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Insulin is made from proinsulin, but the extent to which fasting/feeding controls the homeostatically regulated proinsulin pool in pancreatic ß-cells remains largely unknown. Here, we first examined ß-cell lines (INS1E and Min6, which proliferate slowly and are routinely fed fresh medium every 2-3 days) and found that the proinsulin pool size responds to each feeding within 1 to 2 h, affected both by the quantity of fresh nutrients and the frequency with which they are provided. We observed no effect of nutrient feeding on the overall rate of proinsulin turnover as quantified from cycloheximide-chase experiments. We show that nutrient feeding is primarily linked to rapid dephosphorylation of translation initiation factor eIF2α, presaging increased proinsulin levels (and thereafter, insulin levels), followed by its rephosphorylation during the ensuing hours that correspond to a fall in proinsulin levels. The decline of proinsulin levels is blunted by the integrated stress response inhibitor, ISRIB, or by inhibition of eIF2α rephosphorylation with a general control nonderepressible 2 (not PERK) kinase inhibitor. In addition, we demonstrate that amino acids contribute importantly to the proinsulin pool; mass spectrometry shows that ß-cells avidly consume extracellular glutamine, serine, and cysteine. Finally, we show that in both rodent and human pancreatic islets, fresh nutrient availability dynamically increases preproinsulin, which can be quantified without pulse-labeling. Thus, the proinsulin available for insulin biosynthesis is rhythmically controlled by fasting/feeding cycles.
Assuntos
Células Secretoras de Insulina , Nutrientes , Proinsulina , Humanos , Insulina/biossíntese , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Nutrientes/farmacologia , Proinsulina/biossíntese , Proinsulina/metabolismo , Estresse Fisiológico , Transdução de Sinais , Linhagem Celular , Regulação para CimaRESUMO
Hundreds of RNAs are enriched in the projections of neuronal cells. For the vast majority of them, though, the sequence elements that regulate their localization are unknown. To identify RNA elements capable of directing transcripts to neurites, we deployed a massively parallel reporter assay that tested the localization regulatory ability of thousands of sequence fragments drawn from endogenous mouse 3' UTRs. We identified peaks of regulatory activity within several 3' UTRs and found that sequences derived from these peaks were both necessary and sufficient for RNA localization to neurites in mouse and human neuronal cells. The localization elements were enriched in adenosine and guanosine residues. They were at least tens to hundreds of nucleotides long as shortening of two identified elements led to significantly reduced activity. Using RNA affinity purification and mass spectrometry, we found that the RNA-binding protein Unk was associated with the localization elements. Depletion of Unk in cells reduced the ability of the elements to drive RNAs to neurites, indicating a functional requirement for Unk in their trafficking. These results provide a framework for the unbiased, high-throughput identification of RNA elements and mechanisms that govern transcript localization in neurons.
Assuntos
Neurônios , Sequências Reguladoras de Ácido Ribonucleico , Regiões 3' não Traduzidas/genética , Animais , Humanos , Camundongos , Neurônios/metabolismo , Nucleotídeos/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Análise de Sequência de RNARESUMO
BACKGROUND: The thymus is a glandular organ that is essential for the formation of the adaptive immune system by educating developing T cells. The thymus is most active during childhood and involutes around the time of adolescence, resulting in a severe reduction or absence of naive T-cell output. The ability to generate a patient-derived human thymus would provide an attractive research platform and enable the development of novel cell therapies. OBJECTIVES: This study sought to systematically evaluate signaling pathways to develop a refined direct differentiation protocol that generates patient-derived thymic epithelial progenitor cells from multiple induced pluripotent stem cells (iPSCs) that can further differentiate into functional patient-derived thymic epithelial cells on transplantation into athymic nude mice. METHODS: Directed differentiation of iPSC generated TEPs that were transplanted into nude mice. Between 14 and 19 weeks posttransplantation, grafts were removed and analyzed by flow cytometry, quantitative PCR, bulk RNA sequencing, and single-cell RNA sequencing for markers of thymic-cell and T-cell development. RESULTS: A direct differentiation protocol that allows the generation of patient-derived thymic epithelial progenitor cells from multiple iPSC lines is described. On transplantation into athymic nude mice, patient-derived thymic epithelial progenitor cells further differentiate into functional patient-derived thymic epithelial cells that can facilitate the development of T cells. Single-cell RNA sequencing analysis of iPSC-derived grafts shows characteristic thymic subpopulations and patient-derived thymic epithelial cell populations that are indistinguishable from TECs present in primary neonatal thymus tissue. CONCLUSIONS: These findings provide important insights and resources for researchers focusing on human thymus biology.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Linfócitos T/fisiologia , Timo/citologia , Animais , Diferenciação Celular , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Humanos , Camundongos , Análise de Sequência de RNA , Timo/fisiologiaRESUMO
BACKGROUND: Varicella zoster virus (VZV) vasculopathy is characterized by persistent arterial inflammation leading to stroke. Studies show that VZV induces amyloid formation that may aggravate vasculitis. Thus, we determined if VZV central nervous system infection produces amyloid. METHODS: Aß peptides, amylin, and amyloid were measured in cerebrospinal fluid (CSF) from 16 VZV vasculopathy subjects and 36 stroke controls. To determine if infection induced amyloid deposition, mock- and VZV-infected quiescent primary human perineurial cells (qHPNCs), present in vasculature, were analyzed for intracellular amyloidogenic transcripts/proteins and amyloid. Supernatants were assayed for amyloidogenic peptides and ability to induce amyloid formation. To determine amylin's function during infection, amylin was knocked down with small interfering RNA and viral complementary DNA (cDNA) was quantitated. RESULTS: Compared to controls, VZV vasculopathy CSF had increased amyloid that positively correlated with amylin and anti-VZV antibody levels; Aß40 was reduced and Aß42 unchanged. Intracellular amylin, Aß42, and amyloid were seen only in VZV-infected qHPNCs. VZV-infected supernatant formed amyloid fibrils following addition of amyloidogenic peptides. Amylin knockdown decreased viral cDNA. CONCLUSIONS: VZV infection increased levels of amyloidogenic peptides and amyloid in CSF and qHPNCs, indicating that VZV-induced amyloid deposition may contribute to persistent arterial inflammation in VZV vasculopathy. In addition, we identified a novel proviral function of amylin.
Assuntos
Peptídeos beta-Amiloides , Amiloide , Arterite , Herpes Zoster , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Fragmentos de Peptídeos , Amiloide/líquido cefalorraquidiano , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Arterite/líquido cefalorraquidiano , Arterite/diagnóstico , Arterite/virologia , DNA Complementar , DNA Viral , Herpes Zoster/líquido cefalorraquidiano , Herpes Zoster/diagnóstico , Herpesvirus Humano 3 , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/líquido cefalorraquidiano , Fragmentos de Peptídeos/líquido cefalorraquidiano , Acidente Vascular CerebralRESUMO
BACKGROUND: Herpes zoster is linked to amyloid-associated diseases, including dementia, macular degeneration, and diabetes mellitus, in epidemiological studies. Thus, we examined whether varicella-zoster virus (VZV)-infected cells produce amyloid. METHODS: Production of intracellular amyloidogenic proteins (amylin, amyloid precursor protein [APP], and amyloid-ß [Aß]) and amyloid, as well as extracellular amylin, Aß, and amyloid, was compared between mock- and VZV-infected quiescent primary human spinal astrocytes (qHA-sps). The ability of supernatant from infected cells to induce amylin or Aß42 aggregation was quantitated. Finally, the amyloidogenic activity of viral peptides was examined. RESULTS: VZV-infected qHA-sps, but not mock-infected qHA-sps, contained intracellular amylin, APP, and/or Aß, and amyloid. No differences in extracellular amylin, Aß40, or Aß42 were detected, yet only supernatant from VZV-infected cells induced amylin aggregation and, to a lesser extent, Aß42 aggregation into amyloid fibrils. VZV glycoprotein B (gB) peptides assembled into fibrils and catalyzed amylin and Aß42 aggregation. CONCLUSIONS: VZV-infected qHA-sps produced intracellular amyloid and their extracellular environment promoted aggregation of cellular peptides into amyloid fibrils that may be due, in part, to VZV gB peptides. These findings suggest that together with host and other environmental factors, VZV infection may increase the toxic amyloid burden and contribute to amyloid-associated disease progression.
Assuntos
Peptídeos beta-Amiloides , Astrócitos , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Infecção pelo Vírus da Varicela-Zoster/metabolismo , Aciclovir/farmacologia , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Antivirais/farmacologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/virologia , Células Cultivadas , Espaço Extracelular/metabolismo , Humanos , Espaço Intracelular/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismoRESUMO
Directed differentiation of human pluripotent stem cells into functional insulin-producing beta-like cells holds great promise for cell replacement therapy for patients suffering from diabetes. This approach also offers the unique opportunity to study otherwise inaccessible aspects of human beta cell development and function in vitro. Here, we show that current pancreatic progenitor differentiation protocols promote precocious endocrine commitment, ultimately resulting in the generation of non-functional polyhormonal cells. Omission of commonly used BMP inhibitors during pancreatic specification prevents precocious endocrine formation while treatment with retinoic acid followed by combined EGF/KGF efficiently generates both PDX1(+) and subsequent PDX1(+)/NKX6.1(+) pancreatic progenitor populations, respectively. Precise temporal activation of endocrine differentiation in PDX1(+)/NKX6.1(+) progenitors produces glucose-responsive beta-like cells in vitro that exhibit key features of bona fide human beta cells, remain functional after short-term transplantation, and reduce blood glucose levels in diabetic mice. Thus, our simplified and scalable system accurately recapitulates key steps of human pancreas development and provides a fast and reproducible supply of functional human beta-like cells.
Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular , Células-Tronco Embrionárias/fisiologia , Células Secretoras de Insulina/fisiologia , Pâncreas/citologia , Animais , Glicemia/metabolismo , Células Cultivadas , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/terapia , Células-Tronco Embrionárias/citologia , Glucose/farmacologia , Humanos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/transplante , Camundongos , Camundongos SCID , Camundongos Transgênicos , EstreptozocinaRESUMO
The in vivo assessment of epigenetic changes during mouse pancreatic betacell differentiation reveals surprising differences to directed, in vitro differentiation of human embryonic stem cells. New findings reported in this issue of The EMBO Journal further identify Ezh2 as a critical determinant of endocrine progenitor number and could instruct improved protocols for stem cell-based therapies.
Assuntos
Células Endócrinas/citologia , Histonas/metabolismo , Ilhotas Pancreáticas/citologia , Histona Desmetilases com o Domínio Jumonji/genética , Complexo Repressor Polycomb 2/fisiologia , Animais , Proteína Potenciadora do Homólogo 2 de Zeste , HumanosRESUMO
The generation of stem cell-derived beta-like cells (sBC) holds extreme promise as not only an abundant insulin-producing cell source for replacement therapy of Type 1 diabetes (T1D), but also as an invaluable model system for investigating human beta cell development, immunogenicity, and function. Several groups have developed methodology to direct differentiate human pluripotent stem cells into pancreatic cell populations that include glucose responsive sBC. Nevertheless, the process of generating sBC poses significant experimental challenges. It involves lengthy differentiation periods, experiences substantial variability in efficiency, and inconsistencies in obtaining functional sBC. Here, we described a simple and effective cryopreservation approach for sBC cultures that yields homogeneous sBC clusters that are enriched for insulin expressing cells while simultaneous depleting proliferative progenitors. Thawed sBC display enhanced glucose-stimulated insulin release compared to controls in vitro and can effectively engraft and function in vivo. Collectively, this approach alleviates current challenges with inefficient and variable sBC generation while improving their functional state. We anticipate that these findings can inform ongoing clinical application of sBC for the treatment of patients with T1D and serve as an important resource for the wider diabetes field that will allow for accelerated research discoveries.
RESUMO
Despite significant research, mechanisms underlying the failure of islet beta cells that result in type 2 diabetes (T2D) are still under investigation. Here, we report that Sox9, a transcriptional regulator of pancreas development, also functions in mature beta cells. Our results show that Sox9-depleted rodent beta cells have defective insulin secretion, and aging animals develop glucose intolerance, mimicking the progressive degeneration observed in T2D. Using genome editing in human stem cells, we show that beta cells lacking SOX9 have stunted first-phase insulin secretion. In human and rodent cells, loss of Sox9 disrupts alternative splicing and triggers accumulation of non-functional isoforms of genes with key roles in beta cell function. Sox9 depletion reduces expression of protein-coding splice variants of the serine-rich splicing factor arginine SRSF5, a major splicing enhancer that regulates alternative splicing. Our data highlight the role of SOX9 as a regulator of alternative splicing in mature beta cell function.
Assuntos
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Ilhotas Pancreáticas , Animais , Humanos , Processamento Alternativo/genética , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Splicing de RNARESUMO
Targeting of current therapies to treat or prevent loss of pancreatic islet ß-cells in Type 1 Diabetes (T1D) may provide improved efficacy and reduce off target effects. Current efforts to target the ß-cell are limited by a lack of ß-cell specific targets and the inability to test multiple targeting moieties with the same delivery vehicle. Here we fabricate a novel tailorable polycaprolactone nanocapsule (NC) where multiple different targeting peptides can be interchangeably attached for ß-cell specific delivery. Incorporation of a cationic surfactant in the NC shell allows for the attachment of Exendin-4 and an antibody for ectonucleoside triphosphate diphosphohydrolase 3 (ENTPD3) for ß-cell specific targeting. The average NC size ranges from 250-300nm with a polydispersity index under 0.2. The NCs are non-toxic, stable in media culture, and can be lyophilized and reconstituted. NCs coated with targeting peptide were taken up by human cadaveric islet ß-cells and human stem cell-derived ß-like cells (sBC) in vitro with a high level of specificity. Furthermore, NCs successfully delivered both hydrophobic and hydrophilic cargo to human ß-cells. Finally, Exendin-4 coated NCs were stable and targeted the mouse pancreatic islet ß-cell in vivo . Our unique NC design allows for the interchangeable coating of targeting peptides for future screening of targets with improved cell specificity. The ability to target and deliver thera-peutics to human pancreatic ß-cells opens avenues for improved therapies and treatments to help the delay onset, prevent, or reverse T1D.
RESUMO
In vitro expansion of ß-cells from adult human pancreatic islets would overcome donor ß-cell shortage for cell replacement therapy for diabetes. Using a ß-cell-specific labeling system we have shown that ß-cell expansion is accompanied by dedifferentiation resembling epithelial-mesenchymal transition and loss of insulin expression. Epigenetic analyses indicate that key ß-cell genes maintain open chromatin structure in expanded ß-cell-derived (BCD) cells, although they are not transcribed. In the developing pancreas important cell-fate decisions are regulated by NOTCH receptors, which signal through the Hairy and Enhancer of Split 1 (HES1) transcription regulator. We have reported that BCD cell dedifferentiation and proliferation in vitro correlate with reactivation of the NOTCH pathway. Inhibition of HES1 expression using shRNA during culture initiation results in reduced ß-cell replication and dedifferentiation, suggesting that HES1 inhibition may also affect BCD cell redifferentiation following expansion. Here, we used HES1 shRNA to down-regulate HES1 expression in expanded human BCD cells, showing that HES1 inhibition is sufficient to induce BCD cell redifferentiation, as manifested by a significant increase in insulin expression. Combined treatment with HES1 shRNA, cell aggregation in serum-free medium, and a mixture of soluble factors further stimulated the redifferentiation of BCD cells. In vivo analyses demonstrated the ability of the redifferentiated cells to replace ß-cell function in hyperglycemic immunodeficient mice. These findings demonstrate the redifferentiation potential of ex vivo expanded BCD cells and the reproducible differentiating effect of HES1 inhibition in these cells.
Assuntos
Desdiferenciação Celular , Células Secretoras de Insulina/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Adolescente , Adulto , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proliferação de Células , Células Cultivadas , Epigênese Genética/genética , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação da Expressão Gênica/genética , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Humanos , Insulina/biossíntese , Células Secretoras de Insulina/citologia , Masculino , Pessoa de Meia-Idade , Fatores de Transcrição HES-1RESUMO
Cell replacement therapy using stem-cell-derived insulin-producing ß-like cells (sBCs) has been proposed as a practical cure for patients with type one diabetes (T1D). sBCs can correct diabetes in preclinical animal models, demonstrating the promise of this stem cell-based approach. However, in vivo studies have demonstrated that most sBCs, similarly to cadaveric human islets, are lost upon transplantation due to ischemia and other unknown mechanisms. Hence, there is a critical knowledge gap in the current field concerning the fate of sBCs upon engraftment. Here we review, discuss effects, and propose additional potential mechanisms that could contribute toward ß-cell loss in vivo. We summarize and highlight some of the literature on phenotypic loss in ß-cells under both steady, stressed, and diseased diabetic conditions. Specifically, we focus on ß-cell death, dedifferentiation into progenitors, trans-differentiation into other hormone-expressing cells, and/or interconversion into less functional ß-cell subtypes as potential mechanisms. While current cell replacement therapy efforts employing sBCs carry great promise as an abundant cell source, addressing the somewhat neglected aspect of ß-cell loss in vivo will further accelerate sBC transplantation as a promising therapeutic modality that could significantly enhance the life quality of T1D patients.
Assuntos
Diabetes Mellitus Tipo 1 , Células Secretoras de Insulina , Animais , Humanos , Diabetes Mellitus Tipo 1/terapia , Insulina/metabolismo , Células-Tronco/metabolismo , Células Secretoras de Insulina/metabolismo , Diferenciação CelularRESUMO
Transplantation of limited human cadaveric islets into type 1 diabetic patients results in â¼35 months of insulin independence. Direct differentiation of stem cell-derived insulin-producing beta-like cells (sBCs) that can reverse diabetes in animal models effectively removes this shortage constraint, but uncontrolled graft growth remains a concern. Current protocols do not generate pure sBCs, but consist of only 20%-50% insulin-expressing cells with additional cell types present, some of which are proliferative. Here, we show the selective ablation of proliferative cells marked by SOX9 by simple pharmacological treatment in vitro. This treatment concomitantly enriches for sBCs by â¼1.7-fold. Treated sBC clusters show improved function in vitro and in vivo transplantation controls graft size. Overall, our study provides a convenient and effective approach to enrich for sBCs while minimizing the presence of unwanted proliferative cells and thus has important implications for current cell therapy approaches.
Assuntos
Insulina , Pâncreas , Animais , Humanos , Diferenciação Celular , Terapia Baseada em Transplante de Células e Tecidos , Células-TroncoRESUMO
BACKGROUND: T1D is an autoimmune disease in which pancreatic islets of Langerhans are infiltrated by immune cells resulting in the specific destruction of insulin-producing islet beta cells. Our understanding of the factors leading to islet infiltration and the interplay of the immune cells with target beta cells is incomplete, especially in human disease. While murine models of T1D have provided crucial information for both beta cell and autoimmune cell function, the translation of successful therapies in the murine model to human disease has been a challenge. SCOPE OF REVIEW: Here, we discuss current state of the art and consider knowledge gaps concerning the interface of the islet beta cell with immune infiltrates, with a focus on T cells. We discuss pancreatic and immune cell phenotypes and their impact on cell function in health and disease, which we deem important to investigate further to attain a more comprehensive understanding of human T1D disease etiology. MAJOR CONCLUSIONS: The last years have seen accelerated development of approaches that allow comprehensive study of human T1D. Critically, recent studies have contributed to our revised understanding that the pancreatic beta cell assumes an active role, rather than a passive position, during autoimmune disease progression. The T cell-beta cell interface is a critical axis that dictates beta cell fate and shapes autoimmune responses. This includes the state of the beta cell after processing internal and external cues (e.g., stress, inflammation, genetic risk) that that contributes to the breaking of tolerance by hyperexpression of human leukocyte antigen (HLA) class I with presentation of native and neoepitopes and secretion of chemotactic factors to attract immune cells. We anticipate that emerging insights about the molecular and cellular aspects of disease initiation and progression processes will catalyze the development of novel and innovative intervention points to provide additional therapies to individuals affected by T1D.
Assuntos
Diabetes Mellitus Tipo 1 , Células Secretoras de Insulina , Ilhotas Pancreáticas , Humanos , Camundongos , Animais , Diabetes Mellitus Tipo 1/metabolismo , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Pâncreas/metabolismo , Fatores de RiscoRESUMO
The thymus is critical for the establishment of a functional and self-tolerant adaptive immune system but involutes with age, resulting in reduced naive T cell output. Generation of a functional human thymus from human pluripotent stem cells (hPSCs) is an attractive regenerative strategy. Direct differentiation of thymic epithelial progenitors (TEPs) from hPSCs has been demonstrated in vitro, but functional thymic epithelial cells (TECs) only form months after transplantation of TEPs in vivo. We show the generation of TECs in vitro in isogenic stem cell-derived thymic organoids (sTOs) consisting of TEPs, hematopoietic progenitor cells, and mesenchymal cells, differentiated from the same hPSC line. sTOs support T cell development, express key markers of negative selection, including the autoimmune regulator (AIRE) protein, and facilitate regulatory T cell development. sTOs provide the basis for functional patient-specific thymic organoid models, allowing for the study of human thymus function, T cell development, and transplant immunity.
Assuntos
Células-Tronco Pluripotentes , Timo , Humanos , Linfócitos T , Células Epiteliais/metabolismo , Diferenciação Celular/fisiologia , OrganoidesRESUMO
Cellular reprogramming by only small molecules holds enormous potentials for regenerative medicine. However, chemical reprogramming remains a slow process and labour intensive, hindering its broad applications and the investigation of underlying molecular mechanisms. Here, through screening of over 21,000 conditions, we develop a fast chemical reprogramming (FCR) system, which significantly improves the kinetics of cell identity rewiring. We find that FCR rapidly goes through an interesting route for pluripotent reprogramming, uniquely transitioning through a developmentally diapause-like state. Furthermore, FCR critically enables comprehensive characterizations using multi-omics technologies, and has revealed unexpected important features including key regulatory factors and epigenetic dynamics. Particularly, activation of pluripotency-related endogenous retroviruses via inhibition of heterochromatin significantly enhances reprogramming. Our studies provide critical insights into how only environmental cues are sufficient to rapidly reinstate pluripotency in somatic cells, and make notable technical and conceptual advances for solving the puzzle of regeneration.
Assuntos
Diapausa , Células-Tronco Pluripotentes Induzidas , Células-Tronco Pluripotentes , Animais , Reprogramação Celular/genética , Técnicas de Reprogramação Celular , Medicina RegenerativaRESUMO
Islet transplantation has proven to be an effective treatment for type 1 diabetes (T1D) yet is hampered by the shortage of available tissue. Recently, two reports from a Viacyte multicenter clinical trial demonstrate the feasibility, safety, and potential efficacy of transplanting macro-encapsulated human stem cell-derived pancreatic endoderm cells into patients with T1D, highlighting the promise of a stem cell-based therapeutic approach.
Assuntos
Diabetes Mellitus Tipo 1 , Transplante das Ilhotas Pancreáticas , Diferenciação Celular , Diabetes Mellitus Tipo 1/terapia , Endoderma , Humanos , Células-TroncoRESUMO
BACKGROUND: Type 1 diabetes (T1D) is an autoimmune disease in which pancreatic insulin-producing ß cells are specifically destroyed by the immune system. Understanding the initiation and progression of human T1D has been hampered by the lack of appropriate models that can reproduce the complexity and heterogeneity of the disease. The development of platforms combining multiple human pluripotent stem cell (hPSC) derived tissues to model distinct aspects of T1D has the potential to provide critical novel insights into the etiology and pathogenesis of the human disease. SCOPE OF REVIEW: In this review, we summarize the state of hPSC differentiation approaches to generate cell types and tissues relevant to T1D, with a particular focus on pancreatic islet cells, T cells, and thymic epithelium. We present current applications as well as limitations of using these hPSC-derived cells for disease modeling and discuss efforts to optimize platforms combining multiple cell types to model human T1D. Finally, we outline remaining challenges and emphasize future improvements needed to accelerate progress in this emerging field of research. MAJOR CONCLUSIONS: Recent advances in reprogramming approaches to create patient-specific induced pluripotent stem cell lines (iPSCs), genome engineering technologies to efficiently modify DNA of hPSCs, and protocols to direct their differentiation into mature cell types have empowered the use of stem cell derivatives to accurately model human disease. While challenges remain before complex interactions occurring in human T1D can be modeled with these derivatives, experiments combining hPSC-derived ß cells and immune cells are already providing exciting insight into how these cells interact in the context of T1D, supporting the viability of this approach.
Assuntos
Diabetes Mellitus Tipo 1 , Células-Tronco Pluripotentes Induzidas , Células Secretoras de Insulina , Células-Tronco Pluripotentes , Humanos , Diabetes Mellitus Tipo 1/metabolismo , Células-Tronco Pluripotentes/metabolismo , Células Secretoras de Insulina/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Diferenciação CelularRESUMO
There is a critical need for therapeutic approaches that combine renewable sources of replacement beta cells with localized immunomodulation to counter recurrence of autoimmunity in type 1 diabetes (T1D). However, there are few examples of animal models to study such approaches that incorporate spontaneous autoimmunity directed against human beta cells rather than allogenic rejection. Here, we address this critical limitation by demonstrating rejection and survival of transplanted human stem cell-derived beta-like cells clusters (sBCs) in a fully immune competent mouse model with matching human HLA class I and spontaneous diabetes development. We engineered localized immune tolerance toward transplanted sBCs via inducible cell surface overexpression of PD-L1 (iP-sBCs) with and without deletion of all HLA class I surface molecules via beta-2 microglobulin knockout (iP-BKO sBCs). NOD.HLA-A2.1 mice, which lack classical murine MHC I and instead express human HLA-A*02:01, underwent transplantation of 1,000 human HLA-A*02:01 sBCs under the kidney capsule and were separated into HLA-A2 positive iP-sBC and HLA-class I negative iP-BKO sBC groups, each with +/- doxycycline (DOX) induced PD-L1 expression. IVIS imaging showed significantly improved graft survival in mice transplanted with PD-L1 expressing iP-sBC at day 3 post transplantation compared to controls. However, luciferase signal dropped below in vivo detection limits by day 14 for all groups in this aggressive immune competent diabetes model. Nonetheless, histological examination revealed significant numbers of surviving insulin+/PD-L1+ sBCs cells for DOX-treated mice at day 16 post-transplant despite extensive infiltration with high numbers of CD3+ and CD45+ immune cells. These results show that T cells rapidly infiltrate and attack sBC grafts in this model but that significant numbers of PD-L1 expressing sBCs manage to survive in this harsh immunological environment. This investigation represents one of the first in vivo studies recapitulating key aspects of human autoimmune diabetes to test immune tolerance approaches with renewable sources of beta cells.