cartilage in osteochondral lesions.

HDAC8 overexpression in mesenchymal stromal cells from JAK2+ myeloproliferative neoplasms: a new therapeutic target?

Histone deacetylases (HDACs) are involved in epigenetic modulation and their aberrant expression has been demonstrated in myeloproliferative neoplasms (MPN). HDAC8 inhibition has been shown to inhibit JAK2/STAT5 signaling in hematopoietic cells from MPN. Nevertheless, the role of HDAC8 expression in bone marrow-mesenchymal stromal cells (BM-MSC) has not been assessed. In the current work we describe that HDAC8 is significantly over-expressed in MSC from in JAK-2 positive MPN compared to those from healthy-donors (HD-MSC). Using a selective HDAC8 inhibitor (PCI34051), we verified that the subsequent decrease in the protein and mRNA expression of HDAC8 is linked with an increased apoptosis of malignant MSC whereas it has no effects on normal MSC. In addition, HDAC8 inhibition in MPN-MSC also decreased their capacity to maintain neoplastic hematopoiesis, by increasing the apoptosis, cell-cycle arrest and colony formation of JAK2+-hematopoietic cells. Mechanistic studies using different MPN cell lines revealed that PCI34051 induced their apoptosis, which is enhanced when were co-cultured with JAK2V617F-MSC, decreased their colony formation and the phosphorylation of STAT3 and STAT5. In summary, we show for the first time that the inhibition of HDAC8 in MSC from JAK2+ MPN patients selectively decreases their hematopoietic-supporting ability, suggesting that HDAC8 may be a potential therapeutic target in this setting by acting not only on hematopoietic cells but also on the malignant microenvironment.