Passage-dependent accumulation of somatic mutations in mesenchymal stromal cells during in vitro culture revealed by whole genome sequencing.
ABSTRACT: Human mesenchymal stromal cells (MSCs) have served as a major cellular resource for cell-based immunomodulatory and regenerative therapies. However, genomic instability may accumulate during ex vivo expansion of MSCs, thereby increasing the potential of malignant transformation. Here, we performed whole genome sequencing of two peripheral blood-derived MSC lines (MSC1 and MSC2) at various passages (passage 1 [P1] to P9). The majority of single-nucleotide variations (SNVs) occurred in later passages; specifically, 90% and 70% of all SNVs in MSC1 and MSC2 were observed in P9 and P7/P9, respectively. These late-occurring SNVs were enriched with C?>?A transversions and were overrepresented in intronic regions compared to intergenic regions, suggesting that the mutational forces are not constant across the passages. Clonality analyses also distinguished early-occurring, subclonal SNVs from late-occurring, clonally fixed SNVs. In addition, MSCs were largely devoid of copy number alterations (CNAs) (i.e., 0-2 CNAs per passage), with one exception (MSC2-P3) harboring 29 passage-specific CNAs. Our findings suggest that the SNVs found to be abundant at later passages likely resulted from the accumulation of replication stress, which can be associated with proliferation activity. Thus, the genomic instability associated with proliferation records should be considered for clinical applications of MSCs.
Project description:<h4>Background</h4>Currently, there are many promising clinical trials using mesenchymal stem cells (MSCs) in cell-based therapies of numerous diseases. Increasingly, however, there is a concern over the use of MSCs because they home to tumors and can support tumor growth and metastasis. For instance, we established that MSCs in the ovarian tumor microenvironment promoted tumor growth and favored angiogenesis. In parallel studies, we also developed a new approach to induce the conventional mixed pool of MSCs into two uniform but distinct phenotypes we termed MSC1 and MSC2.<h4>Methodology/principal findings</h4>Here we tested the in vitro and in vivo stability of MSC1 and MSC2 phenotypes as well as their effects on tumor growth and spread. In vitro co-culture of MSC1 with various cancer cells diminished growth in colony forming units and tumor spheroid assays, while conventional MSCs or MSC2 co-culture had the opposite effect in these assays. Co-culture of MSC1 and cancer cells also distinctly affected their migration and invasion potential when compared to MSCs or MSC2 treated samples. The expression of bioactive molecules also differed dramatically among these samples. MSC1-based treatment of established tumors in an immune competent model attenuated tumor growth and metastasis in contrast to MSCs- and MSC2-treated animals in which tumor growth and spread was increased. Also, in contrast to these groups, MSC1-therapy led to less ascites accumulation, increased CD45+leukocytes, decreased collagen deposition, and mast cell degranulation.<h4>Conclusion/significance</h4>These observations indicate that the MSC1 and MSC2 phenotypes may be convenient tools for the discovery of critical components of the tumor stroma. The continued investigation of these cells may help ensure that cell based-therapy is used safely and effectively in human disease.
Project description:Nowadays, immune diseases are a large burden in healthcare. Mesenchymal stem cells (MSCs) have prominent ability in immunomodulation and have been applicated on treating many immune-related diseases. However, the clinical outcomes can be disparate and sometimes completely counterproductive beyond explanation of cell heterogeneity. The theory of immunomodulation plasticity in MSCs has then emerged to explain that MSCs can be induced into proinflammatory MSC1 or anti-inflammatory MSC2 responding to different immune environment. It would be safer and more efficient if we could induce MSCs into a certain immune phenotype, in most cases MSC2, prior to medical treatment. In this study, we screened and identified a classical FDA-approved drug, chlorzoxazone (CZ). Unlike traditional method induced by IFN-?, CZ can induce MSC into MSC2 phenotype and enhance the immunosuppressive capacity without elevation of immunogenicity of MSCs. CZ-treated MSCs can better inhibit T cells activation and proliferation, promote expression of IDO and other immune mediators in vitro, and alleviate inflammatory infiltration and tissue damage in acute kidney injury rat model more effectively. Moreover, we discovered that CZ modulates phosphorylation of transcriptional factor forkhead box O3 (FOXO3) independent of classical AKT or ERK signaling pathways, to promote expression of downstream immune-related genes, therefore contributing to augmentation of MSCs immunosuppressive capacity. Our study established a novel and effective approach to induce MSC2, which is ready for clinical application.
Project description:Production of human mesenchymal stem cells for allogeneic cell therapies requires scalable, cost-effective manufacturing processes. Microcarriers enable the culture of anchorage-dependent cells in stirred-tank bioreactors. However, no robust, transferable methodology for microcarrier selection exists, with studies providing little or no reason explaining why a microcarrier was employed. We systematically evaluated 13 microcarriers for human bone marrow-derived MSC (hBM-MSCs) expansion from three donors to establish a reproducible and transferable methodology for microcarrier selection. Monolayer studies demonstrated input cell line variability with respect to growth kinetics and metabolite flux. HBM-MSC1 underwent more cumulative population doublings over three passages in comparison to hBM-MSC2 and hBM-MSC3. In 100 mL spinner flasks, agitated conditions were significantly better than static conditions, irrespective of donor, and relative microcarrier performance was identical where the same microcarriers outperformed others with respect to growth kinetics and metabolite flux. Relative growth kinetics between donor cells on the microcarriers were the same as the monolayer study. Plastic microcarriers were selected as the optimal microcarrier for hBM-MSC expansion. HBM-MSCs were successfully harvested and characterised, demonstrating hBM-MSC immunophenotype and differentiation capacity. This approach provides a systematic method for microcarrier selection, and the findings identify potentially significant bioprocessing implications for microcarrier-based allogeneic cell therapy manufacture.
Project description:Background Mesenchymal stem cells (MSCs) have been recognized for their regenerative and anti-inflammatory capacity which makes them very attractive to cell therapy, especially those ones to treat inflammatory and autoimmune disease. Two different immune-phenotypes have been described for MSCs depending on which Toll-like receptor (TLR) is activated. MSC1 is endowed with a pro-inflammatory phenotype following TLR4 activation with LPS. On the other hand, anti-inflammatory MSC2 is induced by the activation of TLR3 with Poly(I:C). High immunoplasticity of MSCs is a matter of concern in cell-based therapies. In this study, we investigated whether a single stimulus can induce both types of MSCs through a differential activation of TLR4 with LPS. Methods MSCs were activated with LPS following a short exposure of 1-h (MSCs-LPS1h) or long-time exposure for 48?h (MSCs-LPS48h), and then, we evaluated the biological response in vitro, the immunosuppressive capacity of MSCs in vitro, and the therapeutic potential of MSCs in an experimental autoimmune encephalomyelitis (EAE) mouse model. Results Our results showed that 1-h LPS exposure induced a MSC1 phenotype. Indeed, MSCs-LPS1h expressed low levels of NO/iNOS and decreased immunosuppressive capacity in vitro without therapeutic effect in the EAE model. In contrast, MSCs-LPS48h achieved a MSC2-like phenotype with significant increase in the immunosuppressive capacity on T cell proliferation in vitro, together with an improved in the therapeutic effect and higher Treg, compared to unstimulated MSCs. Furthermore, we determine through the MSCs-TLR4KO that the expression of TLR4 receptor is essential for MSCs’ suppressive activity since TLR4 deletion was associated with a diminished suppressive effect in vitro and a loss of therapeutic effect in vivo. Conclusions We demonstrate that MSCs display a high immunoplasticity commanded by a single stimulus, where LPS exposure time regulated the MSC suppressive effect leading into either an enhanced or an impairment therapeutic activity. Our results underscore the importance of phenotype conversion probably related to the TLR4 expression and activation, in the design of future clinical protocols to treat patients with inflammatory and autoimmune diseases.
Project description:It's still unknown how over-hyperplasia of tissue such like new bone formation (NBF) developed in ankylosing spondylitis (AS). We found low level of IL-17A promoted TLR4+MSC1 polarization with suppressed osteogenic differentiation through JAK2/STAT3 pathway, while high level of IL-17A promoted TLR3+MSC2 polarization with enhanced osteogenic differentiation through WNT10b/RUNX2 pathway. Furthermore, both proteoglycan-induced spondylitis (PGISp) mouse model and AS patients without NBF showed MSC1 polarization, up-regulated JAK2/STAT3 pathway and high level of IL-17A (peripherally, but not locally), but those with NBF showed MSC2 polarization, up-regulated WNT10b/RUNX2 pathway and high expression of IL-17A at local site. Results showed NBF of AS was induced by MSC2 polarization that was promoted by high level of IL-17A, and may be treated by suppressing local MSC2 polarization.
Project description:Our laboratory and others reported that the stimulation of specific Toll-like receptors (TLRs) affects the immune modulating responses of human multipotent mesenchymal stromal cells (hMSCs). Toll-like receptors recognize "danger" signals, and their activation leads to profound cellular and systemic responses that mobilize innate and adaptive host immune cells. The danger signals that trigger TLRs are released following most tissue pathologies. Since danger signals recruit immune cells to sites of injury, we reasoned that hMSCs might be recruited in a similar way. Indeed, we found that hMSCs express several TLRs (e.g., TLR3 and TLR4), and that their migration, invasion, and secretion of immune modulating factors is drastically affected by specific TLR-agonist engagement. In particular, we noted diverse consequences on the hMSCs following stimulation of TLR3 when compared to TLR4 by our low-level, short-term TLR-priming protocol.Here we extend our studies on the effect on immune modulation by specific TLR-priming of hMSCs, and based on our findings, propose a new paradigm for hMSCs that takes its cue from the monocyte literature. Specifically, that hMSCs can be polarized by downstream TLR signaling into two homogenously acting phenotypes we classify here as MSC1 and MSC2. This concept came from our observations that TLR4-primed hMSCs, or MSC1, mostly elaborate pro-inflammatory mediators, while TLR3-primed hMSCs, or MSC2, express mostly immunosuppressive ones. Additionally, allogeneic co-cultures of TLR-primed MSCs with peripheral blood mononuclear cells (PBMCs) predictably lead to suppressed T-lymphocyte activation following MSC2 co-culture, and permissive T-lymphocyte activation in co-culture with MSC1.Our study provides an explanation to some of the conflicting reports on the net effect of TLR stimulation and its downstream consequences on the immune modulating properties of stem cells. We further suggest that MSC polarization provides a convenient way to render these heterogeneous preparations of cells more uniform while introducing a new facet to study, as well as provides an important aspect to consider for the improvement of current stem cell-based therapies.
Project description:The immunomodulatory capacity of mesenchymal stem cells (MSCs) is critical for their use in therapeutic applications. MSC response to specific inflammatory cues allows them to switch between a proinflammatory (MSC1) or anti-inflammatory (MSC2) phenotype. Regulatory mechanisms controlling this switch remain to be defined. One characteristic feature of MSC2 is their ability to respond to IFN? with induction of indoleamine 2,3-dioxygenase (IDO), representing the key immunoregulatory molecule released by human MSC. Here, we show that STAT1 and PI3K? pathways interplay regulates IFN?-induced IDO production in MSC. Chemical phosphoinositide 3-kinase (PI3K) pan-inhibition, PI3K?-specific inhibition or shRNA knockdown diminished IFN?-induced IDO production. This effect involved PI3K?-mediated upregulation of STAT1 protein levels and phosphorylation at Ser727. Overexpression of STAT1 or of a constitutively active PI3K? mutant failed to induce basal IDO production, but shifted MSC into an MSC2-like phenotype by strongly enhancing IDO production in response to IFN? as compared to controls. STAT1 overexpression strongly enhanced MSC-mediated T-cell suppression. The same effect could be induced using short-term pretreatment of MSC with a chemical inhibitor of the counter player of PI3K, phosphatase and tensin homolog. Finally, downregulation of STAT1 abrogated the immunosuppressive capacity of MSC. Our results for the first time identify critical upstream signals for the induced production of IDO in MSCs that could be manipulated therapeutically to enhance their immunosuppressive phenotype.
Project description:The purpose of this prospective study was to evaluate the effects of single and repeated intra-articular administration of allogeneic, umbilical cord-derived, neonatal mesenchymal stem cells (MSC) in horses with lameness due to osteoarthritis (OA) of a metacarpophalangeal joint (MPJ). Twenty-eight horses were included. Horses were divided into two groups. Horses in group MSC1 received an MSC injection at M0 and a placebo injection at M1 (1 month after M0). Horses in group MSC2 received MSC injections at M0 and at M1. Joint injections were performed with a blinded syringe. Clinical assessment was performed by the treating veterinarian at M1, M2 and M6 (2 and 6 months after M0), including lameness evaluation, palpation and flexion of the joint. Radiographic examination of the treated joints was performed at inclusion and repeated at M6. Radiographs were anonymized and assessed by 2 ECVDI LA associate members. Short term safety assessment was performed by owner survey. A 2-month rehabilitation program was recommended to veterinarians. There was a significant improvement of the total clinical score for horses in both groups. There was no significant difference in the total clinical score between groups MSC1 and MSC2 at any time point in the study. There was no significant difference in the total radiographic OA score, osteophyte score, joint space width score and subchondral bone score between inclusion and M6. Owner-detected adverse effects to MSC injection were recorded in 18% of the horses. Lameness caused by OA improved significantly over the 6-month duration of the study after treatment with allogeneic neonatal umbilical cord-derived MSCs combined with 8 weeks rest and rehabilitation. There is no apparent clinical benefit of repeated intra-articular administration of MSCs at a 1-month interval in horses with MPJ OA when compared to the effect of a single injection.
Project description:Adult mesenchymal stem cells (MSCs) hold great promise for regenerative medicine because of their self-renewal, multipotency, and trophic and immunosuppressive effects. Due to the rareness and high heterogeneity of freshly isolated MSCs, extensive in-vitro passage is required to expand their populations prior to clinical use; however, senescence usually accompanies and can potentially affect MSC characteristics and functionality. Therefore, a thorough characterization of the variations in phenotype and differentiation potential of in-vitro aging MSCs must be sought.Human bone marrow-derived MSCs were passaged in vitro and cultivated with either DMEM-based or ?MEM-based expansion media. Cells were prepared for subculture every 10 days up to passage 8 and were analyzed for cell morphology, proliferative capacity, and surface marker expression at the end of each passage. The gene expression profile and adipogenic and osteogenic differentiation capability of MSCs at early (passage 4) and late (passage 8) passages were also evaluated.In-vitro aging MSCs gradually lost the typical fibroblast-like spindle shape, leading to elevated morphological abnormality and inhomogeneity. While the DMEM-based expansion medium better facilitated MSC proliferation in the early passages, the cell population doubling rate reduced over time in both DMEM and ?MEM groups. CD146 expression decreased with increasing passage number only when MSCs were cultured under the DMEM-based condition. Senescence also resulted in MSCs with genetic instability, which was further regulated by the medium recipe. Regardless of the expansion condition, MSCs at both passages 4 and 8 could differentiate into adipocyte-like cells whereas osteogenesis of aged MSCs was significantly compromised. For osteogenic induction, use of the ?MEM-based expansion medium yielded longer osteogenesis and better quality.Human MSCs subjected to extensive in-vitro passage can undergo morphological, phenotypic, and genetic changes. These properties are also modulated by the medium composition employed to expand the cell populations. In addition, adipogenic potential may be better preserved over osteogenesis in aged MSCs, suggesting that MSCs at early passages must be used for osteogenic differentiation. The current study presents valuable information for future basic science research and clinical applications leading to the development of novel MSC-based therapeutic strategies for different diseases.
Project description:Because of their unique ability to modulate the immune system, mesenchymal stromal cells (MSCs) are widely studied to develop cell therapies for detrimental immune and inflammatory disorders. However, controlling the final cell phenotype and determining immunosuppressive function following cell amplification in vitro often requires prolonged cell culture assays, all of which contribute to major bottlenecks, limiting the clinical emergence of cell therapies. For instance, the multipotent Wharton's Jelly mesenchymal stem/stromal cells (WJMSC), extracted from human umbilical cord, exhibit immunosuppressive traits under pro-inflammatory conditions, in the presence of interferon-? (IFN?), and tumor necrosis factor-? (TNF?). However, WJMSCs require co-culture bioassays with immune cells, which can take days, to confirm their immunomodulatory function. Therefore, the establishment of robust cell therapies would benefit from fast and reliable characterization assays. To this end, we have explored the metabolic behaviour of WJMSCs in in vitro culture, to identify biomarkers that are specific to the cell passage effect and the loss of their immunosuppressive phenotype. We clearly show distinct metabolic behaviours comparing WJMSCs at the fourth (P4) and the late ninth (P9) passages, although both P4 and P9 cells do not exhibit significant differences in their low immunosuppressive capacity. Metabolomics data were analysed using an in silico modelling platform specifically adapted to WJMSCs. Of interest, P4 cells exhibit a glycolytic metabolism compared to late passage (P9) cells, which show a phosphorylation oxidative metabolism, while P4 cells show a doubling time of 29 h representing almost half of that for P9 cells (46 h). We also clearly show that fourth passage WJMSCs still express known immunosuppressive biomarkers, although, this behaviour shows overlapping with a senescence phenotype.