Combination therapy with Treg and mesenchymal stromal cells enhances potency and attenuation of inflammation after traumatic brain injury compared to monotherapy.
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ABSTRACT:
SUBMITTER: Caplan HW
PROVIDER: S-EPMC8634698 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:ObjectiveThe actin-sequestering proteins, thymosin beta-4 (Tβ4) and hypoxia-inducible factor (HIF)-1α, are known to be associated with angiogenesis after myocardial infarction (MI). Herein, we aimed to identify the mechanism of HIF-1α induction by Tβ4 and investigate the effects of bone marrow mesenchymal stromal cells (BMMSCs) transfected with the Tβ4 gene (TMSB4) in a rat model of MI.MethodsRat BMMSCs were isolated, cultured, and transfected with the TMSB4 gene by using the lentivirus-mediated method. Rats with surgically induced MI were randomly divided into three groups (n = 9/group); after 1 week, the rats were injected at the heart infarcted border zone with TMSB4-overexpressed BMMSCs (BMMSC-TMSB4 O E ), wild-type BMMSCs that expressed normal levels of TMSB4 (BMMSC-TMSB4 W T ), or medium (MI). The fourth group of animals (n = 9) underwent all surgical procedures necessary for MI induction except for the ligation step (Sham). Four weeks after the injection, heart function was measured using transthoracic echocardiography. Infarct size was calculated by TTC staining, and collagen volume was measured by Masson staining. Angiogenesis in the infarcted heart area was evaluated by CD31 immunofluorescence histochemistry. In vitro experiments were carried out to observe the effect of exogenous Tβ4 on HIF-1α and explore the various possible mechanism(s).ResultsIn vivo experiments showed that vascular density 4 weeks after treatment was about twofold higher in BMMSC-TMSB4 O E -treated animals than in BMMSC-TMSB4 W T -treated animals (p < 0.05). The cardiac function and infarct size significantly improved in both cell-treatment groups compared to controls. Notably, the cardiac function and infarct size were most prominent in BMMSC-TMSB4 O E -treated animals (both p < 0.05). HIF-1α and phosphorylated HIF-1α (p-HIF-1α) in vitro were significantly enhanced by exogenous Tβ4, which was nonetheless blocked by the factor-inhibiting HIF (FIH) promoter (YC-1). The expression of prolyl hydroxylase domain proteins (PHD) was decreased upon treatment with Tβ4 and further decreased with the combined treatment of Tβ4 and FG-4497 (a specific PHD inhibitor).ConclusionTMSB4-transfected BMMSCs might significantly improve recovery from myocardial ischemia and promote the generation of HIF-1α and p-HIF-1α via the AKT pathway, and inhibit the degradation of HIF-1α via the PHD and FIH pathways.
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Project description:Human perinatal tissue is an abundant source of mesenchymal stromal cells(MSCs) and lacks the ethical concerns. Perinatal MSCs can be obtained from various tissues as like amnion, chorion, and umbilical cord. Still, little is known of the distinct nature of each MSC type. In this study, we successfully isolated and cultured MSCs from amnion(AMSCs), chorion(CMSCs), and umbilical cord(UC-MSCs). Proliferation potential was different among them, that AMSCs revealed the lowest proliferation rate due to increased Annexin V and senescence-associated ?-galactosidase positive cells. We demonstrated distinct characteristic gene expression according to the source of the original tissue using microarray. In particular, genes associated with apoptosis and senescence including CDKN2A were up-regulated in AMSCs. In CMSCs, genes associated with heart morphogenesis and blood circulation including HTR2B were up-regulated. Genes associated with neurological system processes including NPY were up-regulated in UC-MSCs. Quantitative RT-PCR confirmed the gene expression data. And in vitro differentiation of MSCs demonstrated that CMSCs and UC-MSCs had a more pronounced ability to differentiate into cardiomyocyte and neural cells, respectively. This study firstly demonstrated the innate tissue-specific differentiation potency of perinatal MSCs which can be helpful in choosing more adequate cell sources for better outcome in a specific disease.
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Project description:Mesenchymal stromal cells (MSC) undergo functional maturation upon their migration from bone marrow and introduction to a site of injury. This inflammatory licensing leads to heightened immune regulation via cell-to-cell interaction and the secretion of immunomodulatory molecules, such as anti-inflammatory mediators and antioxidants. Pro-inflammatory cytokines are a recognized catalyst of inflammatory licensing; however, biomechanical forces, such as fluid shear stress, are a second, distinct class of stimuli that incite functional maturation. Here we show mechanotransduction, achieved by exposing MSC to various grades of wall shear stress (WSS) within a scalable conditioning platform, enhances the immunomodulatory potential of MSC independent of classical pro-inflammatory cytokines. A dose-dependent effect of WSS on potency is evidenced by production of prostaglandin E2 (PGE2) and indoleamine 2,3 dioxygenase 1 (IDO1), as well as suppression of tumor necrosis factor-α (TNF- α) and interferon-γ (IFN-γ) production by activated immune cells. Consistent, reproducible licensing is demonstrated in adipose tissue and bone marrow human derived MSC without significant impact on cell viability, cellular yield, or identity. Transcriptome analysis of WSS-conditioned BM-MSC elucidates the broader phenotypic implications on the differential expression of immunomodulatory factors. These results suggest mechanotransduction as a viable, scalable pre-conditioning alternative to pro-inflammatory cytokines. Enhancing the immunomodulatory capacity of MSC via biomechanical conditioning represents a novel cell therapy manufacturing approach.
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Project description:Assays that can characterize MSC immune potency need to be identified for use in advanced clinical trials. MSCs possess a number of putative regenerative and immunomodulatory properties, and an assay matrix approach may best capture involved effector pathways. We have tested two assay systems to measure the potency of MSCs derived from human subjects: MSC secretome analysis and a quantitative RNA-based array for genes specific to immunomodulatory and homing properties of MSCs. Secretome analysis identified a unique cytokine signature that is upregulated by MSCs or downregulated in responder PBMCs and correlated with T cell suppression. Use of interferon-? as a surrogate for the action of activated PBMCs on MSCs served as an alternative for the use of human PBMCs as responder cells in a potency assay. Our approach and results define and simplify the multifunctional or matrix responses of MSCs and may serve as a platform for robust potency analysis.
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Project description:Immunosuppressive ability in human MSC donors has been shown to be variable and may be a limiting factor in MSC therapeutic efficacy in vivo. The importance of cytokine activation of mesenchymal stromal cells (MSCs) to facilitate their immunosuppressive function is well established. This study sought to further understand the interactions between MSCs and the commonly used calcineurin inhibitor cyclosporine A (CsA). The existing literature regarding approaches that use MSCs and cyclosporine are conflicting regarding the effect of CsA on MSC potency and function. Here, we clearly demonstrate that when added at the same time as MSCs, CsA negatively affects MSC suppression of T cell proliferation. However, licencing MSCs with IFNγ before addition of CsA protects MSCs from this negative effect. Notably, adding CsA to MSCs after IFNγ pre-stimulation enhances MSC production of IDO. Mechanistically, we identified that CsA reduces SOCS1 expression to facilitate enhanced IDO production in IFNγ pre-stimulated MSCs. Importantly, CsA exposure to IFNγ pre-stimulated MSC before administration, significantly enhanced the potency of MSCs in a human relevant humanised mouse model of acute Graft versus Host Disease. In summary, this study identified a novel licencing strategy to enhance MSC potency in vitro and in vivo.
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Project description:More than 6.5 million patients are burdened by the physical, cognitive, and psychosocial deficits associated with traumatic brain injury (TBI) in the U.S. Despite extensive efforts to develop neuroprotective therapies for this devastating disorder, there have been no successful outcomes in human clinical trials to date. Retrospective studies have shown that ?-adrenergic receptor blockers, specifically propranolol, significantly decrease mortality of TBI through mechanisms not yet fully elucidated but are thought to counterbalance a hyperadrenergic state resulting from a TBI. Conversely, cellular therapies have been shown to improve long-term behavior following TBI, likely by reducing inflammation. Given the nonredundancy in their therapeutic mechanisms, we hypothesized that a combination of acute propranolol followed by mesenchymal stem cells (MSCs) isolated from human bone marrow would have additive effects in treating a rodent model of TBI. We have found that the treatments are well-tolerated individually and in combination with no adverse events. MSCs decrease BBB permeability at 96 hours after injury, inhibit a significant accumulation of activated microglia/macrophage in the thalamic region of the brain both short and long term, and enhance neurogenesis short term. Propranolol decreases edema and reduces the number of fully activated microglia at 7 days and the number of semiactivated microglia at 120 days. Combinatory treatment improved cognitive and memory functions 120 days following TBI. Therefore, the results here suggest a new, efficacious sequential treatment for TBI may be achieved using the ?-blocker propranolol followed by MSC treatment.Despite continuous efforts, traumatic brain injury (TBI) remains the leading cause of death and disability worldwide in patients under the age of 44. In this study, an animal model of moderate-severe TBI was treated with an acute dose of propranolol followed by a delayed dose of human mesenchymal stem cells (MSCs), resulting in improved short- and long-term measurements. These results have direct translational application. They reinforce the inevitable clinical trial of MSCs to treat TBI by demonstrating, among other benefits, a notable decrease in chronic neuroinflammation. More importantly, these results demonstrate that MSCs and propranolol, which is increasingly being used clinically for TBI, are compatible treatments that improve overall outcome.
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Project description:Traumatic brain injury (TBI) causes both physical disruption of the blood brain barrier (BBB) and altered immune responses that can lead to significant secondary brain injury and chronic inflammation within the central nervous system (CNS). Cell therapies, including mesenchymal stromal cells (MSC), have been shown to restore BBB integrity and augment endogenous splenic regulatory T cells (Treg), a subset of CD4+ T cells that function to regulate immune responses and prevent autoimmunity. We have recently shown that infusion of human cord blood-derived Treg decreased neuroinflammation after TBI in vivo and in vitro. However, while both cells have demonstrated anti-inflammatory and regenerative potential, they likely utilize differing, although potentially overlapping, mechanisms. Furthermore, studies investigating these two cell types together, as a combination therapy, are lacking. In this study, we compared the ability of Treg+MSC combination therapy, as well as MSC and Treg monotherapies, to improve BBB permeability in vivo and suppress inflammation in vitro. While Treg+MSC combination did not significantly augment potency in vivo, our in vitro data demonstrates that combination therapy may augment therapeutic potency and immunosuppressive potential compared to Treg or MSC monotherapy.
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Project description:Potency analysis of mesenchymal stromal cells (MSCs) is required for their use in advanced clinical trials. Assay matrix strategy evaluating more than a single property of MSCs is an emerging strategy in potency analysis. Here we developed an assay matrix approach focusing on the secretory chemokine responses of MSCs using multiplex analytical method. MSCs' innate fitness in secreting matrix of chemokines is correlated with their metabolic fitness in differential degrees. In addition, innately secreting chemokines are correlated among themselves in a unique pattern. MSC's matrix chemokine responses to exogenous stimulation of IFNγ and/or TNFα are distinct. However, the combination of IFNγ and TNFα is superior than individual stimulations in eliciting robust and broad matrix chemokine responses of MSCs. Correlation matrix analysis has identified that chemokine responses to IFNγ and/or TNFα display unique correlative secretion patterns. MSC and peripheral blood mononuclear cells coculture analysis has identified the correlation matrix responses of chemokines that predicted immune suppression. In addition, MSC-mediated blocking of T-cell proliferation predominantly correlates with chemokines in an inverse manner. Knockdown of chemokines has demonstrated that MSC-sourced inherent chemokines do not actively play a role in T-cell suppression and thus are the bystander predictors of T-cell suppression. The present analysis of MSC's matrix chemokine responses can be deployed in the advanced potency analysis of MSCs.
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Project description:Therapeutic efficacy of various mesenchymal stromal cell (MSC) types for orthopaedic applications is currently being investigated. While the concept of MSC therapy is well grounded in the basic science of healing and regeneration, little is known about individual MSC populations in terms of their propensity to promote the repair and/or regeneration of specific musculoskeletal tissues. Two promising MSC sources, adipose and amnion, have each demonstrated differentiation and extracellular matrix (ECM) production in the setting of musculoskeletal tissue regeneration. However, no study to date has directly compared the differentiation potential of these 2 MSC populations.To compare the ability of human adipose- and amnion-derived MSCs to undergo osteogenic and chondrogenic differentiation.Controlled laboratory study.MSC populations from the human term amnion were quantified and characterized via cell counting, histologic assessment, and flow cytometry. Differentiation of these cells in comparison to commercially purchased human adipose-derived mesenchymal stromal cells (hADSCs) in the presence and absence of differentiation media was evaluated via reverse transcription polymerase chain reaction (PCR) for bone and cartilage gene transcript markers and histology/immunohistochemistry to examine ECM production. Analysis of variance and paired t tests were performed to compare results across all cell groups investigated.The authors confirmed that the human term amnion contains 2 primary cell types demonstrating MSC characteristics-(1) human amniotic epithelial cells (hAECs) and (2) human amniotic mesenchymal stromal cells (hAMSCs)-and each exhibited more than 90% staining for MSC surface markers (CD90, CD105, CD73). Average viable hAEC and hAMSC yields at harvest were 2.3 × 106 ± 3.7 × 105 and 1.6 × 106 ± 4.7 × 105 per milliliter of amnion, respectively. As well, hAECs and hAMSCs demonstrated significantly greater osteocalcin ( P = .025), aggrecan ( P < .0001), and collagen type 2 ( P = .044) gene expression compared with hADSCs, respectively, after culture in differentiation medium. Moreover, both hAECs and hAMSCs produced significantly greater quantities of mineralized ( P < .0001) and cartilaginous ( P = .0004) matrix at earlier time points compared with hADSCs when cultured under identical osteogenic and chondrogenic differentiation conditions, respectively.Amnion-derived MSCs demonstrate a greater differentiation potential toward bone and cartilage compared with hADSCs.Amniotic MSCs may be the source of choice in the regenerative treatment of bone or osteochondral musculoskeletal disease. They show significantly higher yields and better differentiation toward these tissues than MSCs derived from adipose.
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Project description:MSCs derived from the umbilical cord tissue, termed UCX, were investigated for their immunomodulatory properties and compared to bone marrow-derived MSCs (BM-MSCs), the gold-standard in immunotherapy. Immunogenicity and immunosuppression were assessed by mixed lymphocyte reactions, suppression of lymphocyte proliferation and induction of regulatory T cells. Results showed that UCX were less immunogenic and showed higher immunosuppression activity than BM-MSCs. Further, UCX did not need prior activation or priming to exert their immunomodulatory effects. This was further corroborated in vivo in a model of acute inflammation. To elucidate the potency differences observed between UCX and BM-MSCs, gene expression related to immune modulation was analysed in both cell types. Several gene expression profile differences were found between UCX and BM-MSCs, namely decreased expression of HLA-DRA, HO-1, IGFBP1, 4 and 6, ILR1, IL6R and PTGES and increased expression of CD200, CD273, CD274, IL1B, IL-8, LIF and TGFB2. The latter were confirmed at the protein expression level. Overall, these results show that UCX seem to be naturally more potent immunosuppressors and less immunogenic than BM-MSCs. We propose that these differences may be due to increased levels of immunomodulatory surface proteins such as CD200, CD273, CD274 and cytokines such as IL1β, IL-8, LIF and TGFβ2.
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