RESUMEN
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.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Células Madre Embrionarias/fisiología , Células Secretoras de Insulina/fisiología , Páncreas/citología , Animales , Glucemia/metabolismo , Células Cultivadas , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/terapia , Células Madre Embrionarias/citología , Glucosa/farmacología , Humanos , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/trasplante , Ratones , Ratones SCID , Ratones Transgénicos , EstreptozocinaRESUMEN
G protein-coupled receptors (GPCRs) are a group of seven-transmembrane receptor proteins that have proven to be successful drug targets. Antibodies are becoming an increasingly promising modality to target these receptors due to their unique properties, such as exquisite specificity, long half-life, and fewer side effects, and their improved pharmacokinetic and pharmacodynamic profiles compared to peptides and small molecules, which results from their more favorable biodistribution. To date, there are only two US Food and Drug Administration-approved GPCR antibody drugs, namely erenumab and mogamulizumab, and this highlights the challenges encountered in identifying functional antibodies against GPCRs. Utilizing Twist's precision DNA writing technologies, we have created a GPCR-focused phage display library with 1 × 1010 diversity. Specifically, we mined endogenous GPCR binding ligand and peptide sequences and incorporated these binding motifs into the heavy chain complementarity-determining region 3 in a synthetic antibody library. Glucagon-like peptide-1 receptor (GLP-1 R) is a class B GPCR that acts as the receptor for the incretin GLP-1, which is released to regulate insulin levels in response to food intake. GLP-1 R agonists have been widely used to increase insulin secretion to lower blood glucose levels for the treatment of type 1 and type 2 diabetes, whereas GLP-1 R antagonists have applications in the treatment of severe hypoglycemia associated with bariatric surgery and hyperinsulinomic hypoglycemia. Here we present the discovery and creation of both antagonistic and agonistic GLP-1 R antibodies by panning this GPCR-focused phage display library on a GLP-1 R-overexpressing Chinese hamster ovary cell line and demonstrate their in vitro and in vivo functional activity.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Glucemia/efectos de los fármacos , Técnicas de Visualización de Superficie Celular , Receptor del Péptido 1 Similar al Glucagón/agonistas , Receptor del Péptido 1 Similar al Glucagón/antagonistas & inhibidores , Control Glucémico , Hipoglucemiantes/farmacología , Incretinas/farmacología , Biblioteca de Péptidos , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacocinética , Sitios de Unión de Anticuerpos , Biomarcadores/sangre , Glucemia/metabolismo , Células CHO , Cricetulus , Receptor del Péptido 1 Similar al Glucagón/genética , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Ensayos Analíticos de Alto Rendimiento , Hipoglucemiantes/metabolismo , Hipoglucemiantes/farmacocinética , Incretinas/genética , Incretinas/metabolismo , Incretinas/farmacocinética , Ligandos , Masculino , Ratones Endogámicos C57BL , Dominios y Motivos de Interacción de Proteínas , Ratas Sprague-DawleyRESUMEN
The low number of CD4+ CD25+ regulatory T cells (Tregs), their anergic phenotype, and diverse antigen specificity present major challenges to harnessing this potent tolerogenic population to treat autoimmunity and transplant rejection. In this study, we describe a robust method to expand antigen-specific Tregs from autoimmune-prone nonobese diabetic mice. Purified CD4+ CD25+ Tregs were expanded up to 200-fold in less than 2 wk in vitro using a combination of anti-CD3, anti-CD28, and interleukin 2. The expanded Tregs express a classical cell surface phenotype and function both in vitro and in vivo to suppress effector T cell functions. Most significantly, small numbers of antigen-specific Tregs can reverse diabetes after disease onset, suggesting a novel approach to cellular immunotherapy for autoimmunity.
Asunto(s)
Autoantígenos/inmunología , Antígenos CD4/análisis , Diabetes Mellitus Tipo 1/terapia , Receptores de Interleucina-2/análisis , Linfocitos T Reguladores/inmunología , Traslado Adoptivo , Animales , Diabetes Mellitus Tipo 1/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/fisiologíaRESUMEN
FcR-binding "classical" anti-CD3 mAb is a potent immunosuppressive drug that alters CD4(+) and CD8(+) T cell function in vivo via anergy induction and programmed cell death (PCD). Anti-CD3-mediated PCD was Fas independent but was mediated by the mitochondria-initiated apoptosis that was abrogated in Bcl-x(L)-transgenic T cells. The PCD was more pronounced in CD28-deficient mice consistent with defective Bcl-x(L) up-regulation. Residual T cells isolated from anti-CD3-treated wild-type, CD28(-/-), and Bcl-x(L)-transgenic mice were hyporesponsive. The hyporesponsiveness was more pronounced in CD28(-/-) and wild-type mice treated with anti-B7-2, suggesting that CD28 interaction with B7-2 regulates T cell responsiveness in anti-CD3-treated animals. Finally, anti-CD3 treatment led to indefinite cardiac allograft survival in wild-type but not Bcl-x(L) animals. Together these results implicate CD28/B7 signaling in the regulation of both anti-CD3-induced T cell depletion and hyporesponsiveness in vivo, but T cell depletion, not hyporesponsiveness, appears to be critical for anti-CD3 mAb-mediated long-term immune regulation.