MSC Subpopulations Research Articles

Improved therapeutic consistency and efficacy of CD317+ MSCs through stabilizing TSG6 by PTX3

BackgroundPreviously, we have demonstrated that the batch variations of human platelet lysate (conventional MSC expansion medium) induce MSC heterogeneity and therapeutic inconsistency. On the other hand, the MSCs expanded with chemical defined medium have improved therapeutic consistency.MethodsIn the current study, we studied the MSC subpopulation composition and variation in different types and batches of MSC expansion medium with scRNA-seq analysis.ResultsMSCs expanded with different batches of media have higher levels of heterogeneity from the perspective of cell subpopulation composition at transcriptome levels and therapeutic inconsistency. The CD317+ subpopulation has enhanced immune suppression activities. And the percentage of CD317+ MSCs within MSCs is tightly correlated with its immune suppression activities, and also contributes to the heterogeneity and therapeutic inconsistency of MSCs. the CD317+ MSCs have increased expression levels of PTX3, which might stabilize the TSG6 protein and improve the therapeutic effectsConclusionsThus, purifying CD317+ MSCs is one efficient strategy to reduce MSC heterogeneity and increase the therapeutic consistency of MSCs.

Evaluation of human adipose-derived mesenchymal stromal cell Toll-like receptor priming and effects on interaction with prostate cancer cells

Background aimsMesenchymal stromal cells (MSCs) are a multipotent cell population of clinical interest because of their ability to migrate to injury and tumor sites, where they may participate in tissue repair and modulation of immune response. Although the processes regulating MSC function are incompletely understood, it has been shown that stimulation of Toll-like receptors (TLRs) can alter MSC activity. More specifically, it has been reported that human bone marrow-derived MSCs can be “polarized” by TLR priming into contrasting immunomodulatory functions, with opposite (supportive or suppressive) roles in tumor progression and inflammation. Adipose-derived MSCs (ASCs) represent a promising alternative MSC subpopulation for therapeutic development because of their relative ease of isolation and higher abundance compared with their bone marrow-derived counterparts; however, the polarization of ASCs remains unreported. MethodsIn this study, we evaluated the phenotypic and functional consequences of short-term, low-level stimulation of ASCs with TLR3 and TLR4 agonists. ResultsIn these assays, we identified transient gene expression changes resembling the reported pro-inflammatory and anti-inflammatory MSC phenotypes. Furthermore, these priming strategies led to changes in the functional properties of ASCs, affecting their ability to migrate and modulate immune-mediated responses to prostate cancer cells in vitro. ConclusionsTLR3 stimulation significantly decreased ASC migration, and TLR4 stimulation increased ASC immune-mediated killing potential against prostate cancer cells.

TNFAIP6 defines the MSC subpopulation with enhanced immune suppression activities

BackgroundMesenchymal stromal/stem cells (MSCs) have been intensively investigated in both pre-clinical and clinical studies. However, the therapeutic efficacy varies resulting from the heterogenicity of MSCs. Therefore, purifying the specific MSC subpopulation with specialized function is necessary for their therapeutic applications.MethodsThe large-scale RNA sequencing analysis was performed to identify potential cell markers for the mouse MSCs. Then, the immune suppression activities of the purified MSC subpopulation were assessed in vitro and in vivo.ResultsThe TNFAIP6 (tumor necrosis factor alpha-induced protein 6) has been identified as a potential cell marker for mouse MSCs, irrespective of tissue origin and laboratory origin. The TNFAIP6+ mouse MSCs showed enhanced immune suppression activities and improved therapeutic effects on the mouse model of acute inflammation, resulting from faster response to immune stimulation.ConclusionsTherefore, we have demonstrated that the TNFAIP6+ MSC subpopulation has enhanced immune suppression capabilities.

Manipulating Osteogenic/Adipogenic Balance in Mesenchymal Stromal Cells (MSCs) for Hematopoietic Applications

Aims: Bone marrow-derived mesenchymal stromal cells (BM-MSCs) have the potential to improve hematopoietic transplantation by supporting hematopoietic stem and progenitor cell (HSPC) expansion as well as by promoting HSPC engraftment to the BM. However, culture expanded MSCs becomes a mixture of progenitor cells committed to different cell lineages, each with different potencies for any one desired application. Clinically, the accumulation of adipogenic MSCs in the BM is believed to contribute towards age-related impairment of regeneration and hematopoiesis; this observation, together with our understanding of the inverse relationship between MSC osteogenic and adipogenic differentiation prompted a more in depth study of the ability of these different MSC lineages to support hematopoietic clinical applications. Clinically relevant methods for engineering a pure cell population from heterogeneous MSC culture will also be discussed.Methods: MSCs were derived from the healthy BM of patients aged 30-50 years. At various stages of osteogenic and adipogenic differentiation in vitro , cultures were evaluated for their ability to support cord blood (CB) CD34+ HSC expansion and stimulate regenerative processes such as angiogenesis and cell proliferation. qPCR, coupled with secretome analysis was performed to discover gene expression markers for MSCs with potent hematopoietic supportive capabilities. Finally, due to the lack of unique surface markers for identification of MSC subpopulations, we utilized multivariate biophysical analysis (microfluidic sorting, atomic force microscopy, etc.), correlating these quantitative measures with biomolecular markers as well as in vitro and in vivo functionality. Novel label-free cell separation strategies that take advantage of unique biophysical traits in a potent MSC subpopulation for HSPC transplantation was also developed to enable clinical translation.Results: Differentiated osteoblasts (OB) as well as pre-OB demonstrated a higher ability to support CB CD34+ HSC expansion compared to undifferentiated MSCs or MSCs in the adipogenic state. Over a period of 10 days, the total HSPC fold expansion was 44.4 ± 4.83, 57.8 ± 10.67, 40.2 ± 4.65, 34.2 ± 6.18 and 13.8 ± 5.50 for OB, pre-OB, MSCs, pre-AD and AD, respectively (n = 5). Absolute expanded CD34+ HSCs was highest with pre-OB cultures (~1.7 fold, n = 5) compared to undifferentiated MSCs. Adipogenic MSCs had much reduced capabilities for supporting HSPC expansion. In vitro angiogenic and proliferation assays showed the greatest degree of HUVEC sprouting and proliferation (~ 2.6 fold greater than undifferentiated MSCs) when exposed to a pre-OB secretome. Pre-OBs are distinct in their transcriptome (up-regulated osteopontin, runx2, angiopoietin-1, FGF1, etc) but no surface marker reliably distinguishes them from other MSCs. We subsequently found a minimal set of three biophysical markers: cell diameter, membrane stiffness and nuclear membrane fluctuations, which collectively are predictive of MSC subpopulations (multipotent vs committed progenitors). We hypothesized and experimentally verified that a biophysically derived pre-OB subpopulation, defined biophysically as MSCs with >20 μm diameter, >375Pa stiffness and <1.2 % nuclear fluctuation, exhibits significantly greater efficacy over other MSC subpopulations for promoting bone marrow repair (~2-3 fold improvements in medial survival following BM injury) and expansion of HSPCs (~1.5-2 fold increase in HSPC numbers). Following co-transplantation with HSPCs, biophysically derived pre-OB promoted faster engraftment of donor cells in the peripheral blood by week 4 (~ 9% vs < 5% in other MSC groups, P<0.05).Conclusion: These results demonstrate a correlation between MSCs with high osteogenic activity and the capabilities for BM repair as well as hematopoietic support, making such cells ideal for augmentation of HSC transplantations. In the absence of reliable surface markers for isolation of these pre-OBs, we developed a clinically relevant biophysical isolation strategy for derivation of pre-OBs that can markedly improve the efficacy of MSC-based approaches for augmentation of hematopoietic transplants. DisclosuresNo relevant conflicts of interest to declare.

More Human BM-MSC With Similar Subpopulation Composition and Functional Characteristics Can Be Produced With a GMP-Compatible Fabric Filter System Compared to Density Gradient Technique.

BackgroundMesenchymal stromal cells (MSCs), multipotent progenitors that can be isolated from a variety of different tissues, are becoming increasingly important as cell therapeutics targeting immunopathologies and tissue regeneration. Current protocols for MSC isolation from bone marrow (BM) rely on density gradient centrifugation (DGC), and the production of sufficient MSC doses is a critical factor for conducting clinical MSC trials. Previously, a Good Manufacturing Practice (GMP)–compatible non-woven fabric filter device system to isolate MSCs was developed to increase the MSC yield from the BM. The aim of our study was to compare high-resolution phenotypic and functional characteristics of BM-MSCs isolated with this device and with standard DGC technology.MethodsHuman BM samples from 5 donors were analyzed. Each sample was divided equally, processing by DGC, and with the filter device. Stem cell content was assessed by quantification of colony-forming units fibroblasts (CFU-F). Immunophenotype was analyzed by multicolor flow cytometry. In vitro trilineage differentiation potential, trophic factors, and IDO-1 production were assessed. Functionally, immunomodulatory potential, wound healing, and angiogenesis were assayed in vitro.ResultsThe CFU-F yield was 15-fold higher in the MSC preparations isolated with the device compared to those isolated by DGC. Consequently, the MSC yield that could be manufactured at passage 3 per mL collected BM was more than 10 times higher in the device group compared to DGC (1.65 × 109 vs. 1.45 × 108). The immunomodulatory potential and IDO-1 production showed donor-to-donor variabilities without differences between fabric filter-isolated and DGC-isolated MSCs. The results from the wound closure assays, the tube formation assays, and the trilineage differentiation assays were similar between the groups with respect to the isolation method. Sixty-four MSC subpopulations could be quantified with CD140a+CD119+CD146+ as most common phenotype group, and CD140a+CD119+CD146+MSCA-1–CD106–CD271– and CD140a+CD119+CD146–MSCA-1–CD106–CD271– as most frequent MSC subpopulations. As trophic factors hepatocyte growth factor, epidermal growth factor, brain-derived neurotrophic factor, angiopoietin-1, and vascular endothelial growth factor A could be detected in both groups with considerable variability between donors, but independent of the respective MSC isolation technique.ConclusionThe isolation of MSCs using a GMP-compatible fabric filter system device resulted in higher yield of CFU-F, producing substantially more MSCs with similar subpopulation composition and functional characteristics as MSCs isolated by DGC.

Ex vivo-expanded highly pure ABCB5+ mesenchymal stromal cells as Good Manufacturing Practice-compliant autologous advanced therapy medicinal product for clinical use: process validation and first in-human data

Background aimMesenchymal stromal cells (MSCs) hold promise for the treatment of tissue damage and injury. However, MSCs comprise multiple subpopulations with diverse properties, which could explain inconsistent therapeutic outcomes seen among therapeutic attempts. Recently, the adenosine triphosphate-binding cassette transporter ABCB5 has been shown to identify a novel dermal immunomodulatory MSC subpopulation. MethodsThe authors have established a validated Good Manufacturing Practice (GMP)-compliant expansion and manufacturing process by which ABCB5+ MSCs can be isolated from skin tissue and processed to generate a highly functional homogeneous cell population manufactured as an advanced therapy medicinal product (ATMP). This product has been approved by the German competent regulatory authority to be tested in a clinical trial to treat therapy-resistant chronic venous ulcers. ResultsAs of now, 12 wounds in nine patients have been treated with 5 × 105 autologous ABCB5+ MSCs per cm2 wound area, eliciting a median wound size reduction of 63% (range, 32–100%) at 12 weeks and early relief of pain. ConclusionsThe authors describe here their GMP- and European Pharmacopoeia-compliant production and quality control process, report on a pre-clinical dose selection study and present the first in-human results. Together, these data substantiate the idea that ABCB5+ MSCs manufactured as ATMPs could deliver a clinically relevant wound closure strategy for patients with chronic therapy-resistant wounds.

Signature quality attributes of CD146+ mesenchymal stem/stromal cells correlate with high therapeutic and secretory potency.

CD146+ bone marrow-derived mesenchymal stem/stromal cells (BM-MSCs) play key roles in the perivascular niche, skeletogenesis, and hematopoietic support; however, comprehensive evaluation of therapeutic potency has yet to be determined. In this study, in vitro inflammatory priming to crude human BM-MSCs (n = 8) captured a baseline of signature responses, including enriched CD146+ with coexpression of CD107aHigh , CXCR4High , and LepRHigh , transcriptional profile, enhanced secretory capacity, and robust immunomodulatory secretome and function, including immunopotency assays (IPAs) with stimulated immune cells. These signatures were significantly more pronounced in CD146+ (POS)-sorted subpopulation than in the CD146- (NEG). Mechanistically, POS BM-MSCs showed a markedly higher secretory capacity with significantly greater immunomodulatory and anti-inflammatory protein production upon inflammatory priming compared with the NEG BM-MSCs. Moreover, IPAs with stimulated peripheral blood mononuclear cells and T lymphocytes demonstrated robust immunosuppression mediated by POS BM-MSC while inducing significant frequencies of regulatory T cells. in vivo evidence showed that POS BM-MSC treatment promoted pronounced M1-to-M2 macrophage polarization, ameliorating inflammation/fibrosis of knee synovium and fat pad, unlike treatment with NEG BM-MSCs. These data correlate the expression of CD146 with innately higher immunomodulatory and secretory capacity, and thus therapeutic potency. This high-content, reproducible evidence suggests that the CD146+ (POS) MSC subpopulation are the mediators of the beneficial effects achieved using crude BM-MSCs, leading to translational implications for improving cell therapy and manufacturing.

Osteogenic differentiation potential of porcine bone marrow mesenchymal stem cell subpopulations selected in different basal media.

ABSTRACTMultipotent porcine mesenchymal stem cells (pMSC) are invaluable for research and therapeutic use in regenerative medicine. Media used for derivation and expansion of pMSC may play an important role for the selection of MSC subpopulation at an early stage and thereby, the specific basal medium may also affect differentiation potential of these cells. The present study was undertaken to evaluate the effects of αMEM, aDMEM, M199, αMEM/M199, aDMEM/M199 and αMEM/aDMEM media on (1) porcine bone marrow MSC derivation; (2) expression of number of osteogenic markers (ALP, COL1A1, SPP1 and BGLAP) at 5th and 10th passage in pMSC before differentiation; and (3) differentiation of pMSC (at 5th passage) to osteogenic lineage. Morphological changes and matrix formation in osteogenic cells were evaluated by microscopic examination. Calcium deposits in osteocytes were confirmed by Alizarin Red S staining. Based on expression of different markers, it was evident that selection of bone marrow pMSC subpopulations was independent of basal media used. However, the differentiation of those pMSCs, specifically to osteogenic lineage, was dependent on the medium used for expansion of pMSC at the pre-differentiation stage. We demonstrated here that the pMSC grown in combined αMEM/aDMEM (1:1) medium expressed number of osteogenic markers and these pMSC underwent osteogenic differentiation most efficiently, in comparison to porcine mesenchymal stem cells grown in other media. In conclusion, osteogenic differentiation potential of pMSC maintained in αMEM/aDMEM medium was observed significantly higher compared to cells cultivated in other media and therefore, the combined medium αMEM/aDMEM (1:1) may preferentially be used for expansion of pMSC, if needed for osteogenic differentiation.

Transcriptional Landscape of the Microenvironment in Bone Marrow Fibrosis at Single Cell Level

Although the molecular alterations in hematopoietic cells which drive the development of myeloproliferative neoplasms (MPN) have been largely defined, reactive cellular alterations in the non-hematopoietic compartment remain rather obscure and have not been studied at single cell level. We therefore profiled enriched non-hematopoietic bone marrow cells by scRNAseq in bone marrow (BM) fibrosis compared to healthy marrow. BM fibrosis was induced by transplantation of hematopoietic stem and progenitor cells (HSPCs) with overexpression of Thrombopoietin (ThPO) into lethally irradiated mice. As ThPO-overexpression robustly leads to reticulin fibrosis in all mice (100%), we were able to study 1) pre-fibrosis (5 weeks after transplantation; reticulin fibrosis grade 0) and 2) manifest bone marrow fibrosis (10 weeks after transplantation, reticulin grade 2-3). The analysis revealed a total of 8 distinct clusters: 1-4) subpopulations of mesenchymal stromal cells (MSC-1: adipogenic, MSC-2: osteogenic, MSC-3: transition, MSC-4: interferonhigh), 5) osteoblastic lineage cells (OLCs), 6) arterial endothelial cells (ECs) and 7-8) Schwann cell precursors (SCP-1: non-myelinating SCPs; SCP-2: myelinating SCPs). Exposure to ThPO overexpressing HSPCs resulted in an overrepresentation of adipogenic MSCs at the expense of all other MSC subclusters. Differential gene expression analysis revealed a functional reprogramming of the "adipogenic" expanding MSCs with down-regulation of hematopoiesis-support and induction of a secretory phenotype including upregulation of various extracellular matrix (ECM) proteins driving fibrosis. Interestingly, only two MSC subclusters gained significant ECM expression indicating myofibroblast differentiation. Expansion of OLCs in BM fibrosis suggested a differentiation of the underrepresented MSC subpopulations into osteolineage cells which was confirmed by pseudotime analysis. Myelinating SCPs, highly expressing interleukin-33 (IL-33), showed the largest expansion in fibrosis. IL-33 is described to play a significant role in solid organ fibrosis by having both pro- and anti-fibrotic effects. Nerve injury triggers the expansion of myelinating and non-myelinating Schwann cells to promote repair, suggesting that mSCPs increase as compensatory and regenerative mechanism for the previously described MPN-induced sympathetic neuropathy. Dissection of cellular and molecular alterations in pre-fibrosis and manifest fibrosis demonstrated that only one MSC subpopulation was already significantly expanded in the pre-fibrotic phase, but only showed minor transcriptional changes. The upregulation of ECM proteins, osteogenesis as well as proinflammatory genes were hallmark features of manifest fibrosis. Interestingly, the overrepresentation of IL-33 expressing mSCPs was more pronounced in the pre-fibrotic phase, indicating that the expansion is a regenerative phenomenon failing in the stages of manifest fibrosis. Our findings were validated in the clinically relevant JAK2(V617F)-induced model of myelofibrosis. In conclusion, we here identified two distinct MSC subsets that are pro-fibrotic and contribute to osteosclerosis in PMF. The functional reprogramming of these MSCs in the bone marrow niche was accompanied by expansion of mSCPs with regenerative capacities, most likely caused by neural damage and Schwann cell death triggered by mutant HSCs. Disclosures Crysandt: Amgem: Other: travel grant; Pfizer: Other: travel grant; Gilead: Other: travel grant; Incyte: Membership on an entity's Board of Directors or advisory committees; celgene: Other: travel grant. Koschmieder:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers-Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Shire: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Ariad: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; AOP Pharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; CTI: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis Foundation: Research Funding.

Harnessing MSC Osteoprogenitor Subpopulations to Augment Hematopoietic Transplantations

Adjuvant MSC infusions could improve transplantation outcomes via suppression of graft-vs-host reactions and enhancement of HSPC engraftment. However, bone marrow (BM) MSCs are a heterogeneous cell population, particularly when culture expanded to obtain therapeutically relevant cell numbers. A systematic study of expanded subpopulations is hindered by the lack of unique surface markers. In lieu of immunophenotype, we hypothesized and verified that biophysical properties of MSCs can be a useful strategy for isolating unique subpopulations. Here, we will describe a clinically relevant biophysical strategy for deriving different MSC subpopulations and discuss their potential utility in the context of hematopoietic transplantation. Cell diameter, membrane stiffness and nuclear membrane fluctuations are collectively predictive of at least two MSC subpopulations (multipotent and osteoprogenitors). Both MSC subpopulations are distinct in their transcriptome (different signatures of VEGF, IL8, ANG1, etc) and differentiation potential (osteogenic and adipogenic) but no surface marker reliably distinguishes them apart. In functional assays, osteoprogenitor vs multipotent MSCs show stronger capacities for CD34+CD38−HSPC expansion (Day 10: 42.7 ± 6.21 vs 29.1 ± 8.27, n = 5, respectively), but no significant differences (n = 3 biological repeats) in their ability to suppress CD8+T-cell proliferation in mixed lymphocyte reactions. When used as a rescue therapy for lethal irradiation, osteoprogenitors improved survival rates by 30-40% compared to multipotent MSCs. Part of the recovery process was prompted by MSC secretome mediated stimulation of BM tissue repair, including increased cell proliferation, reduced inflammation and the restoration of niche environments. These paracrine effects may be beneficial in fostering favorable post-myeloablative BM environments for donor HSPCs to home, engraft and survive. In murine transplantation models, we observed a higher rate of early engraftment following adjuvant therapy with osteoprogenitors. Week 4 CD45 chimerism in peripheral blood was 2- to 3- fold higher (∼11% vs ∼4%) in the osteoprogenitor treated group compared to the other MSC groups. These results demonstrate heterogeneity in expanded MSCs populations that would lead to inconsistencies in their overall properties. Newer biophysical technologies have the potential for isolating useful subpopulations defined by key quality attributes for specific forms of MSC-therapy; these capabilities may advance MSC-based therapy for regenerative medicine. Here, we demonstrate the augmentation of HSPC transplants in murine models using a biophysically isolated osteogenic MSC subpopulation (osteoprogenitors) which led to the fastest engraftment rates; this may be due to their superior abilities for promoting BM tissue regeneration as well as hematopoietic expansion.

Human bone marrow-derived MSCs spontaneously express specific Schwann cell markers.

In peripheral nerve injuries, Schwann cells (SC) play pivotal roles in regenerating damaged nerve. However, the use of SC in clinical cell-based therapy is hampered due to its limited availability. In this study, we aim to evaluate the effectiveness of using an established induction protocol for human bone marrow derived-MSC (hBM-MSCs) transdifferentiation into a SC lineage. A relatively homogenous culture of hBM-MSCs was first established after serial passaging (P3), with profiles conforming to the minimal criteria set by International Society for Cellular Therapy (ISCT). The cultures (n = 3) were then subjected to a series of induction media containing β-mercaptoethanol, retinoic acid, and growth factors. Quantitative RT-PCR, flow cytometry, and immunocytochemistry analyses were performed to quantify the expression of specific SC markers, that is, S100, GFAP, MPZ and p75 NGFR, in both undifferentiated and transdifferentiated hBM-MSCs. Based on these analyses, all markers were expressed in undifferentiated hBM-MSCs and MPZ expression (mRNA transcripts) was consistently detected before and after transdifferentiation across all samples. There was upregulation at the transcript level of more than twofolds for NGF, MPB, GDNF, p75 NGFR post-transdifferentiation. This study highlights the existence of spontaneous expression of specific SC markers in cultured hBM-MSCs, inter-donor variability and that MSC transdifferentiation is a heterogenous process. These findings strongly oppose the use of a single marker to indicate SC fate. The heterogenous nature of MSC may influence the efficiency of SC transdifferentiation protocols. Therefore, there is an urgent need to re-define the MSC subpopulations and revise the minimal criteria for MSC identification.

Mesenchymal Stem Cell Functionalization for Enhanced Therapeutic Applications.

Culture expansion of MSCs has detrimental effects on various cell characteristics and attributes (e.g., phenotypic changes and senescence), which, in addition to inherent interdonor variability, negatively impact the standardization and reproducibility of their therapeutic potential. The identification of innate distinct functional MSC subpopulations, as well as the description of ex vivo protocols aimed at maintaining phenotypes and enhancing specific functions have the potential to overcome these limitations. The incorporation of those approaches into cell-based therapy would significantly impact the field, as more reproducible clinical outcomes may be achieved.

Microarray analysis after adipose derived mesenchymal stem cells injection in monosodium iodoacetate-induced osteoarthritis rats.

Osteoarthritis (OA) is a degenerative joint disease characterized by abrasion, and ultimately, destruction of the articular cartilage and trabecular bone loss. OA is still considered a devastating disease, which requires an aggressive therapeutic approach. Despite the therapeutic potential of human adipose-derived mesenchymal stem cells (AD-MSCs), the molecular parameters needed to define the "stemness" remain largely unknown. Using high-density oligonucleotide microarrays, the differential gene expression profiles between a fraction of human adipose-derived (AD) mononuclear cells and its MSC subpopulation were obtained. Of particular interest was a subset of 58 genes preferentially expressed at sevenfold or higher in the group treated with human AD-MSCs. This subset contained numerous genes involved in the inflammatory response, immune response, lipid metabolism, cell death, cell proliferation, and DNA repair. Additionally, four protein networks were constructed. The interaction network consisted of 46 proteins encoded by up-regulated genes. However, the interaction network also consisted of 38 proteins encoded by down-regulated genes. My results provide a basis for a more reproducible and reliable quality control using genotypic analysis for the definition of human AD-MSCs. Therefore, these results will provide a basis for studies on molecular mechanisms controlling the core properties of human MSCs.

In vitro cell surface markers are insufficient to identify in vivo/in situ multipotent synovial mesenchymal stem cells isolated from normal or osteoarthritic knees

Purpose: Osteoarthritis (OA) is a chronic disease that results in the degeneration of the articular cartilage, but also effects all tissues present in the joint. Current treatments for OA patients focus on pain/symptom relief, however, the core persisting issue of cartilage degeneration remains unmodified. Synovial mesenchymal stem cells derived from the synovium (sMSCs), are an attractive source for articular cartilage regeneration since they demonstrate robust chondrogenic capacity. However, it is now becoming accepted that MSC populations can demonstrate significant phenotypically heterogeneity, which in turn, may affect their potential therapeutic efficacy. Furthermore, it remains unknown if this heterogeneity is natural (e.g. multiple MSC sub-populations present) or if MSCs are influenced by factors in vivo (such as the disease stage of the individual) and/or in vitro (such as culture microenvironment). Additionally, the methods used to define MSCs, such as marker profile could be influenced by varying laboratory/cell culture conditions suggesting that cell surface markers present on MSCs in vitro could be distinct from those that are present in vivo. Therefore, it may be possible that in vitro defined MSC markers may be unreliable to use as a standard to isolate MSCs directly from in vivo/in situ which would be necessary to understand and characterize MSC heterogeneity. A more complete understanding of MSC heterogeneity would allow us to identify and purify only sMSCs that have both self-renewal capacity as well as tri-lineage potential. To address this gap in knowledge we re-assessed the cell surface markers in vivo that are widely used to identify and purify MSCs in vitro. To accomplish this aim and also address questions on MSC heterogeneity, we examined the cell surface marker profiles (in vivo/in situ vs. in vitro) between clonal sMSCs isolated from OA and normal individuals. Additionally, we correlated the cell surface profile in situ with the tri-lineage potential to determine if the differentiation potential of sMSCs can be predicted based on their in situ cell surface expression profile. Methods: Synovium tissue was collected from knees of late stage OA patients (n = 15) and normal controls (n = 21). These biopsies were digested with collagenase type IV and the cell suspension was stained for: MSCs markers (CD90, CD44, CD73, CD271, CD105), a macrophage marker (CD68), and a viability marker (FVS510). An unbiased index sorted approach was utilized where 1 cell/well (per 96 well plate) was sorted based on the absence of CD68 and FVS510. Expanded clones were differentiated into adipocytes, chondrocytes and osteoblasts. Differentiation was analyzed via qPCR and histology. Each in vitro expanded clonal population was also re-examined for surface marker expression. Results: Figure 1 displays the differentiation capacity of 4 clones isolated from a single OA patient. Regardless of the in situ marker profiles expression, all sMSC clones, when cultured in vitro, converged to a unified marker profile of: CD90+, CD44+, CD73+ and CD105+. The in situ marker profile that was correlated with multipotent differentiation capacity of sMSCs was found to be similar between OA and normal clones (CD90+, CD44+, and CD73+). A second cohort of patients was recruited to validate the in situ MSC profile, however, it was found that isolation of CD90+, CD44+, and CD73+ triple positive cells didn't guarantee multipotent differentiation potential as tested by qPCR and histology analysis. Conclusions: This study has demonstrated that using in vitro defined MSC markers to select sMSCs populations directly from human synovial tissue is not specific and the markers currently used by the field are not capable of distinguishing MSCs with or without full differentiation capacity if employed in situ/in vivo. Furthermore, after reviewing in situ vs. in vitro surface marker expression data, it was found that all sMSC sub-populations present in situ (based on marker expression) converge to an indiscriminate profile after culturing. Overall, this suggests that at least some of the MSC markers employed are likely artifacts of the cell culturing process. Thus, the results of this project raise a potentially larger question regarding the identification of MSCs with these standard markers. Do these markers truly describe MSCs? This question should be thoroughly addressed before MSC therapies are continued to be delivered to patients suffering from OA.

Microarray analysis after adipose derived mesenchymal stem cells injection in monosodium iodoacetate-induced osteoarthritis rats

Osteoarthritis (OA) is a degenerative joint disease characterized by abrasion, and ultimately, destruction of the articular cartilage and trabecular bone loss. OA is still considered a devastating disease, which requires an aggressive therapeutic approach. Despite the therapeutic potential of human adipose-derived mesenchymal stem cells (AD-MSCs), the molecular parameters needed to define the “stemness” remain largely unknown. Using high-density oligonucleotide microarrays, the differential gene expression profiles between a fraction of human adipose-derived (AD) mononuclear cells and its MSC subpopulation were obtained. Of particular interest was a subset of 58 genes preferentially expressed at sevenfold or higher in the group treated with human AD-MSCs. This subset contained numerous genes involved in the inflammatory response, immune response, lipid metabolism, cell death, cell proliferation, and DNA repair. Additionally, four protein networks were constructed. The interaction network consisted of 46 proteins encoded by up-regulated genes. However, the interaction network also consisted of 38 proteins encoded by down-regulated genes. My results provide a basis for a more reproducible and reliable quality control using genotypic analysis for the definition of human AD-MSCs. Therefore, these results will provide a basis for studies on molecular mechanisms controlling the core properties of human MSCs.

VCAM-1+ placenta chorionic villi-derived mesenchymal stem cells display potent pro-angiogenic activity.

IntroductionMesenchymal stem cells (MSCs) represent a heterogeneous cell population that is promising for regenerative medicine. The present study was designed to assess whether VCAM-1 can be used as a marker of MSC subpopulation with superior angiogenic potential.MethodsMSCs were isolated from placenta chorionic villi (CV). The VCAM-1+/− CV-MSCs population were separated by Flow Cytometry and subjected to a comparative analysis for their angiogenic properties including angiogenic genes expression, vasculo-angiogenic abilities on Matrigel in vitro and in vivo, angiogenic paracrine activities, cytokine array, and therapeutic angiogenesis in vascular ischemic diseases.ResultsAngiogenic genes, including HGF, ANG, IL8, IL6, VEGF-A, TGFβ, MMP2 and bFGF, were up-regulated in VCAM-1+CV-MSCs. Consistently, angiogenic cytokines especially HGF, IL8, angiogenin, angiopoitin-2, μPAR, CXCL1, IL-1β, IL-1α, CSF2, CSF3, MCP-3, CTACK, and OPG were found to be significantly increased in VCAM-1+ CV-MSCs. Moreover, VCAM-1+CV-MSCs showed remarkable vasculo-angiogenic abilities by angiogenesis analysis with Matrigel in vitro and in vivo and the conditioned medium of VCAM-1+ CV-MSCs exerted markedly pro-proliferative and pro-migratory effects on endothelial cells compared to VCAM-1−CV-MSCs. Finally, transplantation of VCAM-1+CV-MSCs into the ischemic hind limb of BALB/c nude mice resulted in a significantly functional improvement in comparison with VCAM-1−CV-MSCs transplantation.ConclusionsVCAM-1+CV-MSCs possessed a favorable angiogenic paracrine activity and displayed therapeutic efficacy on hindlimb ischemia. Our results suggested that VCAM-1+CV-MSCs may represent an important subpopulation of MSC for efficient therapeutic angiogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0297-0) contains supplementary material, which is available to authorized users.

Where is the common ground between bone marrow mesenchymal stem/stromal cells from different donors and species?

Mesenchymal stem/stromal cells (MSCs) feature promising potential for cellular therapies, yet significant progress in development of MSC therapeutics and assays is hampered because of remarkable MSC heterogeneity in vivo and in vitro. This heterogeneity poses challenges for standardization of MSC characterization and potency assays as well as for MSC study comparability and manufacturing. This review discusses promising marker combinations for prospective MSC subpopulation enrichment and expansion, and reflects MSC phenotype changes due to environment and age. In order to address animal modelling in MSC biology, comparison of mouse and human MSC markers highlights current common ground of MSCs between species.

The Influence of Oxygen on the Proliferative Capacity and Differentiation Potential of Lacrimal Gland-Derived Mesenchymal Stem Cells.

The application of lacrimal gland-derived mesenchymal stem cells (LG-MSC) for the regeneration of lacrimal gland tissue could result in a novel therapy for dry-eye syndrome. To optimize the culture conditions, the purpose of this study was to evaluate the influence of low oxygen on phenotype, differentiation potential, proliferative, and regenerative capacity of murine LG-MSC. Murine LG-MSC were cultured in 21% and 5% oxygen and characterized by flow cytometry, cell sorter assisted proliferation-, and colony forming unit-assays. Reactive oxygen species (ROS) levels as well as lineage differentiation were evaluated. The effect of conditioned medium of LG-MSC from both oxygen conditions (CM MSC 21%, respectively, CM MSC 5%) on lacrimal gland epithelial cells (LG-EC) was examined in wound healing and proliferation assays. Cells under both culture conditions revealed differentiation potential and presented a MSC-specific flow cytometric phenotype. In 5% oxygen, cells yielded less ROS, showed a stable morphology, higher colony forming potential, and an increased proliferation capacity. Five percent oxygen significantly increased the number of CD44+ LG-MSC. Furthermore, CM MSC 5% significantly enhanced migration and proliferation in LG-EC. In vitro expansion in low oxygen preserves the proliferation capacity and differentiation potential of LG-MSC and increases the effects of conditioned medium on migration and proliferation in LG-EC. Therefore, expansion in low oxygen seems to be an excellent method, to obtain vital MSC. Also, an increased number of LG-MSC expressing CD44 was observed under low oxygen, which might be a valuable marker to identify a potent MSC subpopulation.

Progerin expression disrupts critical adult stem cell functions involved in tissue repair.

Vascular disease is one of the leading causes of death worldwide. Vascular repair, essential for tissue maintenance, is critically reduced during vascular disease and aging. Efficient vascular repair requires functional adult stem cells unimpaired by aging or mutation. One protein candidate for reducing stem cell?mediated vascular repair is progerin, an alternative splice variant of lamin A. Progerin results from erroneous activation of cryptic splice sites within the LMNA gene, and significantly increases during aging. Mutations triggering progerin overexpression cause the premature aging disorder Hutchinson-Gilford Progeria Syndrome (HGPS), in which patients die at approximately 13-years of age due to atherosclerosis-induced disease. Progerin expression affects tissues rich in cells that can be derived from marrow stromal cells (MSCs. Studies using various MSC subpopulations and models have led to discrepant results. Using a well-defined, immature subpopulation of MSCs, Marrow Isolated Adult Multilineage Inducible (MIAMI) cells, we find progerin significantly disrupts expression and localization of self-renewal markers, proliferation, migration, and membrane elasticity. One potential treatment, farnesyltransferase inhibitor, ameliorates some of these effects. Our results confirm proposed progerin-induced mechanisms and suggest novel ways in which progerin disturbs critical stem cell functions collectively required for proper tissue repair, offering promising treatment targets for future therapies.

Functional potentials of human hematopoietic progenitor cells are maintained by mesenchymal stromal cells and not impaired by plerixafor

Background aimsMesenchymal stromal cells (MSCs) resemble an essential component of the bone marrow niche for maintenance of stemness of hematopoietic progenitor cells (HPCs). Perturbation of the C-X-C chemokine receptor type 4 (CXCR4)/stromal cell-derived factor-1α (SDF-1α) axis by plerixafor (AMD3100) mobilizes HPCs from their niche; however, little is known about how plerixafor affects interaction of HPCs and MSCs in vitro. MethodsWe monitored cell division kinetics, surface expression of CD34 and CXCR4, migration behavior and colony-forming frequency of HPCs on co-culture with MSCs either with or without exposure to plerixafor. ResultsCo-culture with MSCs significantly accelerated cell division kinetics of HPCs. Despite this, the proportion of CD34+ cells was significantly increased on co-culture, whereas the expression of CXCR4 was reduced. In addition, co-culture with MSCs led to significantly higher colony-forming capacity and enhanced migration rate of HPCs compared with mono-culture conditions. The composition of MSC sub-populations—and conversely their hematopoiesis supportive functions—may be influenced by culture conditions. We compared the stromal function of MSCs isolated with three different culture media. Overall, the supporting potentials of these MSC preparations were quite similar. Perturbation by the CXCR4-antagonist plerixafor reduced the cell division kinetics of HPCs on co-culture with MSCs. However, the progenitor cell potential of the HPCs as reflected by colony-forming capacity was not affected by plerixafor. ConclusionsThese results support the notion that the CXCR4/SDF-1α axis is critical for HPC-MSC interaction with regard to migration, adhesion and regulation of proliferation but not for maintenance of primitive progenitor cells.