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1.
Diabet Med ; 40(12): e15227, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37728506

RESUMO

AIMS: Human islet transplantation as a therapy for type 1 diabetes is compromised by the loss of functional beta cells in the immediate post-transplantation period. Mesenchymal stromal cells (MSCs) and MSC-derived secretory peptides improve the outcomes of islet transplantation in rodent models of diabetes. Here, we utilized a mouse model for human islet transplantation and assessed the effects of a cocktail of MSC-secreted peptides (screened by MSC-secretome for human islet GPCRs) on the functional survival of human islets. METHODS: Human islets from nine donors (Age: 36-57; BMI: 20-35) were treated with a cocktail of human recombinant annexin A1 (ANXA1), stromal cell-derived factor-1 (SDF-1/CXCL12) and complement component C3 (C3a). Glucose-stimulated insulin secretion (GSIS) was assessed in static incubation, and cytokine-induced apoptosis was assessed by measuring caspase 3/7 activity. mRNA expression levels were determined by qPCR. Human islet function in vivo was assessed using a novel model for human islet transplantation into a T1D mouse model. Human islet function in vivo was assessed using islet transplantation under the kidney capsule of immunodeficient mice prior to STZ destruction of endogenous mouse beta cells to model T1DM. RESULTS: Pretreatment with a cocktail of MSC-secreted peptides increased GSIS in vitro and protected against cytokine-induced apoptosis in human islets isolated from nine donors. Animals transplanted with either treated or untreated human islets remained normoglycaemic for up to 28 days after STZ-administration to ablate the endogenous mouse beta cells, whereas non-transplanted animals showed significantly increased blood glucose immediately after STZ administration. Removal of the human islet graft by nephrectomy resulted in rapid increases in blood glucose to similar levels as the non-transplanted controls. Pretreating human islets with the MSC-derived cocktail significantly improved glucose tolerance in graft recipients, consistent with enhanced functional survival of the treated islets in vivo. CONCLUSION: Pretreating human islets before transplantation with a defined cocktail of MSC-derived molecules could be employed to improve the quality of human islets for transplantation therapy for type 1 diabetes.


Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas , Células-Tronco Mesenquimais , Humanos , Camundongos , Animais , Adulto , Pessoa de Meia-Idade , Ilhotas Pancreáticas/metabolismo , Insulina/metabolismo , Glicemia/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Experimental/terapia , Transplante das Ilhotas Pancreáticas/métodos , Células-Tronco Mesenquimais/metabolismo , Glucose/farmacologia , Glucose/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças
2.
Stem Cells Transl Med ; 10(5): 674-680, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33544449

RESUMO

Islet transplantation is an emerging treatment for type 1 diabetes which offers the prospect of physiological control of blood glucose and reductions in acute hypoglycaemic episodes. However, current protocols are limited by a rapid decline in islet functional viability during the isolation process, culture period, and post-transplantation. Much of this can be attributed to the deleterious effects of hypoxic and cytokine stressors on ß cells. One experimental strategy to improve the functional viability of islets is coculture or cotransplantation with mesenchymal stromal cells (MSCs). Numerous studies have shown that MSCs have the capacity to improve islet survival and insulin secretory function, and the mechanisms of these effects are becoming increasingly well understood. In this review, we will focus on recent studies demonstrating the capacity for MSCs to protect islets from hypoxia- and cytokine-induced stress. Islets exposed to acute hypoxia (1%-2% O2 ) or to inflammatory cytokines (including IFN-γ, TNF-α, and IL-B) in vitro undergo apoptosis and a rapid decline in glucose-stimulated insulin secretion. Coculture of islets with MSCs, or with MSC-conditioned medium, protects from these deleterious effects, primarily with secreted factors. These protective effects are distinct from the immunomodulatory and structural support MSCs provide when cotransplanted with islets. Recent studies suggest that MSCs may support secretory function by the physical transfer of functional mitochondria, particularly to metabolically compromised ß cells. Understanding how MSCs respond to stressed islets will facilitate the development of MSC secretome based, cell-free approaches to supporting islet graft function during transplantation by protecting or repairing ß cells.


Assuntos
Ilhotas Pancreáticas , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Técnicas de Cocultura , Citocinas/metabolismo , Humanos , Hipóxia/metabolismo , Insulina , Ilhotas Pancreáticas/metabolismo , Transplante das Ilhotas Pancreáticas , Células-Tronco Mesenquimais/metabolismo , Secretoma
3.
Stem Cells ; 38(4): 574-584, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31912945

RESUMO

Pretransplant islet culture is associated with the loss of islet cell mass and insulin secretory function. Insulin secretion from islet ß-cells is primarily controlled by mitochondrial ATP generation in response to elevations in extracellular glucose. Coculture of islets with mesenchymal stromal cells (MSCs) improves islet insulin secretory function in vitro, which correlates with superior islet graft function in vivo. This study aimed to determine whether the improved islet function is associated with mitochondrial transfer from MSCs to cocultured islets. We have demonstrated mitochondrial transfer from human adipose MSCs to human islet ß-cells in coculture. Fluorescence imaging showed that mitochondrial transfer occurs, at least partially, through tunneling nanotube (TNT)-like structures. The extent of mitochondrial transfer to clinically relevant human islets was greater than that to experimental mouse islets. Human islets are subjected to more extreme cellular stressors than mouse islets, which may induce "danger signals" for MSCs, initiating the donation of MSC-derived mitochondria to human islet ß-cells. Our observations of increased MSC-mediated mitochondria transfer to hypoxia-exposed mouse islets are consistent with this and suggest that MSCs are most effective in supporting the secretory function of compromised ß-cells. Ensuring optimal MSC-derived mitochondria transfer in preculture and/or cotransplantation strategies could be used to maximize the therapeutic efficacy of MSCs, thus enabling the more widespread application of clinical islet transplantation.


Assuntos
Diabetes Mellitus Experimental/terapia , Células Secretoras de Insulina/metabolismo , Transplante das Ilhotas Pancreáticas/métodos , Células-Tronco Mesenquimais/metabolismo , Mitocôndrias/metabolismo , Animais , Células Cultivadas , Humanos , Camundongos
4.
Methods Mol Biol ; 2076: 265-280, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31586334

RESUMO

Islet transplantation is a potential treatment for Type 1 diabetes; however, improvements need to be made before it could become clinically widely available. In preclinical studies, the mouse is often used to model islet transplantation, with most studies aiming to improve transplantation outcome by manipulating the islets prior to transplantation or by treating the recipient mouse. Here, we describe the process of islet transplantation in the mouse, including how one can make the mouse diabetic, isolate donor islets, and transplant the islets into two different sites. Finally, we discuss how to assess the outcome of the transplantation in order to determine whether the experimental intervention has been beneficial.


Assuntos
Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas , Animais , Glicemia , Separação Celular/métodos , Diabetes Mellitus Experimental , Rejeição de Enxerto , Sobrevivência de Enxerto , Xenoenxertos , Insulina/sangue , Ilhotas Pancreáticas/citologia , Transplante das Ilhotas Pancreáticas/efeitos adversos , Transplante das Ilhotas Pancreáticas/métodos , Camundongos , Transplante Homólogo , Resultado do Tratamento
5.
Stem Cells Transl Med ; 8(9): 935-944, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31066521

RESUMO

Islet transplantation has the potential to cure type 1 diabetes, but current transplantation protocols are not optimal and there is extensive loss of islet ß-cell insulin secretory function during the immediate post-transplantation period. Studies using experimental models of diabetes have shown that the coculture of islets with mesenchymal stromal cells (MSCs) prior to transplantation improves graft function, but several variables differed among research groups (e.g., type of MSCs used and the treatment conditions). We have therefore assessed the effects of MSCs on mouse and human islets by investigating the importance of tissue source for MSCs, the coculture protocol configuration and length, the effect of activated MSCs, and different ß-cell secretory stimuli. MSCs derived from adipose tissue (aMSCs) were the most effective at supporting ß-cell insulin secretion in both mouse and human islets, in a direct contact coculture configuration. Preculture with aMSCs enhanced both phases of glucose-induced insulin secretion and further enhanced secretory responses to the non-nutrients carbachol and arginine. These effects required a coculture period of 48-72 hours and were not dependent on activation of the MSCs. Thus, direct contact coculture with autologous, adipose-derived MSCs for a minimum of 48 hours before implantation is likely to be an effective addition to human islet transplantation protocols. Stem Cells Translational Medicine 2019;8:935&944.


Assuntos
Ilhotas Pancreáticas/citologia , Células-Tronco Mesenquimais/citologia , Tecido Adiposo/citologia , Animais , Quimiocina CXCL9/genética , Quimiocina CXCL9/metabolismo , Técnicas de Cocultura , Glucose/farmacologia , Humanos , Secreção de Insulina/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Pâncreas/citologia , Fator de Necrose Tumoral alfa/farmacologia
6.
Cytotherapy ; 20(12): 1427-1436, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30377040

RESUMO

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) enhance islet function both in vitro and in vivo, at least in part by secreting ligands that activate islet G-protein coupled receptors (GPCRs). We assessed whether pre-treatment with a defined "cocktail" of MSC-secreted GPCR ligands enhances islet functional survival in vitro and improves the outcomes of islet transplantation in an experimental model of diabetes. METHODS: Isolated islets were cultured for 48 h with ANXA1, SDF-1 or C3a, alone or in combination. Glucose-stimulated insulin secretion (GSIS) and cytokine-induced apoptosis were measured immediately after the 48 h culture period and at 24 h or 72 h following removal of the ligands from the culture media. Islets were syngeneically transplanted underneath the kidney capsule of streptozotocin-induced diabetic C57BL/6 mice and blood glucose levels monitored for 28 days. RESULTS: Pre-culturing islets with a cocktail of ANXA1/SDF-1/C3a potentiated GSIS and protected islet cells from cytokine-induced apoptosis in vitro. These effects were maintained for up to 72 h after the removal of the factors from the culture medium, suggesting a sustained protection of islet graft functional survival during the immediate post-transplantation period. Islets pre-treated with the cocktail of MSC secretory factors were more effective in reducing blood glucose in diabetic mice, consistent with their improved functional survival in vivo. DISCUSSION: Pre-culturing islets with a cocktail of MSC secretory products offers a well-defined, cell-free approach to improve clinical islet transplantation outcomes while avoiding many of the safety, regulatory and logistical hurdles of incorporating MSCs into transplantation protocols.


Assuntos
Quimiocina CXCL12/farmacologia , Complemento C3a/farmacologia , Transplante das Ilhotas Pancreáticas/métodos , Células-Tronco Mesenquimais/metabolismo , Animais , Anexina A1/genética , Anexina A1/metabolismo , Anexina A1/farmacologia , Apoptose/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Complemento C3a/genética , Complemento C3a/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/terapia , Glucose/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Receptores Acoplados a Proteínas G/metabolismo
7.
Curr Opin Pharmacol ; 43: 34-39, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30103073

RESUMO

Allogeneic islet transplantation as a therapy for Type 1 Diabetes (T1D) is restricted by the limited availability of donor islets, loss of functional islets during pre-transplantation culture in vitro and further extensive loss during the immediate post-transplantation period when islet function and survival is compromised by the hypoxic, inflammatory host environment. In the longer term pathogenic T cell responses drive autoimmunity and chronic allograft rejection. Experimental studies have demonstrated that mesenchymal stromal cells (MSCs) have significant potential to improve the outcomes of clinical islet transplantation. This review explores the potential for MSCs and their 'secretome' to influence donor islet cell function and survival, as well as the host niche. We discuss the possibility of harnessing the therapeutic benefits of MSCs in a cell-free strategy to offer a well-defined, cell-free approach to improve the outcomes of clinical islet transplantation.


Assuntos
Glicemia/metabolismo , Diabetes Mellitus Tipo 1/cirurgia , Transplante das Ilhotas Pancreáticas/métodos , Ilhotas Pancreáticas/cirurgia , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais , Animais , Biomarcadores/sangue , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/patologia , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto , Humanos , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Transplante das Ilhotas Pancreáticas/efeitos adversos , Transplante de Células-Tronco Mesenquimais/efeitos adversos , Células-Tronco Mesenquimais/imunologia , Células-Tronco Mesenquimais/metabolismo , Fenótipo , Recuperação de Função Fisiológica , Regeneração , Fatores de Risco
9.
Clin Sci (Lond) ; 131(23): 2835-2845, 2017 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-29101297

RESUMO

AIMS: The aims of the present study were (i) to determine whether the reported beneficial effects of mesenchymal stromal cells (MSCs) on mouse islet function extend to clinically relevant human tissues (islets and MSCs), enabling translation into improved protocols for clinical human islet transplantation; and (ii) to identify possible mechanisms through which human MSCs influence human islet function. MATERIALS AND METHODS: Human islets were co-cultured with human adipose tissue-derived MSCs (hASCs) or pre-treated with its products - extracellular matrix (ECM) and annexin A1 (ANXA1). Mouse islets were pre-treated with mouse MSC-derived ECM. Islet insulin secretory function was assessed in vitro by radioimmunoassay. Quantitative RT-PCR was used to screen human adipMSCs for potential ligands of human islet G-protein-coupled receptors. RESULTS: We show that co-culture with hASCs improves human islet secretory function in vitro, as measured by glucose-stimulated insulin secretion, confirming previous reports using rodent tissues. Furthermore, we demonstrate that these beneficial effects on islet function can be partly attributed to the MSC-derived products ECM and ANXA1. CONCLUSIONS: Our results suggest that hASCs have the potential to improve the quality of human islets isolated for transplantation therapy of Type 1 diabetes. Furthermore, it may be possible to achieve improvements in human islet quality in a cell-free culture system by using the MSC-derived products ANXA1 and ECM.


Assuntos
Matriz Extracelular/fisiologia , Ilhotas Pancreáticas/fisiologia , Células-Tronco Mesenquimais/fisiologia , Tecido Adiposo/citologia , Animais , Anexina A1/metabolismo , Anexina A1/farmacologia , Técnicas de Cocultura , Humanos , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Ligantes , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos C57BL , Receptores Odorantes/metabolismo
10.
Diabetes ; 65(1): 129-39, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26470781

RESUMO

We have previously demonstrated that coculture of islets with mesenchymal stromal cells (MSCs) enhanced islet insulin secretory capacity in vitro, correlating with improved graft function in vivo. To identify factors that contribute to MSC-mediated improvements in islet function, we have used an unbiased quantitative RT-PCR screening approach to identify MSC-derived peptide ligands of G-protein-coupled receptors that are expressed by islets cells. We demonstrated high expression of annexin A1 (ANXA1) mRNA by MSCs and confirmed expression at the protein level in lysates and MSC-conditioned media by Western blot analysis and ELISA. Preculturing islets with exogenous ANXA1 enhanced glucose-stimulated insulin secretion (GSIS), thereby mimicking the beneficial influence of MSC preculture in vitro. Small interfering RNA-mediated knockdown of ANXA1 in MSCs reduced their capacity to potentiate GSIS. MSCs derived from ANXA1(-/-) mice had no functional capacity to enhance GSIS, in contrast to wild-type controls. Preculturing islets with ANXA1 had modest effects on their capacity to regulate blood glucose in streptozotocin-induced diabetic mice, indicating that additional MSC-derived factors are required to fully mimic the beneficial effects of MSC preculture in vivo. These findings demonstrate the feasibility of harnessing the MSC secretome as a defined, noncellular strategy to improve the efficiency of clinical islet transplantation protocols.


Assuntos
Anexina A1/genética , Glicemia/metabolismo , Diabetes Mellitus Experimental/genética , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Células-Tronco Mesenquimais/metabolismo , RNA Mensageiro/metabolismo , Animais , Anexina A1/metabolismo , Western Blotting , Técnicas de Cocultura , Diabetes Mellitus Experimental/metabolismo , Ensaio de Imunoadsorção Enzimática , Técnicas de Silenciamento de Genes , Técnicas In Vitro , Secreção de Insulina , Transplante de Células-Tronco Mesenquimais , Camundongos , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real
11.
Eur J Immunol ; 45(3): 843-53, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25408265

RESUMO

In humans, tolerance to renal transplants has been associated with alterations in B-cell gene transcription and maintenance of the numbers of circulating transitional B cells. Here, we use a mouse model of transplantation tolerance to investigate the contribution of B cells to allograft survival. We demonstrate that transfer of B cells from mice rendered tolerant to MHC class I mismatched skin grafts can prolong graft survival in a dose-dependent and antigen-specific manner to a degree similar to that afforded by graft-specific regulatory T (Treg) cells. Tolerance in this model was associated with an increase in transitional-2 (T2) B cells. Only T2 B cells from tolerized mice, not naïve T2 nor alloantigen experienced T2, were capable of prolonging skin allograft survival, and suppressing T-cell activation. Tolerized T2 B cells expressed lower levels of CD86, increased TIM-1, and demonstrated a preferential survival in vivo. Furthermore, we demonstrate a synergistic effect between tolerized B cells and graft-specific Treg cells. IL-10 production by T2 B cells did not contribute to tolerance, as shown by transfer of B cells from IL-10(-/-) mice. These results suggest that T2 B cells in tolerant patients may include a population of regulatory B cells that directly inhibit graft rejection.


Assuntos
Sobrevivência de Enxerto/imunologia , Ativação Linfocitária , Células Precursoras de Linfócitos B/imunologia , Transplante de Pele , Linfócitos T Reguladores/imunologia , Tolerância ao Transplante , Aloenxertos , Animais , Sobrevivência de Enxerto/genética , Interleucina-10/genética , Interleucina-10/imunologia , Camundongos , Camundongos Knockout
12.
Cell Med ; 7(1): 37-47, 2014 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-26858891

RESUMO

We have recently shown that preculturing islets with kidney-derived mesenchymal stromal cells (MSCs) improves transplantation outcome in streptozotocin-diabetic mice implanted with a minimal mass of islets beneath the kidney capsule. In the present study, we have extended our previous observations to investigate whether preculturing islets with MSCs can also be used to enhance islet function at the clinically used intraportal site. We have used MSCs derived from adipose tissue, which are more readily accessible than alternative sources in human subjects and can be expanded to clinically efficacious numbers, to preculture islets throughout this study. The in vivo efficacy of grafts consisting of islets precultured alone or with MSCs was tested using a syngeneic streptozotocin-diabetic minimal islet mass model at the clinically relevant intraportal site. Blood glucose concentrations were monitored for 1 month. The vascularization of islets precultured alone or with MSCs was investigated both in vitro and in vivo, using immunohistochemistry. Islet insulin content was measured by radioimmunoassay. The effect of preculturing islets with MSCs on islet function in vitro was investigated using static incubation assays. There was no beneficial angiogenic influence of MSC preculture, as demonstrated by the comparable vascularization of islets precultured alone or with MSCs, both in vitro after 3 days and in vivo 1 month after islet transplantation. However, the in vitro insulin secretory capacity of MSC precultured islets was superior to that of islets precultured alone. In vivo, this was associated with improved glycemia at 7, 14, 21, and 28 days posttransplantation, in recipients of MSC precultured islets compared to islets precultured alone. The area of individual islets within the graft-bearing liver was significantly higher in recipients of MSC precultured islets compared to islets precultured alone. Our experimental studies suggest that preculturing islets with MSCs represents a favorable strategy for improving the efficiency of clinical islet transplantation.

13.
PLoS One ; 8(2): e57844, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23451276

RESUMO

We have previously shown that co-transplantation of islets and Mesenchymal Stem Cells (MSCs) improves islet graft function and revascularisation, which was associated with the maintenance of normal islet morphology. The aim of the current study was to determine whether maintaining islet morphology in the absence of additional islet-helper cells would improve transplantation outcome in diabetic mice. Islets were isolated from C57BL/6 mice. Recipient streptozotocin-diabetic C57BL/6 mice were transplanted with a minimal mass of 150 islets as a single pellet or islets that were either manually dispersed or dispersed within a matrigel plug beneath the kidney capsule. Blood glucose concentrations were monitored for one month. Islet graft morphology and vascularisation were analysed by histology. Islets dispersed either alone or within matrigel plugs maintained near normal morphology, in contrast to pelleted islets, where individual islets fused to form large endocrine aggregates. The vascularisation of manually dispersed islets and islets dispersed within matrigel plugs was increased relative to respective control pelleted islet grafts. After one month 1/6 mice transplanted with pelleted islets cured compared to 5/6 mice transplanted with manually dispersed islets. The curative capacity of islets dispersed in matrigel was also better than that of pelleted islets (5/8 islet-matrigel implanted mice vs. 1/7 mice transplanted with pelleted islets cured by one month). Therefore, this study demonstrates that the maintenance of islet morphology is associated with improved graft function and revascularisation in diabetic mice.


Assuntos
Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/cirurgia , Transplante das Ilhotas Pancreáticas/métodos , Ilhotas Pancreáticas/patologia , Animais , Glicemia/metabolismo , Colágeno/metabolismo , Colágeno/fisiologia , Diabetes Mellitus Experimental/induzido quimicamente , Combinação de Medicamentos , Ilhotas Pancreáticas/cirurgia , Laminina/metabolismo , Laminina/fisiologia , Masculino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Proteoglicanas/metabolismo , Proteoglicanas/fisiologia
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