Mesenchymal Stromal Cells Populations Research Articles

Combination Therapy of Mesenchymal Stromal Cells and Interleukin-4 Attenuates Rheumatoid Arthritis in a Collagen-Induced Murine Model.

Rheumatoid arthritis (RA) is a disease of the joints that causes decreased quality of life. Mesenchymal stromal cells (MSCs) have immunosuppressive properties, with possible use in the treatment of RA. Similarly, interleukin (IL)-4 has been shown as a potential RA treatment. However, their combination has not been explored before. Therefore, this study aimed to investigate the effect of a combination therapy of MSCs and IL-4 in the treatment of RA, using a murine collagen-induced arthritis (CIA) model. Arthritis was induced in Balb/c mice by two intradermal injections of type II collagen (CII), at days 0 and 21. CIA mice were randomly assigned to four groups; group I received an intravenous injection of mouse bone marrow-derived MSCs, while group II received an intraperitoneal injection of IL-4. Group III received a combined treatment of MSC and IL-4, while group IV served as a CIA diseased control group receiving phosphate buffer saline (PBS). A fifth group of healthy mice served as the normal healthy control. To assess changes induced by different treatments, levels of RA-associated inflammatory cytokines and biomarkers were measured in the serum, knee joints, and synovial tissue, using ELISA and Real Time-qPCR. Histopathological features of knee joints were analyzed for all groups. Results showed that combined MSC and IL-4 treatment alleviated signs of synovitis in CIA mice, reverting to the values of healthy controls. This was evident by the decrease in the levels of rheumatic factor (RF), C-reactive protein (CRP) and anti-nuclear antibodies (ANA) by 64, 80, and 71%, respectively, compared to the diseased group. Moreover, tumor necrosis factor-alpha (TNF- α) and monocyte chemoattractant protein-1 (MCP-1) levels decreased by 63 and 68%, respectively. Similarly, our gene expression data showed improvement in mice receiving combined therapy compared to other groups receiving single treatment, where cartilage oligomeric matrix protein (Comp), tissue inhibitor metalloproteinase-1 (Timp1), matrix metalloproteinase1 (Mmp-1), and IL-1 receptor (Il-1r) gene expression levels decreased by 75, 70, and 78%, respectively. Collectively, treatment with a combined therapy of MSC and IL-4 might have an efficient therapeutic effect on arthritis. Thus, further studies are needed to assess the potential of different MSC populations in conjugation with IL-4 in the treatment of experimental arthritis.

Expression of Musashi-1 During Osteogenic Differentiation of Oral MSC: An In Vitro Study.

Background: Musashi-1 (MSI1) is a negative regulator of mesenchymal stromal cell (MSC) differentiation which in turn favors cell proliferation. However, little is known about its expression by MSC from the oral cavity and in the context of osteogenic differentiation. Aim: The aim of this study was to analyze the expression of MSI1 in the context of osteogenic differentiation of MSC derived from the oral cavity. Material/methods: For this in vitro study, MSC were isolated from six different origins of the oral cavity. They were extensively characterized in terms of proliferative and clonogenicity potential, expression of stemness genes (MYC, NANOG, POU5F1, and SOX2), expression of surface markers (CD73, CD90, CD105, CD14, CD31, CD34, and CD45) and adipo-, chondro- and osteogenic differentiation potential. Then, osteogenic differentiation was induced and the expression of MSI1 mRNA and other relevant markers of osteogenic differentiation, including RUNX2 and Periostin, were also evaluated. Results: Cell populations from the alveolar bone (pristine or previously grafted with xenograft), dental follicle, dental germ, dental pulp, and periodontal ligament were obtained. The analysis of proliferative and clonogenicity potential, expression of the stemness genes, expression of surface markers, and differentiation potential showed similar characteristics to those of previously published MSC from the umbilical cord. Under osteogenic differentiation conditions, all MSC populations formed calcium deposits and expressed higher SPARC. Over time, the expression of MSI1 followed different patterns for the different MSC populations. It was not significantly different than the expression of RUNX2. In contrast, the expression of MSI1 and POSTN and RUNX2 were statistically different in most MSC populations. Conclusion: In the current study, a similar expression pattern of MSI1 and RUNX2 during in vitro osteogenic differentiation was identified.

Accumulating Transcriptome Drift Precedes Cell Aging in Human Umbilical Cord-Derived Mesenchymal Stromal Cells Serially Cultured to Replicative Senescence.

In preclinical studies, mesenchymal stromal cells (MSCs) exhibit robust potential for numerous applications. To capitalize on these benefits, cell manufacturing and delivery protocols have been scaled up to facilitate clinical trials without adequately addressing the impact of these processes on cell utility nor inevitable regulatory requirements for consistency. Growing evidence indicates that culture-aged MSCs, expanded to the limits of replicative exhaustion to generate human doses, are not equivalent to early passage cells, and their use may underpin reportedly underwhelming or inconsistent clinical outcomes. Here, we sought to define the maximum expansion boundaries for human umbilical cord-derived MSCs, cultured in chemically defined xeno- and serum-free media, that yield consistent cell batches comparable to early passage cells. Two male and two female donor populations, recovered from cryostorage at mean population doubling level (mPDL) 10, were serially cultivated until replicative exhaustion (senescence). At each passage, growth kinetics, cell morphology, and transcriptome profiles were analyzed. All MSC populations displayed comparable growth trajectories through passage 9 (P9; mPDL 45) and variably approached senescence after P10 (mPDL 49). Transcription profiles of 14,500 human genes, generated by microarray, revealed a nonlinear evolution of culture-adapted MSCs. Significant expression changes occurred only after P5 (mPDL 27) and accumulated rapidly after P9 (mPDL 45), preceding other cell aging metrics. We report that cryobanked umbilical cord-derived MSCs can be reliably expanded to clinical human doses by P4 (mPDL 23), before significant transcriptome drift, and thus represent a mesenchymal cell source suited for clinical translation of cellular therapies. Stem Cells Translational Medicine 2019;8:945&958.

Human umbilical cord perivascular cells: A novel source of the organophosphate antidote butyrylcholinesterase

Human butyrylcholinesterase (BChE) is a well-characterized bioscavenger with significant potential as a prophylactic or post-exposure treatment for organophosphate poisoning. Despite substantial efforts, BChE has proven technically challenging to produce in recombinant systems. Recombinant BChE tends to be insufficiently or incorrectly glycosylated, and consequently exhibits a truncated half-life, compromised activity, or is immunogenic. Thus, expired human plasma remains the only reliable source of the benchmark BChE tetramer, but production is costly and time intensive and presents possible blood-borne disease hazards. Here we report a human BChE production platform that produces functionally active, tetrameric BChE enzyme, without the addition of external factors such as polyproline peptides or chemical or gene modification required by other systems. Human umbilical cord perivascular cells (HUCPVCs) are a rich population of mesenchymal stromal cells (MSCs) derived from Wharton's jelly. We show that HUCPVCs naturally and stably secrete BChE during culture in xeno- and serum-free media, and can be gene-modified to increase BChE output. However, BChE secretion from HUCPVCs is limited by innate feedback mechanisms that can be interrupted by addition of miR 186 oligonucleotide mimics or by competitive inhibition of muscarinic cholinergic signalling receptors by addition of atropine. By contrast, adult bone marrow-derived mesenchymal stromal cells neither secrete measurable levels of BChE naturally, nor after gene modification. Further work is required to fully characterize and disable the intrinsic ceiling of HUCPVC-mediated BChE secretion to achieve commercially relevant enzyme output. However, HUCPVCs present a unique opportunity to produce both native and strategically engineered recombinant BChE enzyme in a human platform with the innate capacity to secrete the benchmark human plasma form.

Analysis of the Interaction and Proliferative Activity of Adenocarcinoma, Peripheral Blood Mononuclear and Mesenchymal Stromal Cells after Co-Cultivation In Vitro

The tumor microenvironment is a heterogeneous population of cells actively involved in the process of growth and development of a tumor. Research has demonstrated the interactions between the different populations of cells are critical for the formation of the tumor microenvironment and, if recapitulated experimentally, can be used to produce more effective models for preclinical screening of anticancer drugs. In this study, we demonstrate co-culturing HeLa adenocarcinoma cells, peripheral blood mononuclear cells, and mesenchymal stromal cells results in changes in the proliferative activity of the peripheral blood mononuclear cells and mesenchymal stromal cell populations. This data supports the further development of in vitro co-culture systems utilizing these cell types for pre-clinical screening of anticancer drugs.

Bone marrow stromal cells from β-thalassemia patients have impaired hematopoietic supportive capacity.

BACKGROUND. The human bone marrow (BM) niche contains a population of mesenchymal stromal cells (MSCs) that provide physical support and regulate hematopoietic stem cell (HSC) homeostasis. β-Thalassemia (BT) is a hereditary disorder characterized by altered hemoglobin beta-chain synthesis amenable to allogeneic HSC transplantation and HSC gene therapy. Iron overload (IO) is a common complication in BT patients affecting several organs. However, data on the BM stromal compartment are scarce.METHODS. MSCs were isolated and characterized from BM aspirates of healthy donors (HDs) and BT patients. The state of IO was assessed and correlated with the presence of primitive MSCs in vitro and in vivo. Hematopoietic supportive capacity of MSCs was evaluated by transwell migration assay and 2D coculture of MSCs with human CD34+ HSCs. In vivo, the ability of MSCs to facilitate HSC engraftment was tested in a xenogenic transplant model, whereas the capacity to sustain human hematopoiesis was evaluated in humanized ossicle models.RESULTS. We report that, despite iron chelation, BT BM contains high levels of iron and ferritin, indicative of iron accumulation in the BM niche. We found a pauperization of the most primitive MSC pool caused by increased ROS production in vitro which impaired MSC stemness properties. We confirmed a reduced frequency of primitive MSCs in vivo in BT patients. We also discovered a weakened antioxidative response and diminished expression of BM niche–associated genes in BT-MSCs. This caused a functional impairment in MSC hematopoietic supportive capacity in vitro and in cotransplantation models. In addition, BT-MSCs failed to form a proper BM niche in humanized ossicle models.CONCLUSION. Our results suggest an impairment in the mesenchymal compartment of BT BM niche and highlight the need for novel strategies to target the niche to reduce IO and oxidative stress before transplantation.FUNDING. This work was supported by the SR-TIGET Core grant from Fondazione Telethon and by Ricerca Corrente.

Dental Pulp-Derived Stem Cells: A Promising Source for Regenerative Medicine

Regenerative medicine has the potential to replace tissues and organs damaged using stem cells (SCs) and their derivatives. Mesenchymal stromal cells (MSCs) are adult cells, multipotent and functionally defined as having self-renewal capacity, differentiation capacity in several cell lines and extensive paracrine and immunomodulatory activity. It has been described the presence of different types of MSCs populations in teeth. Dental pulp-derived SCs (DPSCs) are an important source of SC for cell therapy, can easily be collected and presents a low risk for the patient besides the great availability of material (e.g.: third molar). DPSCs have differentiation potential in multiple lineages like MSCs obtained from other tissues. The use of MSCs on a large scale in the research depends on the quality of the cells that will promote the regeneration of an injured tissue. An important aspect of standardization is the isolation method. The isolated and cultivated DPSCs present fibroblast-like morphology, plastic adherence and demonstrate the immunophenotypic profile suggested by International Society for Cellular Therapy. DPSCs derive from neural crest and have the potential to differentiate into neurons and promote the survival of neuronal cells from the injured site. The characteristics (differentiation and surface markers) presented by DPSCs are very similar with stem cells of other tissues. However, for DPSC to be used on a large scale in regenerative medicine it is necessary to standardize the isolation methodology.

Autofluorescence is a Reliable in vitro Marker of Cellular Senescence in Human Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSC) are used in cell therapies, however cellular senescence increases heterogeneity of cell populations and leads to uncertainty in therapies’ outcomes. The determination of cellular senescence is time consuming and logistically intensive. Here, we propose the use of endogenous autofluorescence as real-time quantification of cellular senescence in human MSC, based on label-free flow cytometry analysis. We correlated cell autofluorescence to senescence using senescence-associated beta-galactosidase assay (SA-β-Gal) with chromogenic (X-GAL) and fluorescent (C12FDG) substrates, gene expression of senescence markers (such as p16INK4A, p18INK4C, CCND2 and CDCA7) and telomere length. Autofluorescence was further correlated to MSC differentiation assays (adipogenesis, chondrogenesis and osteogenesis), MSC stemness markers (CD90/CD106) and cytokine secretion (IL-6 and MCP-1). Increased cell autofluorescence significantly correlated with increased SA-β-Gal signal (both X-GAL and C12FDG substrates), cell volume and cell granularity, IL-6/MCP-1 secretion and with increased p16INK4A and CCND2 gene expression. Increased cell autofluorescence was negatively associated with the expression of the CD90/CD106 markers, osteogenic and chondrogenic differentiation potentials and p18INK4C and CDCA7 gene expression. Cell autofluorescence correlated neither with telomere length nor with adipogenic differentiation potential. We conclude that autofluorescence can be used as fast and non-invasive senescence assay for comparing MSC populations under controlled culture conditions.

Aldehyde dehydrogenase activity of Wharton jelly mesenchymal stromal cells: isolation and characterization.

Mesenchymal stromal cells (MSCs) are promising tools in regenerative medicine and targeted therapies. Although different origins have been described, there is still huge need to find a valuable source harboring specific subpopulations of MSCs with precise therapeutic functions. Here, we isolated by fluorescence activated cell sorting technique, two populations of Wharton's jelly (WJ)-MSCs based on their aldehyde dehydrogenase (ALDH) activity. Two different ALDH activities (low vs. high) were thus observed. We then analyzed their gene expression profile for stemness, phenotype, response to hypoxia, angiogenesis, hematopoietic support, immunomodulation and multilineage differentiation abilities (osteogenesis, adipogenesis, and chondrogenesis). According to ALDH activity, many differences in the mRNA expression of these populations were noticed. In conclusion, we provide evidences that WJ harbors two distinct populations of MSCs with different ALDH activity. These populations seem to display specific functional competences that may be interesting for concise therapeutic applications.

Improving hematopoietic recovery through modeling and modulation of the mesenchymal stromal cell secretome

BackgroundEfficient and sustained hematopoietic recovery after hematopoietic stem cell or bone marrow transplantation is supported by paracrine signaling from specific subpopulations of mesenchymal stromal cells (MSCs). Here, we considered whether in vitro mechanopriming of human MSCs could be administered to predictively and significantly improve in vivo hematopoietic recovery after irradiation injury.MethodsFirst, we implemented regression modeling to identify eight MSC-secreted proteins that correlated strongly with improved rescue from radiation damage, including hematopoietic recovery, in a murine model of hematopoietic failure. Using these partial least squares regression (PLSR) model parameters, we then predicted recovery potential of MSC populations that were culture expanded on substrata of varying mechanical stiffness. Lastly, we experimentally validated these predictions using an in vitro co-culture model of hematopoiesis and using new in vivo experiments for the same irradiation injury model used to generate survival predictions.ResultsMSCs grown on the least stiff (elastic moduli ~ 1 kPa) of these polydimethylsiloxane (PDMS) substrata secreted high concentrations of key proteins identified in regression modeling, at concentrations comparable to those secreted by minor subpopulations of MSCs shown previously to be effective in supporting such radiation rescue. We confirmed that these MSCs expanded on PDMS could promote hematopoiesis in an in vitro co-culture model with hematopoietic stem and progenitor cells (HSPCs). Further, MSCs cultured on PDMS of highest stiffness (elastic moduli ~ 100 kPa) promoted expression of CD123+ HSPCs, indicative of myeloid differentiation. Systemic administration of mechanoprimed MSCs resulted in improved mouse survival and weight recovery after bone marrow ablation, as compared with both standard MSC expansion on stiffer materials and with biophysically sorted MSC subpopulations. Additionally, we observed recovery of white blood cells, platelets, and red blood cells, indicative of complete recovery of all hematopoietic lineages.ConclusionsThese results demonstrate that computational techniques to identify MSC biomarkers can be leveraged to predict and engineer therapeutically effective MSC phenotypes defined by mechanoprimed secreted factors, for translational applications including hematopoietic recovery.

Syndecan-2-positive, Bone Marrow-derived Human Mesenchymal Stromal Cells Attenuate Bacterial-induced Acute Lung Injury and Enhance Resolution of Ventilator-induced Lung Injury in Rats.

WHAT THIS ARTICLE TELLS US THAT IS NEW: BACKGROUND:: Human mesenchymal stromal cells demonstrate promise for acute respiratory distress syndrome, but current studies use highly heterogenous cell populations. We hypothesized that a syndecan 2 (CD362)-expressing human mesenchymal stromal cell subpopulation would attenuate Escherichia coli-induced lung injury and enhance resolution after ventilator-induced lung injury. In vitro studies determined whether CD362 human mesenchymal stromal cells could modulate pulmonary epithelial inflammation, wound healing, and macrophage phagocytosis. Two in vivo rodent studies determined whether CD362 human mesenchymal stromal cells attenuated Escherichia coli-induced lung injury (n = 10/group) and enhanced resolution of ventilation-induced injury (n = 10/group). CD362 human mesenchymal stromal cells attenuated cytokine-induced epithelial nuclear factor kappa B activation, increased epithelial wound closure, and increased macrophage phagocytosis in vitro. CD362 human mesenchymal stromal cells attenuated Escherichia coli-induced injury in rodents, improving arterial oxygenation (mean ± SD, 83 ± 9 vs. 60 ± 8 mmHg, P < 0.05), improving lung compliance (mean ± SD: 0.66 ± 0.08 vs. 0.53 ± 0.09 ml · cm H2O, P < 0.05), reducing bacterial load (median [interquartile range], 1,895 [100-3,300] vs. 8,195 [4,260-8,690] colony-forming units, P < 0.05), and decreasing structural injury compared with vehicle. CD362 human mesenchymal stromal cells were more effective than CD362 human mesenchymal stromal cells and comparable to heterogenous human mesenchymal stromal cells. CD362 human mesenchymal stromal cells enhanced resolution after ventilator-induced lung injury in rodents, restoring arterial oxygenation (mean ± SD: 113 ± 11 vs. 89 ± 11 mmHg, P < 0.05) and lung static compliance (mean ± SD: 0.74 ± 0.07 vs. 0.45 ± 0.07 ml · cm H2O, P < 0.05), resolving lung inflammation, and restoring histologic structure compared with vehicle. CD362 human mesenchymal stromal cells efficacy was at least comparable to heterogenous human mesenchymal stromal cells. A CD362 human mesenchymal stromal cell population decreased Escherichia coli-induced pneumonia severity and enhanced recovery after ventilator-induced lung injury.

Abstract 2140: Mesenchymal stromal cells expressing a PEAK1/Cripto axis sustain pro-survival NF-κB signaling in adjacent tumor cells to promote disease progression and therapy resistance

Abstract The cellular and molecular heterogeneity of solid tumors, like breast cancer, is a significant hurdle in the effort to develop effective therapies. The complex breast cancer microenvironment (BCME) includes mesenchymal stromal cells (MSCs) that engage in paracrine/juxtacrine signaling with adjacent tumor cells to support disease progression. Since PEAK1 (Pseudopodium-Enriched Atypical Kinase One) is a cytoskeleton-associated kinase that regulates growth factor-integrin signaling crosstalk in mesenchymal cell types and recent evidence demonstrates that stromal expression of PEAK1 correlates with breast cancer recurrence, we hypothesized that PEAK1 expression in the mesenchymal compartment of the BCME promotes tumorigenesis. We report that PEAK1 is expressed in patient-derived cancer-associated fibroblasts (CAFs) from all breast cancer subtypes. Additionally, PEAK1hi CAF conditioned media promoted breast cancer cell (BCC) proliferation, migration and therapy resistance in vitro. We established in vitro co-culture and in vivo co-xenografting models for evaluating the effects of defined MSC populations on BCC proliferation/survival, metastasis and therapy response. Co-xenografting PEAK1hi CAFs or MSCs with BCCs increased the mass of HER2-positive and ER-positive primary tumors. In agreement with these data, co-culturing MSCs with BCCs promoted cancer cell proliferation and resistance to lapatinib or tamoxifen treatment, while MSC conditioned media supported BCC expansion under serum-free conditions in vitro. Notably, PEAK1 knockdown in MSCs abrogated their ability to promote tumor growth in vivo and therapy/stress resistance in vitro. Using the highly multiplexed cyclic immunofluorescence (CycIF) platform, we analyzed tumor cell states in these MSC-BCC co-cultures at different time points following lapatinib treatment. We demonstrate that MSCs sustain NF-κB (p65) signaling and anti-apoptotic gene expression within BCCs in the presence of lapatinib. Finally, we discovered that PEAK1 is required for MSC expression of Cripto, a GPI-anchored glycoprotein previously reported to activate NF-κB signaling in trans. Taken together, this work identifies new PEAK1-dependent stroma-tumor signaling vulnerabilities that may be exploited for improving patient responses to current therapeutic interventions. Citation Format: Sarkis Hamalian, Robert Güth, Ioannis Zervantonakis, Erika Duell, Jia-Ren Lin, Preston Shisgal, Justin Molnar, Cameron Geller, Megan Agajanian, Julia Tchou, Peter K. Sorger, Joan S. Brugge, Jonathan A. Kelber. Mesenchymal stromal cells expressing a PEAK1/Cripto axis sustain pro-survival NF-κB signaling in adjacent tumor cells to promote disease progression and therapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2140.

Forced expression of CD200 improves the differentiation capability and immunoregulatory functions of mesenchymal stromal cells.

In order to identify specific mesenchymal stromal cell (MSC) populations with enhanced therapeutic efficacy, we evaluated the functional changes associated with the stable expression of CD200, which is associated with immune regulatory function and osteogenic differentiation, in human bone marrow-derived MSCs (CD200/MSCs). We detected significantly greater osteogenesis and chondrogenesis in CD200/MSCs than in mock-transfected MSCs. In addition, the immune regulatory function of MSCs in mixed lymphocyte reactions was enhanced by CD200 gene transfection. In CD200/MSCs, the secretion of inflammatory cytokines, i.e., IL-6 and IL-8, was reduced, and levels of the anti-inflammatory factors IL-10, FOXP3, and indoleamine 2,3-dioxygenase 1 were elevated. Finally, CD200 transfection increased the stemness of MSCs, as evidenced by greater colony numbers in colony-forming unit fibroblast assays and analyses of NANOG and OCT-4 expression. These results suggest that CD200/MSCs have therapeutic applications, and further in-depth research should focus on the development of a clinically applicable cell-based therapeutic strategy.

Reproductive stage and sex steroid hormone levels influence the expression of mesenchymal stromal cell (MSC) markers in the equine endometrium

Mesenchymal stem or stromal cells (MSCs) play key roles in tissue homeostasis. In the cyclic equine endometrium, this may be regulated by changes in serum concentrations of sex steroid hormones. This study was designed to investigate the changes in endometrial expression of MSC markers during reproductive cycles in mares and the influence of sex steroid hormones on endometrial MSC proliferation in vitro. Endometrial biopsies were collected from pony mares at different reproductive stages (estrus; day 5 and 13 after ovulation; seasonal anestrus; 20 h and 7days post-partum; n = 5 per stage) and were analyzed by RT-qPCR. MSC (CD29, CD44, CD73, CD90, CD105) and perivascular (CD146, NG2) markers were present in all samples irrespective of reproductive stage. Transcript levels of most markers were present at lowest levels on day 5 after ovulation and at 20 h post-partum. MSCs isolated from endometrial tissue (n = 6 mares) were cultured in the presence of progesterone (0.01–100 μM) and estradiol 17β (0.1–1 μM), and cell proliferation was analyzed using alamarBlue® assay. Relative to cells incubated in steroid-depleted media, both progesterone and estradiol 17β moderately increased cell proliferation (1.1- and 1.2-fold, respectively) independently of the concentration used. In conclusion, our results suggest that levels of MSC markers in equine endometrium dynamically change across reproductive cycles and that MSC populations are in part regulated by sex steroids.

Mesenchymal stromal cell lineages in synovium and their contribution to osteoarthritis and cartilage repair

In recent years, landmark studies using genetic lineage tracing models in mice have identified and functionally characterised skeletal stem and progenitor cell populations in bone marrow, providing important insights in the regulation of bone homeostasis and repair, as well as the control of haematopoiesis. By comparison, the stem and progenitor cells that are resident in the synovial joint and maintain and repair joint tissues such as articular cartilage remained poorly defined, but recent work from our lab and others is starting to shed light on this. Lineage tracing of progenitor cells in cartilage has provided important new insights into the process of articular cartilage formation and maintenance. Our work has focused on characterising stem and progenitor cells in the synovial membrane, and how these cells respond to cartilage injury. Tracing of cells from the embryonic joint interzone, using Gdf5 as driver, showed that Gdf5-lineage cells persist in adult knee synovium up to at least one year of age as a mesenchymal stromal cell population with progenitor activity, which is largely distinct from the skeletal stem/progenitor cell populations identified in bone marrow. After purification, Gdf5-lineage cells display a differentiation potency in vitro that is skewed towards formation of cartilage and a synovial lining-like layer, while showing poor osteogenic ability. Cartilage injury in vivo triggers Gdf5-lineage cells in synovium to undergo proliferation leading to synovial lining hyperplasia, a process that is dependent on the activity of the transcriptional co-factor Yap in these cells. Gdf5-lineage cells also contribute to repair of traumatic joint surface defects, though they do not seem to be the sole cell population able to populate the repair tissue. We further find that in pathology, using the destabilisation of the medial meniscus model of osteoarthritis, Gdf5-lineage cells contribute to chondro-osteophyte formation and subchondral bone remodeling. In conclusion, recent findings are starting to unravel distinct mesenchymal stromal cell subsets in the adult synovium, identifying the key players in joint pathophysiology and promising therapeutic targets for cartilage repair and treatment of osteoarthritis.

CD317 profiling distinguishes bone marrow stromal cell subtypes with distinct functional impact upon T cell proliferation and polarisation

Purpose: The development of enhanced therapeutic interventions using human mesenchymal stromal cells (MSCs) for regenerative medicine is a priority in osteoarthritis research. MSCs are a heterogeneous cell population with immunomodulatory and anti-inflammatory properties. We have identified a novel biomarker (CD317) that allows the isolation of MSCs subpopulations with tissue regenerative properties (Y201, CD317-) and non-differentiating MSCs with an enriched pro-inflammatory transcriptional profile (Y202, CD317+) that make up an average of 9% of the total MSCs population. We investigated the immunomodulatory function of these subpopulations to understand their role in disease processes and potential impact on regenerative therapies. Methods: MSC subpopulations (Y201 and Y202) were co-cultured with human T cells to examine the effects on proliferation and polarisation. Primary T cell populations labelled with VPD450 proliferation dye and activated with CD3/CD28 beads were cultured for 5 days with MSCs (either Y201 or Y202) or alone. T cells without activation were used as controls. After 5 days, CD4+ T cells were assessed for reduction in VPD450 intensity and cultures examined for T helper polarisation. CD4+ cells were evaluated for interferon gamma, interleukin 17a, interleukin 4 and nuclear protein FOXP3. These T helper subsets are representative of pro-inflammatory (Th1, Th17) and anti-inflammatory (Th2, regulatory T cells (Tregs)) cells respectively. Results: T cells cultured alone underwent increased proliferative cycles (mean 6.283±0.383) compared with those co-cultured with Y201 (1.95±0.17, P<0.001) and Y202 (3.76±0.55, P<0.005). Y202 MSCs were less effective at suppressing T cell proliferation compared to Y201 MSCs (P<0.05). A proliferative index was calculated to represent proportions of cells undergoing multiple divisions. This index reduced from T cell alone (7.60±1.00) when T cells are co-cultured with Y201 (1.47±0.11, P<0.001) or Y202 (3.16±0.40, P<0.005). When assessed for T helper polarisation, a highly significant proportion of CD4+ cells did not express any of the markers examined (80.29±2.33%) compared to co-cultures with Y201 (21.23±6.60%) or Y202 (7.54±4.60%) (P<0.001). T cells co-cultured with Y201 and Y202 MSCs differentiated predominantly towards anti-inflammatory Th2 cell types (55.32±8.95% and 59.72±6.02% respectively) compared with control T cells alone (9.70±0.51%, P<0.005). However, Y202 induced an increase in pro-inflammatory Th1 cells (14.43±2.03%) compared to Y201 (7.04±0.62%) and T cells cultured alone (5.23±1.96%) (P<0.05). Both Y202 and Y201 MSCs also increased pro-inflammatory Th17 polarisation (6.61±1.00%, 7.40±1.40% respectively) compared to T cells alone (1.78±0.24, P<0.05). Only Y202 MSCs prompted a significant increase in Treg production (11.63±2.55%) compared to T cells alone (3.01±0.68%) (P<0.05). These results demonstrate clear and distinct functional interactions between Y201 and Y202 MSCs and T cell populations. Conclusions: These data demonstrate that the transcriptional profile of CD317+ Y202 MSCs translates to functional outcomes through direct impact upon the cells of the immune system. Y202 MSCs encourage increased polarisation of T helper cell subsets with a more pro-inflammatory profile compared to Y201. Further investigation of the impact of CD317+ MSCs on the immune system is underway to assess the impact of these cells in osteoarthritis and autoimmunity. Stem cell based therapies do not account for the 9% of MSCs that are CD317+. The inclusion of a cell fraction of non-migratory, non-differentiating, proliferative and pro-inflammatory population of cells in cell therapies could contribute to poor tissue regenerative responses in clinical interventions. These results strengthen the case for negative selection of MSCs using biomarker CD317 for tissue regenerative applications and highlight the potential for an association between CD317+ MSCs and autoimmune disorders.

Distinct protein signatures of acute myeloid leukemia bone marrow-derived stromal cells are prognostic for patient survival.

Mesenchymal stromal cells (MSC) support acute myeloid leukemia (AML) cell survival in the bone marrow (BM) microenvironment. Protein expression profiles of AML-derived MSC are unknown. Reverse phase protein array analysis was performed to compare expression of 151 proteins from AML-MSC (n=106) with MSC from healthy donors (n=71). Protein expression differed significantly between the two groups with 19 proteins over-expressed in leukemia stromal cells and 9 over-expressed in normal stromal cells. Unbiased hierarchical clustering analysis of the samples using these 28 proteins revealed three protein constellations whose variation in expression defined four MSC protein expression signatures: Class 1, Class 2, Class 3, and Class 4. These cell populations appear to have clinical relevance. Specifically, patients with Class 3 cells have longer survival and remission duration compared to other groups. Comparison of leukemia MSC at first diagnosis with those obtained at salvage (i.e. relapse/refractory) showed differential expression of 9 proteins reflecting a shift toward osteogenic differentiation. Leukemia MSC are more senescent compared to their normal counterparts, possibly due to the overexpressed p53/p21 axis as confirmed by high β-galactosidase staining. In addition, overexpression of BCL-XL in leukemia MSC might give survival advantage under conditions of senescence or stress and overexpressed galectin-3 exerts profound immunosuppression. Together, our findings suggest that the identification of specific populations of MSC in AML patients may be an important determinant of therapeutic response.

Tissue-resident mesenchymal stromal cells: Implications for tissue-specific antifibrotic therapies.

Recent scientific findings support the notion that fibrosis is driven by tissue-specific cellular and molecular mechanisms. Analysis of seemingly equivalent mesenchymal stromal cell (MSC) populations residing in different organs revealed unique properties and lineage capabilities that vary from one anatomical location to another. We review recently characterized tissue-resident MSC populations with a prominent role in fibrosis and highlight therapeutically relevant molecular pathways regulating their activity in chronic disease.

Isolation and Characterisation of Mesenchymal Stem Cells from Rat Bone Marrow and the Endosteal Niche: A Comparative Study.

Within bone, mesenchymal stromal cells (MSCs) exist within the bone marrow stroma (BM-MSC) and the endosteal niche, as cells lining compact bone (CB-MSCs). This study isolated and characterised heterogeneous MSC populations from each niche and subsequently investigated the effects of extensive cell expansion, analysing population doublings (PDs)/cellular senescence, colony-forming efficiencies (CFEs), MSC cell marker expression, and osteogenic/adipogenic differentiation. CB-MSCs and BM-MSCs demonstrated similar morphologies and PDs, reaching 100 PDs. Both populations exhibited consistent telomere lengths (12–17 kb), minimal senescence, and positive telomerase expression. CB-MSCs (PD15) had significantly lower CFEs than PD50. CB-MSCs and BM-MSCs both expressed MSC (CD73/CD90/CD105); embryonic (Nanog) and osteogenic markers (Runx2, osteocalcin) but no hematopoietic markers (CD45). CB-MSCs (PD15) strongly expressed Oct4 and p16INK4A. At early PDs, CB-MSCs possessed a strong osteogenic potency and low potency for adipogenesis, whilst BM-MSCs possessed greater overall bipotentiality for osteogenesis and adipogenesis. At PD50, CB-MSCs demonstrated reduced potency for both osteogenesis and adipogenesis, compared to BM-MSCs at equivalent PDs. This study demonstrates similarities in proliferative and mesenchymal cell characteristics between CB-MSCs and BM-MSCs, but contrasting multipotentiality. Such findings support further comparisons of human CB-MSCs and BM-MSCs, facilitating selection of optimal MSC populations for regenerative medicine purposes.

Circulating Progenitor Cell Response to Exercise in Wheelchair Racing Athletes.

Circulating progenitor cells (CPC) are a heterogeneous population of stem/progenitor cells in peripheral blood that participate in tissue repair. CPC mobilization has been well characterized in able-bodied persons but has not been previously investigated in wheelchair racing athletes. The purpose of this study was to characterize CPC and CPC subpopulation mobilization in elite wheelchair racing athletes in response to acute, upper-extremity aerobic exercise to determine whether CPC responses are similar to ambulatory populations. Eight participants (three females; age = 27.5 ± 4.0 yr, supine height = 162.5 ± 18.6 cm, weight = 53.5 ± 10.9 kg, V˙O2peak = 2.4 ± 0.62 L·min, years postinjury = 21.5 ± 6.2 yr) completed a 25-km time trial on a road course. Blood sampling occurred before and immediately after exercise for quantification of CPC (CD34), hematopoietic stem and progenitor cells (HSPC) (CD34/CD45), hematopoietic stem cells (HSC) (CD34/CD45/CD38), CD34 adipose tissue (AT)-derived mesenchymal stromal cells (MSC) (CD45/CD34/CD105/CD31), CD34 bone marrow (BM)-derived MSC (CD45/CD34/CD105/CD31), and endothelial progenitor cells (EPC) (CD45/CD34/VEGFR2) via flow cytometry. Blood lactate was measured before and after trial as an indicator of exercise intensity. CPC concentration increased 5.7-fold postexercise (P = 0.10). HSPC, HSC, EPC, and both MSC populations were not increased postexercise. Baseline HSPC populations were significantly positively correlated to absolute V˙O2peak (rho = 0.71, P < 0.05) with HSC trending to positively correlate to V˙O2peak (rho = 0.62, P = 0.10). AT-MSC populations were trending to be negatively correlated to baseline V˙O2peak (rho = -0.62, P = 0.058). The change in CPC, EPC, and AT-MSC pre- and postexercise significantly positively correlated to the change in lactate concentrations (rho = 0.91 P = 0.002, 0.71 P = 0.047, 0.81 P = 0.02, respectively, all P < 0.05). These data suggest that CPC content in wheelchair racing athletes is related to cardiorespiratory fitness, and responses to exercise are positively related to exercise intensity.