Maria F. Dutreil

Interleukin 1 receptor antagonist mediates the antiinflammatory and antifibrotic effect of mesenchymal stem cells during lung injury

Mesenchymal stem cells (MSCs) have been exploited as cellular vectors to treat a wide array of diseases but the mechanisms responsible for their therapeutic effect remain indeterminate. Previously, we reported that MSCs inhibit bleomycin (BLM)-induced inflammation and fibrosis within the lungs of mice. Interrogation of the MSC transcriptome identified interleukin 1 receptor antagonist (IL1RN) as a potential mediator of this effect. Fractionation studies indicated that MSCs are the principal source of IL1RN in murine bone marrow and that its expression is restricted to a unique subpopulation of cells. Moreover, MSC-conditioned media was shown to block proliferation of an IL-1α-dependent T cell line and inhibit production of TNF-α by activated macrophages in vitro. Studies conducted in mice revealed that MSC administration was more effective than recombinant IL1RN delivered via adenoviral infection or osmotic pumps in inhibiting BLM-induced increases in TNF-α, IL-1α, and IL1RN mRNA in lung, IL1RN protein in bronchoalveolar lavage (BAL) fluid, and trafficking of lymphocytes and neutrophils into the lung. Therefore, MSCs protect lung tissue from BLM-induced injury by blocking TNF-α and IL-1, two fundamental proinflammatory cytokines in lung. Identification of IL1RN-expressing human MSC subpopulations may provide a novel cellular vector for treating chronic inflammatory diseases in humans.

Biological Activities Encoded by the Murine Mesenchymal Stem Cell Transcriptome Provide a Basis for Their Developmental Potential and Broad Therapeutic Efficacy

We used serial analysis of gene expression to catalog the transcriptome of murine mesenchymal stem cells (MSCs) enriched from bone marrow by immunodepletion. Interrogation of this database, results of which are delineated in the appended databases, revealed that immunodepleted murine MSCs (IDmMSCs) highly express transcripts encoding connective tissue proteins and factors modulating T‐cell proliferation, inflammation, and bone turnover. Categorizing the transcriptome based on gene ontologies revealed the cells also expressed mRNAs encoding proteins that regulate mesoderm development or that are characteristic of determined mesenchymal cell lineages, thereby reflecting both their stem cell nature and differentiation potential. Additionally, IDmMSCs also expressed transcripts encoding proteins regulating angiogenesis, cell motility and communication, hematopoiesis, immunity and defense as well as neural activities. Immunostaining and fluorescence‐activated cell sorting analysis revealed that expression of various regulatory proteins was restricted to distinct subpopulations of IDmMSCs. Moreover, in some cases, these proteins were absent or expressed at reduced levels in other murine MSC preparations or cell lines. Lastly, by comparing their transcriptome to that of 17 other murine cell types, we also identified 43 IDmMSC‐specific transcripts, the nature of which reflects their varied functions in bone and marrow. Collectively, these results demonstrate that IDmMSC express a diverse repertoire of regulatory proteins, which likely accounts for their demonstrated efficacy in treating a wide variety of diseases. The restricted expression pattern of these proteins within populations suggests that the cellular composition of marrow stroma and its associated functions are more complex than previously envisioned.

Leptin produced by obese adipose stromal/stem cells enhances proliferation and metastasis of estrogen receptor positive breast cancers

IntroductionThe steady increase in the incidence of obesity among adults has been paralleled with higher levels of obesity-associated breast cancer. While recent studies have suggested that adipose stromal/stem cells (ASCs) isolated from obese women enhance tumorigenicity, the mechanism(s) by which this occurs remains undefined. Evidence suggests that increased adiposity results in increased leptin secretion from adipose tissue, which has been shown to increased cancer cell proliferation. Previously, our group demonstrated that ASCs isolated from obese women (obASCs) also express higher levels of leptin relative to ASCs isolated from lean women (lnASCs) and that this obASC-derived leptin may account for enhanced breast cancer cell growth. The current study investigates the impact of inhibiting leptin expression in lnASCs and obASCs on breast cancer cell (BCC) growth and progression.MethodsEstrogen receptor positive (ER+) BCCs were co-cultured with leptin shRNA lnASCs or leptin shRNA obASCs and changes in the proliferation, migration, invasion, and gene expression of BCCs were investigated. To assess the direct impact of leptin inhibition in obASCs on BCC proliferation, MCF7 cells were injected alone or mixed with control shRNA obASCs or leptin shRNA obASCs into SCID/beige mice.ResultsER+ BCCs were responsive to obASCs during direct co-culture, whereas lnASCs were unable to increase ER+ BCC growth. shRNA silencing of leptin in obASCs negated the enhanced proliferative effects of obASC on BCCs following direct co-culture. BCCs co-cultured with obASCs demonstrated enhanced expression of epithelial-to-mesenchymal transition (EMT) and metastasis genes (SERPINE1, MMP-2, and IL-6), while BCCs co-cultured with leptin shRNA obASCs did not display similar levels of gene induction. Knockdown of leptin significantly reduced tumor volume and decreased the number of metastatic lesions to the lung and liver. These results correlated with reduced expression of both SERPINE1 and MMP-2 in tumors formed with MCF7 cells mixed with leptin shRNA obASCs, when compared to tumors formed with MCF7 cells mixed with control shRNA obASCs.ConclusionThis study provides mechanistic insight as to how obesity enhances the proliferation and metastasis of breast cancer cells; specifically, obASC-derived leptin contributes to the aggressiveness of breast cancer in obese women.

Age-and Dose-Related Effects on MSC Engraftment Levels and Anatomical Distribution in the Central Nervous Systems of Nonhuman Primates : Identification of Novel MSC Subpopulations That Respond to Guidance Cues in Brain

Mesenchymal stem cells (MSCs) have demonstrated efficacy as therapeutic vectors in rodent models of neurological diseases, but few studies have evaluated their safety and efficacy in a relevant large animal model. Previously, we reported that MSCs transplanted to the central nervous systems (CNS) of adult rhesus macaques engrafted at low levels without adversely affecting animal health, behavior, or motor function. Herein, we injected MSCs intracranially into 10 healthy infant macaques and quantified their engraftment levels and mapped their anatomical distribution in brain by real‐time polymerase chain reaction using an sry gene‐specific probe. These analyses revealed that MSC engraftment levels in brain were on average 18‐fold higher with a maximal observed difference of 180‐fold in neonates as compared with that reported previously for young adult macaques. Moreover, engraftment levels were 30‐fold higher after injection of a low versus high cell dose and engrafted MSCs were nonrandomly distributed throughout the infant brain and localized to specific anatomical regions. Identification of unique subpopulations of macaque and human MSCs that express receptor proteins known to regulate tangential migration of interneurons may explain their migration patterns in brain. Extensive monitoring of infant transplant recipients using a battery of age appropriate tests found no evidence of any long‐term adverse effects on the health or social, behavioral, cognitive, or motor abilities of animals up to 6 months post‐transplant. Therefore, direct intracranial injection represents a safe means to deliver therapeutic levels of MSCs to the CNS. Moreover, expressed guidance receptors on MSC subpopulations may regulate migration of cells in the host brain.

Human Adipose Stromal/Stem Cells from Obese Donors Show Reduced Efficacy in Halting Disease Progression in the Experimental Autoimmune Encephalomyelitis Model of Multiple Sclerosis

Multiple sclerosis is an autoimmune disease that affects the white matter of the central nervous system and involves inflammation and demyelination. The recent advances in our understanding of adipose‐derived stromal/stem cells (ASCs) and the utilization of these cells in clinical settings to treat diseases have made it essential to identify the most effective ASCs for therapy. Studies have not yet investigated the impact of obesity on the therapeutic efficacy of ASCs. Obesity is characterized by adipocyte hyperplasia and hypertrophy and can extend to metabolic and endocrine dysfunction. Investigating the impact obesity has on ASC biology will determine whether these cells are suitable for use in regenerative medicine. The therapeutic efficacy of ASCs isolated from lean subjects (body mass index [BMI] < 25; lnASCs) and obese subjects (BMI > 30; obASCs) were determined in murine experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. Compared with the EAE disease‐modifying effects of lnASCs, obASCs consistently failed to alleviate clinical symptoms or inhibit inflammation in the central nervous system. When activated, obASCs expressed higher mRNA levels of several pro‐inflammatory cytokines compared with lnASCs. Additionally, conditioned media (CM) collected from the obASCs markedly enhanced the proliferation and differentiation of T cells; whereas, CM from lnASC did not. These results indicate that obesity reduces, or eliminates, the anti‐inflammatory effects of human ASCs such that they may not be a suitable cell source for the treatment of autoimmune diseases. The data suggest that donor demographics may be particularly important when identifying suitable stem cells for treatment. Stem Cells 2016;34:614–626

Adipose Stromal Vascular Fraction‐Mediated Improvements at Late‐Stage Disease in a Murine Model of Multiple Sclerosis

Multiple sclerosis (MS) is a common neurodegenerative disease and remains an unmet clinical challenge. In MS, an autoimmune response leads to immune cell infiltration, inflammation, demyelination, and lesions in central nervous system (CNS) tissues resulting in tremors, fatigue, and progressive loss of motor function. These pathologic hallmarks are effectively reproduced in the murine experimental autoimmune encephalomyelitis (EAE) model. The stromal vascular fraction (SVF) of adipose tissue is composed of adipose‐derived stromal/stem cells (ASC), adipocytes, and various leukocytes. The SVF can be culture expanded to generate ASC lines. Clinical trials continue to demonstrate the safety and efficacy of ASC therapies for treating several diseases. However, little is known about the effectiveness of the SVF for neurodegenerative diseases, such as MS. At late‐stage disease, EAE mice show severe motor impairment. The goal for these studies was to test the effectiveness of SVF cells and ASC in EAE mice after the onset of neuropathology. The clinical scoring, behavior, motor function, and histopathologic analyses revealed significant improvements in EAE mice treated with the SVF or ASC. Moreover, SVF treatment mediated more robust improvements to CNS pathology than ASC treatment based on significant modulations of inflammatory factors. The most pronounced changes following SVF treatment were the high levels of interleukin‐10 in the peripheral blood, lymphoid and CNS tissues along with the induction of regulatory T cells in the lymph nodes which indicate potent immunomodulatory effects. The data indicate SVF cells effectively ameliorated the EAE immunopathogenesis and supports the potential use of SVF for treating MS. Stem Cells 2017;35:532–544

Obesity inhibits the osteogenic differentiation of human adipose-derived stem cells

BackgroundCraniomaxillofacial defects secondary to trauma, tumor resection, or congenital malformations are frequent unmet challenges, due to suboptimal alloplastic options and limited autologous tissues such as bone. Significant advances have been made in the application of adipose-derived stem/stromal cells (ASCs) in the pre-clinical and clinical settings as a cell source for tissue engineering approaches. To fully realize the translational potential of ASCs, the identification of optimal donors for ASCs will ensure the successful implementation of these cells for tissue engineering approaches. In the current study, the impact of obesity on the osteogenic differentiation of ASCs was investigated.MethodsASCs isolated from lean donors (body mass index <25; lnASCs) and obese donors (body mass index >30; obASCs) were induced with osteogenic differentiation medium as monolayers in an estrogen-depleted culture system and on three-dimensional scaffolds. Critical size calvarial defects were generated in male nude mice and treated with scaffolds implanted with lnASCs or obASCs.ResultslnASCs demonstrated enhanced osteogenic differentiation in monolayer culture system, on three-dimensional scaffolds, and for the treatment of calvarial defects, whereas obASCs were unable to induce similar levels of osteogenic differentiation in vitro and in vivo. Gene expression analysis of lnASCs and obASCs during osteogenic differentiation demonstrated higher levels of osteogenic genes in lnASCs compared to obASCs.ConclusionCollectively, these results indicate that obesity reduces the osteogenic differentiation capacity of ASCs such that they may have a limited suitability as a cell source for tissue engineering.

ENDOGENOUS EXPRESSION OF INTERLEUKIN-1 RECEPTOR ANTAGONIST ISOFORMS BY MURINE MESENCHYMAL STEM CELLS.: 251

Mesenchymal stem cells (MSCs) are pluripotent cells derived from the bone marrow possessing a very high potential to regulate the cellular microenvironment due to expression of a vast array of cytokines, chemokines, and adhesion factors. The objective of this study was to characterize the expression and biological activity of interleukin-1 receptor antagonist (IL-1rn) in immunodepleted murine mesenchymal stem cells (IDmMSCs). We hypothesized that expression of IL-1rn by IDmMSCs may explain, in part, their immunomodulatory activity and ability to ameliorate various injuries/disease states. IL-1rn production of the IDmMSCs was compared with a multipotent murine MSC line, CRL-12424. Screening of an IDmMSC cDNA library by PCR confirmed expression of transcripts corresponding to IL-1rn. To directly evaluate protein expression, IDmMSCs were isolated from bone marrow collected from the long bones of FVB/n mice via immunodepletion. Enzyme-linked immunosorbent assays (ELISAs) quantitatively demonstrated that IDmMSCs produced high levels of the secreted isoform of IL-1rn (sIL-1rn). Production of sIL-1rn in IDmMSCs was fivefold higher than the CRL-12424 cells, with the average secretion of the IDmMSCs totaling 1 ng/mL/cell. Immunofluorescence staining further revealed that intracellular IL-1rn (icIL-1rn) expression was restricted to specific subpopulations of IDmMSCs, and FACS analysis confirmed that 24% of IDmMSCs expressed the protein. Interestingly, FACS analysis of the depleted cell populations demonstrated no expression of icIL-1rn, indicating that expression is specific and limited to the IDmMSCs subpopulation. An in vitro T-cell proliferation assay was employed to demonstrate that conditioned medium from IDmMSCs, containing IL-1rn, inhibited the ability of IL-1α to induce proliferation of the T cells. The results of the assay showed that sIL-1rn in the conditioned media of IDmMSCs completely inhibited T-cell proliferation. Collectively, these results demonstrate that subpopulations of IDmMSCs express high levels of IL-1rn and that this protein can inhibit T-cell proliferation. By counteracting the effects of IL-1 in vivo, IL-1rn may regulate T-cell proliferation and bone metabolism. Additionally, these findings implicate that IL-1rn expression contributes to the immunomodulatory effects of MSCs, as demonstrated in the amelioration of various injuries such as bleomycin-induced lung injury, perhaps via immunomodulation of the cellular microenvironment.

155 MURINE MESENCHYMAL STEM CELLS BLOCK T-CELL PROLIFERATION VIA PRODUCTION OF INTERLEUKIN-1 RECEPTOR ANTAGONIST.

The objective of this study was to characterize the expression of interleukin-1 receptor antagonist (IL-1rn) in immunodepleted murine mesenchymal stem cells (IDmMSCs). To better characterize IDmMSCs, we also catalogued their transcriptome via serial analysis of gene expression (SAGE). Interrogation of the SAGE database revealed an abundance of SAGE tags corresponding specifically to mRNA encoding IL-1rn, a potent anti-inflammatory protein that antagonizes the activity of interleukin-1 (IL-1). Expression of IL-1rn by IDmMSCs may explain, in part, their immunomodulatory activity and ability to ameliorate various injuries. Consequently, we characterized the expression of IL-1rn in IDmMSCs and evaluated its biological activity using a T-cell proliferation assay. Screening an IDmMSC cDNA library by PCR confirmed that the cells expressed transcripts corresponding to IL-1rn, thereby validating the SAGE data. To directly evaluate protein expression, IDmMSCs were isolated from bone marrow collected from the long bones of FVB/n mice via immunodepletion. Briefly, whole bone marrow cells were cultured in a-MEM supplemented with 10% FBS in a humidified chamber with 5 % CO2 at 378C for 4 days, fed with fresh media, and incubated an additional 3-5 days. Cells were then immunodepleted by incubation with M-280 Dynabeads conjugated to anti-CD11b, CD34, and CD45 antibodies. Enzyme-linked immunosorbent assays (ELISA) quantitatively demonstrated that IDmMSCs secreted high levels of IL-1rn protein, 1ng/mL/cell (p < .01). Immunofluorescence staining further revealed that IL-1rn expression was restricted to specific subpopulations of IDmMSCs, and FACS analysis confirmed that approximately 24% of IDmMSCs expressed the protein. An in vitro T-cell proliferation assay was employed to demonstrate that IL-1rn secreted by IDmMSCs inhibited the ability of IL-1 to induce proliferation of the T cells (p < .01). Collectively, these results demonstrate that subpopulations of IDmMSCs express high levels of IL-1rn and that this protein can inhibit T-cell proliferation. By counteracting the effects of IL-1 in vivo, IL-1rn may regulate T-cell proliferation and bone metabolism. Additionally, these findings imply that IL-1rn expression contributes to the immunomodulatory effects of MSCs as demonstrated in the amelioration of various injuries such as bleomycin-induced lung injury.