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Early Immunomodulatory Effects of Implanted Human Perivascular Stromal Cells During Bone Formation.

ABSTRACT: Human perivascular stem/stromal cells (PSC) are a multipotent mesodermal progenitor cell population defined by their perivascular residence. PSC are most commonly derived from subcutaneous adipose tissue, and recent studies have demonstrated the high potential for clinical translation of this fluorescence-activated cell sorting-derived cell population for bone tissue engineering. Specifically, purified PSC induce greater bone formation than unpurified stroma taken from the same patient sample. In this study, we examined the differences in early innate immune response to human PSC or unpurified stroma (stromal vascular fraction [SVF]) during the in vivo process of bone formation. Briefly, SVF or PSC from the same patient sample were implanted intramuscularly in the hindlimb of severe combined immunodeficient (SCID) mice using an osteoinductive demineralized bone matrix carrier. Histological examination of early inflammatory infiltrates was examined by hematoxylin and eosin and immunohistochemical staining (Ly-6G, F4/80). Results showed significantly greater neutrophilic and macrophage infiltrates within and around SVF in comparison to PSC-laden implants. Differences in early postoperative inflammation among SVF-laden implants were associated with reduced osteogenic differentiation and bone formation. Similar findings were recapitulated with PSC implantation in immunocompetent mice. Exaggerated postoperative inflammation was associated with increased IL-1?, IL-1?, IFN-?, and TNF-? gene expression among SVF samples, and conversely increased IL-6 and IL-10 expression among PSC samples. These data document a robust immunomodulatory effect of implanted PSC, and an inverse correlation between host inflammatory cell infiltration and stromal progenitor cell-mediated ossification.

SUBMITTER: Meyers CA

PROVIDER: S-EPMC5833257 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Early Immunomodulatory Effects of Implanted Human Perivascular Stromal Cells During Bone Formation.

Meyers Carolyn A CA Xu Jiajia J Zhang Lei L Asatrian Greg G Ding Catherine C Yan Noah N Broderick Kristen K Sacks Justin J Goyal Raghav R Zhang Xinli X Ting Kang K Péault Bruno B Soo Chia C James Aaron W AW

Tissue engineering. Part A 20170815 5-6

Human perivascular stem/stromal cells (PSC) are a multipotent mesodermal progenitor cell population defined by their perivascular residence. PSC are most commonly derived from subcutaneous adipose tissue, and recent studies have demonstrated the high potential for clinical translation of this fluorescence-activated cell sorting-derived cell population for bone tissue engineering. Specifically, purified PSC induce greater bone formation than unpurified stroma taken from the same patient sample. I ...[more]

PMID: 28683667

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Project description:BackgroundMesenchymal stromal cells (MSCs) have broad potential as a cell therapy including for the treatment of drug-resistant inflammatory conditions with abnormal T cell proliferation such as graft-versus-host disease (GVHD). Clinical success, however, has been complicated by the heterogeneity of culture-expanded MSCs as well as donor variability. Here, we devise culture conditions that promote expansion of MSCs with enhanced immunomodulatory functions both in vitro and in animal models of GVHD.MethodsHuman bone marrow-derived MSCs were expanded at high-confluency (MSCHC) and low-confluency state (MSCLC). Their immunomodulatory properties were evaluated with in vitro co-culture assays based on suppression of activated T cell proliferation and secretion of pro-inflammatory cytokines from activated T cells. Metabolic state of these cells was determined, while RNA sequencing was performed to explore transcriptome of these MSCs. Ex vivo expanded MSCHC or MSCLC was injected into human peripheral blood mononuclear cells (PBMC)-induced GVHD mouse model to determine their in vivo therapeutic efficacy based on clinical grade scoring, human CD45+ blood count and histopathological examination.ResultsAs compared to MSCLC, MSCHC significantly reduced both the proliferation of anti-CD3/CD28-activated T cells and secretion of pro-inflammatory cytokines upon MSCHC co-culture across several donors even in the absence of cytokine priming. Mechanistically, metabolic analysis of MSCHC prior to co-culture with activated T cells showed increased glycolytic metabolism and lactate secretion compared to MSCLC, consistent with their ability to inhibit T cell proliferation. Transcriptome analysis further revealed differential expression of immunomodulatory genes including TRIM29, BPIFB4, MMP3 and SPP1 in MSCHC as well as enriched pathways including cytokine-cytokine receptor interactions, cell adhesion and PI3K-AKT signalling. Lastly, we demonstrate in a human PBMC-induced GVHD mouse model that delivery of MSCHC showed greater suppression of inflammation and improved outcomes compared to MSCLC and saline controls.ConclusionOur study provides evidence that ex vivo expansion of MSCs at high confluency alters the metabolic and transcriptomic states of these cells. Importantly, this approach maximizes the production of MSCs with enhanced immunomodulatory functions without priming, thus providing a non-invasive and generalizable strategy for improving the use of MSCs for the treatment of inflammatory diseases.

Project description:BACKGROUND:Mesenchymal stromal cells (MSCs), due to their regenerative and immunomodulatory properties, are therapeutically used for diseases, including heart failure. As early gestational-phase embryonic tissues exhibit extraordinary regenerative potential, fetal MSCs exposed to inflammation offer a unique opportunity to evaluate molecular mechanisms underlying preferential healing, and investigate their inherent abilities to communicate with the immune system during development. The principal aim of this study was to evaluate the effects of interferon-? (IFN?) on the immunomodulatory effects of first-trimester human fetal cardiac (hfc)-MSCs. METHODS:hfcMSCs (gestational week 8) were exposed to IFN?, with subsequent analysis of the whole transcriptome, based on RNA sequencing. Exploration of surface-expressed immunoregulatory mediators and modulation of T cell responses were performed by flow cytometry. Presence and activity of soluble mediators were assessed by ELISA or high-performance liquid chromatography. RESULTS:Stimulation of hfcMSCs with IFN? revealed significant transcriptional changes, particularly in respect to the expression of genes belonging to antigen presentation pathways, cell cycle control, and interferon signaling. Expression of immunomodulatory genes and associated functional changes, including indoleamine 2,3-dioxygenase activity, and regulation of T cell activation and proliferation via programmed cell death protein (PD)-1 and its ligands PD-L1 and PD-L2, were significantly upregulated. These immunoregulatory molecules diminished rapidly upon withdrawal of inflammatory stimulus, indicating a high degree of plasticity by hfcMSCs. CONCLUSIONS:To our knowledge, this is the first study performing a systematic evaluation of inflammatory responses and immunoregulatory properties of first-trimester cardiac tissue. In summary, our study demonstrates the dynamic responsiveness of hfcMSCs to inflammatory stimuli. Further understanding as to the immunoregulatory properties of hfcMSCs may be of benefit in the development of novel stromal cell therapeutics for cardiovascular disease.

Project description:BackgroundMesenchymal stromal cells (MSC) hold promise for both cell replacement and immune modulation strategies owing to their progenitor and non-progenitor functions, respectively. Characterization of MSC from different sources is an important and necessary step before clinical use of these cells is widely adopted. Little is known about the biology and function of canine MSC compared to their mouse or human counterparts. This knowledge-gap impedes development of canine evidence-based MSC technologies.Hypothesis and objectivesWe hypothesized that canine adipose tissue (AT) and bone marrow (BM) MSC (derived from the same dogs) will have similar differentiation and immune modulatory profiles. Our objectives were to evaluate progenitor and non-progenitor functions as well as other characteristics of AT- and BM-MSC including 1) proliferation rate, 2) cell surface marker expression, 3) DNA methylation levels, 4) potential for trilineage differentiation towards osteogenic, adipogenic, and chondrogenic cell fates, and 5) immunomodulatory potency in vitro.Results1) AT-MSC proliferated at more than double the rate of BM-MSC (population doubling times in days) for passage (P) 2, AT: 1.69, BM: 3.81; P3, AT: 1.80, BM: 4.06; P4, AT: 2.37, BM: 5.34; P5, AT: 3.20, BM: 7.21). 2) Canine MSC, regardless of source, strongly expressed cell surface markers MHC I, CD29, CD44, and CD90, and were negative for MHC II and CD45. They also showed moderate expression of CD8 and CD73 and mild expression of CD14. Minor differences were found in expression of CD4 and CD34. 3) Global DNA methylation levels were significantly lower in BM-MSC compared to AT-MSC. 4) Little difference was found between AT- and BM-MSC in their potential for adipogenesis and osteogenesis. Chondrogenesis was poor to absent for both sources in spite of adding varying levels of bone-morphogenic protein to our standard transforming growth factor (TGF-β3)-based induction medium. 5) Immunomodulatory capacity was equal regardless of cell source when tested in mitogen-stimulated lymphocyte reactions. Priming of MSC with pro-inflammatory factors interferon-gamma and/or tumour necrosis factor did not increase the lymphocyte suppressive properties of the MSC compared to untreated MSC.Conclusions/significanceNo significant differences were found between AT- and BM-MSC with regard to their immunophenotype, progenitor, and non-progenitor functions. Both MSC populations showed strong adipogenic and osteogenic potential and poor chondrogenic potential. Both significantly suppressed stimulated peripheral blood mononuclear cells. The most significant differences found were the higher isolation success and proliferation rate of AT-MSC, which could be realized as notable benefits of their use over BM-MSC.

Dataset Information

Early Immunomodulatory Effects of Implanted Human Perivascular Stromal Cells During Bone Formation.

Publications

Early Immunomodulatory Effects of Implanted Human Perivascular Stromal Cells During Bone Formation.

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