RESUMEN

The magnitude of CAR T cell expansion has been associated with clinical efficacy. Although cytokines can augment CAR T cell proliferation, systemically administered cytokines can result in toxicities. To gain the benefits of cytokine signaling while mitigating toxicities, we designed constitutively active synthetic cytokine receptor chimeras (constitutive Turbodomains) that signal in a CAR T cell-specific manner. The modular design of Turbodomains enables diverse cytokine signaling outputs from a single homodimeric receptor chimera and allows multiplexing of different cytokine signals. Turbodomains containing an IL-2/15Rß-derived signaling domain closely mimicked IL-15 signaling and enhanced CAR T cell potency. Allogeneic TurboCAR T cells targeting BCMA showed no evidence of aberrant proliferation yet displayed enhanced expansion and antitumor activity, prolonging survival and preventing extramedullary relapses in mouse models. These results illustrate the potential of constitutive Turbodomains to achieve selective potentiation of CAR T cells and demonstrate the safety and efficacy of allogeneic BCMA TurboCAR T cells, supporting clinical evaluation in multiple myeloma.

Asunto(s)

Trasplante de Células Madre Hematopoyéticas , Receptores Quiméricos de Antígenos , Animales , Ratones , Receptores Quiméricos de Antígenos/genética , Inmunoterapia Adoptiva/métodos , Antígeno de Maduración de Linfocitos B , Recurrencia Local de Neoplasia , Linfocitos T , Citocinas

RESUMEN

Multiple myeloma remains an incurable plasma cell malignancy despite the rapidly evolving treatment landscape. Chimeric antigen receptor T cells targeted against BCMA have recently shown great promise in relapsed refractory multiple myeloma; however, all patients ultimately still progress from their disease. Lack of CAR T-cell persistence, impaired T-cell fitness in autologous CAR T-cell products and the presence of an immunosuppressive bone marrow (BM) microenvironment are contributory factors to treatment failure. We generated anti-BCMA CAR T cells from healthy donors (HD) and patients with multiple myeloma at different stages of disease to compare their T-cell profile, fitness, and cytotoxic activity in preclinical studies. We also used an ex vivo assay with multiple myeloma BM biopsies from distinct genomic subgroups to test the efficacy of HD-derived CAR T cells in a clinically relevant model. HD volunteers showed increased T-cell counts, higher CD4/CD8 ratio, and expanded naïve T-cell population compared with patients with multiple myeloma. After anti-BCMA CAR T-cell production, patients with relapsed multiple myeloma had lower frequencies of CAR+ T cells, decreased central memory phenotype, and increased checkpoint inhibitory markers compared with HD-derived products, which compromised their expansion and cytotoxicity against multiple myeloma cells in vitro. Importantly, HD-derived CAR T cells efficiently killed primary multiple myeloma cells within the BM microenvironment of different multiple myeloma genomic subgroups and their cytotoxic activity could be boosted with gamma secretase inhibitors. In conclusion, allogeneic anti-BCMA CAR T cells are a potential therapeutic strategy for patients with relapsed multiple myeloma and should be further developed in the clinic. Significance: Multiple myeloma is an incurable cancer of the plasma cells. A new therapy with anti-BCMA CAR T cells - the patient's own T cells genetically engineered to find and kill myeloma cancer cells - has shown encouraging results. Unfortunately, patients still relapse. In this study, we propose to use T cells from HD volunteers, which have a stronger T-cell fitness, higher cancer killing capacity, and are ready to be administered when needed.

Asunto(s)

Trasplante de Células Madre Hematopoyéticas , Mieloma Múltiple , Receptores Quiméricos de Antígenos , Humanos , Mieloma Múltiple/terapia , Receptores Quiméricos de Antígenos/genética , Inhibidores y Moduladores de Gamma Secretasa , Recurrencia Local de Neoplasia , Linfocitos T , Microambiente Tumoral

RESUMEN

Patients with relapsed or refractory acute myeloid leukemia (AML) have a dismal prognosis and limited treatment options. Chimeric antigen receptor (CAR) T cells have achieved unprecedented clinical responses in patients with B cell leukemias and lymphomas and could prove highly efficacious in AML. However, a significant number of patients with AML may not receive treatment with an autologous product due to manufacturing failures associated with low lymphocyte counts or rapid disease progression while the therapeutic is being produced. We report the preclinical evaluation of an off-the-shelf CAR T cell therapy targeting Fms-related tyrosine kinase 3 (FLT3) for the treatment of AML. Single-chain variable fragments (scFvs) targeting various epitopes in the extracellular region of FLT3 were inserted into CAR constructs and tested for their ability to redirect T cell specificity and effector function to FLT3+ AML cells. A lead CAR, exhibiting minimal tonic signaling and robust activity in vitro and in vivo, was selected and then modified to incorporate a rituximab-responsive off-switch in cis. We found that allogeneic FLT3 CAR T cells, generated from healthy-donor T cells, eliminate primary AML blasts but are also active against mouse and human hematopoietic stem and progenitor cells, indicating risk of myelotoxicity. By employing a surrogate CAR with affinity to murine FLT3, we show that rituximab-mediated depletion of FLT3 CAR T cells after AML eradication enables bone marrow recovery without compromising leukemia remission. These results support clinical investigation of allogeneic FLT3 CAR T cells in AML and other FLT3+ hematologic malignancies.

Asunto(s)

Inmunoterapia Adoptiva , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/terapia , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/inmunología , Tirosina Quinasa 3 Similar a fms/inmunología , Animales , Médula Ósea/inmunología , Médula Ósea/metabolismo , Modelos Animales de Enfermedad , Humanos , Inmunoterapia Adoptiva/efectos adversos , Inmunoterapia Adoptiva/métodos , Leucemia Mieloide Aguda/diagnóstico , Ratones , Receptores Quiméricos de Antígenos/genética , Especificidad del Receptor de Antígeno de Linfocitos T , Linfocitos T/metabolismo , Resultado del Tratamiento , Ensayos Antitumor por Modelo de Xenoinjerto , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores

RESUMEN

FLT3 (FMS-like tyrosine kinase 3), expressed on the surface of acute myeloid leukemia (AML) blasts, is a promising AML target, given its role in the development and progression of leukemia, and its limited expression in tissues outside the hematopoietic system. Small molecule FLT3 kinase inhibitors have been developed, but despite having clinical efficacy, they are effective only on a subset of patients and associated with high risk of relapse. A durable therapy that can target a wider population of AML patients is needed. Here, we developed an anti-FLT3-CD3 immunoglobulin G (IgG)-based bispecific antibody (7370) with a high affinity for FLT3 and a long half-life, to target FLT3-expressing AML blasts, irrespective of FLT3 mutational status. We demonstrated that 7370 has picomolar potency against AML cell lines in vitro and in vivo. 7370 was also capable of activating T cells from AML patients, redirecting their cytotoxic activity against autologous blasts at low effector-to-target (E:T) ratio. Additionally, under our dosing regimen, 7370 was well tolerated and exhibited potent efficacy in cynomolgus monkeys by inducing complete but reversible depletion of peripheral FLT3+ dendritic cells (DCs) and bone marrow FLT3+ stem cells and progenitors. Overall, our results support further clinical development of 7370 to broadly target AML patients.

Asunto(s)

Anticuerpos Biespecíficos/farmacología , Antineoplásicos Inmunológicos/farmacología , Complejo CD3/antagonistas & inhibidores , Hematopoyesis/efectos de los fármacos , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Animales , Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/uso terapéutico , Antineoplásicos Inmunológicos/química , Antineoplásicos Inmunológicos/uso terapéutico , Médula Ósea/efectos de los fármacos , Médula Ósea/metabolismo , Médula Ósea/patología , Complejo CD3/química , Línea Celular Tumoral , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Humanos , Inmunoglobulina G/farmacología , Inmunofenotipificación , Leucemia Mieloide Aguda , Depleción Linfocítica , Macaca fascicularis , Ratones , Modelos Moleculares , Dominios Proteicos/efectos de los fármacos , Relación Estructura-Actividad , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Tirosina Quinasa 3 Similar a fms/química

RESUMEN

Clinical success of autologous CD19-directed chimeric antigen receptor T cells (CAR Ts) in acute lymphoblastic leukemia and non-Hodgkin lymphoma suggests that CAR Ts may be a promising therapy for hematological malignancies, including multiple myeloma. However, autologous CAR T therapies have limitations that may impact clinical use, including lengthy vein-to-vein time and manufacturing constraints. Allogeneic CAR T (AlloCAR T) therapies may overcome these innate limitations of autologous CAR T therapies. Unlike autologous cell therapies, AlloCAR T therapies employ healthy donor T cells that are isolated in a manufacturing facility, engineered to express CARs with specificity for a tumor-associated antigen, and modified using gene-editing technology to limit T cell receptor (TCR)-mediated immune responses. Here, transcription activator-like effector nuclease (TALEN) gene editing of B cell maturation antigen (BCMA) CAR Ts was used to confer lymphodepletion resistance and reduced graft-versus-host disease (GvHD) potential. The safety profile of allogeneic BCMA CAR Ts was further enhanced by incorporating a CD20 mimotope-based intra-CAR off switch enabling effective CAR T elimination in the presence of rituximab. Allogeneic BCMA CAR Ts induced sustained antitumor responses in mice supplemented with human cytokines, and, most importantly, maintained their phenotype and potency after scale-up manufacturing. This novel off-the-shelf allogeneic BCMA CAR T product is a promising candidate for clinical evaluation.

Asunto(s)

Antígeno de Maduración de Linfocitos B/inmunología , Trasplante de Células/métodos , Inmunoterapia Adoptiva/métodos , Mieloma Múltiple/terapia , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/inmunología , Linfocitos T/trasplante , Animales , Antineoplásicos Inmunológicos/uso terapéutico , Antígeno de Maduración de Linfocitos B/genética , Donantes de Sangre , Línea Celular Tumoral , Trasplante de Células/efectos adversos , Citotoxicidad Inmunológica/genética , Edición Génica , Vectores Genéticos , Enfermedad Injerto contra Huésped/terapia , Humanos , Inmunoterapia Adoptiva/efectos adversos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Mieloma Múltiple/patología , Supervivencia sin Progresión , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Rituximab/uso terapéutico , Linfocitos T/metabolismo , Nucleasas de los Efectores Tipo Activadores de la Transcripción/genética , Transducción Genética , Trasplante Homólogo/métodos

RESUMEN

Mutations in the gene Adenomatous Polyposis Coli or APC appear in most sporadic cases of colorectal cancer and it is the most frequent mutation causing hereditary Familial Adenomatous Polyposis. The detailed molecular mechanism by which APC mutations predispose to the development of colorectal cancer is not completely understood. This is in part due to the lack of accessibility to appropriate models that recapitulate the early events associated with APC mediated intestinal transformation. We have established a novel platform utilizing human induced Pluripotent Stem cells or iPSC from normal or FAP-specific APC mutant individuals and evaluated the effect of the mutation in the cells before and after differentiation into intestinal organoids. In order to minimize genetic background effects, we also established an isogenic platform using TALEN-mediated gene editing. Comparison of normal and APC mutant iPSC revealed a significant defect in cell identity and polarity due to the presence of APC in heterozygosity as well as chromosomal aberrations including abnormal anaphases and centrosome numbers. Importantly, upon specification into intestinal progeny, APC heterozygosity was responsible for a major change in the transcriptional identity of the cells with dysregulation of key signaling pathways, including metabolic reprogramming, abnormal lipid metabolism and intestinal-specific cadherin expression. In conclusion, we have developed a novel iPSC/intestinal model of APC mutagenesis and provide strong evidence that APC in heterozygosity imparts a clear phenotypic and molecular defect, affecting basic cellular functions and integrity, providing novel insights in the earlier events of APC-mediated tumorigenesis.

Asunto(s)

Proteína de la Poliposis Adenomatosa del Colon/genética , Poliposis Adenomatosa del Colon/genética , Células Madre Pluripotentes Inducidas/metabolismo , Mutagénesis , Mutación , Poliposis Adenomatosa del Colon/patología , Diferenciación Celular/genética , Transformación Celular Neoplásica/genética , Células Cultivadas , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Humanos , Células Madre Pluripotentes Inducidas/citología , Intestinos/patología , Organoides/metabolismo , Transducción de Señal/genética

RESUMEN

CAR T-cell therapies hold great promise for treating a range of malignancies but are however challenged by the complexity of their production and by the adverse events related to their activity. Here we report the development of the CubiCAR, a tri-functional CAR architecture that enables CAR T-cell detection, purification and on-demand depletion by the FDA-approved antibody Rituximab. This novel architecture has the potential to streamline the manufacturing of CAR T-cells, allow their tracking and improve their overall safety.

Asunto(s)

Inmunoterapia Adoptiva , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/cirugía , Receptores Quiméricos de Antígenos/inmunología , Rituximab/farmacología , Animales , Línea Celular Tumoral , Humanos , Ratones , Ratones Endogámicos BALB C , Neoplasias Experimentales/patología

RESUMEN

Patients with short bowel syndrome lack sufficient functional intestine to sustain themselves with enteral intake alone. Transplantable vascularized bioengineered intestine could restore nutrient absorption. Here we report the engineering of humanized intestinal grafts by repopulating decellularized rat intestinal matrix with human induced pluripotent stem cell-derived intestinal epithelium and human endothelium. After 28 days of in vitro culture, hiPSC-derived progenitor cells differentiate into a monolayer of polarized intestinal epithelium. Human endothelial cells seeded via native vasculature restore perfusability. Ex vivo isolated perfusion testing confirms transfer of glucose and medium-chain fatty acids from lumen to venous effluent. Four weeks after transplantation to RNU rats, grafts show survival and maturation of regenerated epithelium. Systemic venous sampling and positron emission tomography confirm uptake of glucose and fatty acids in vivo. Bioengineering intestine on vascularized native scaffolds could bridge the gap between cell/tissue-scale regeneration and whole organ-scale technology needed to treat intestinal failure patients.There is a need for humanised grafts to treat patients with intestinal failure. Here, the authors generate intestinal grafts by recellularizing native intestinal matrix with human induced pluripotent stem cell-derived epithelium and human endothelium, and show nutrient absorption after transplantation in rats.

Asunto(s)

Células Madre Pluripotentes Inducidas/citología , Intestinos/citología , Síndrome del Intestino Corto/terapia , Animales , Bioingeniería , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Células Endoteliales/citología , Ácidos Grasos/metabolismo , Glucosa/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/trasplante , Masculino , Ratas , Ratas Sprague-Dawley , Síndrome del Intestino Corto/metabolismo , Ingeniería de Tejidos , Andamios del Tejido , Trasplantes

RESUMEN

How embryonic stem cells (ESCs) commit to specific cell lineages and yield all cell types of a fully formed organism remains a major question. ESC differentiation is accompanied by large-scale histone and DNA modifications, but the relations between these epigenetic categories are not understood. Here we demonstrate the interplay between the histone deacetylase sirtuin 6 (SIRT6) and the ten-eleven translocation enzymes (TETs). SIRT6 targets acetylated histone H3 at Lys 9 and 56 (H3K9ac and H3K56ac), while TETs convert 5-methylcytosine into 5-hydroxymethylcytosine (5hmC). ESCs derived from Sirt6 knockout (S6KO) mice are skewed towards neuroectoderm development. This phenotype involves derepression of OCT4, SOX2 and NANOG, which causes an upregulation of TET-dependent production of 5hmC. Genome-wide analysis revealed neural genes marked with 5hmC in S6KO ESCs, thereby implicating TET enzymes in the neuroectoderm-skewed differentiation phenotype. We demonstrate that SIRT6 functions as a chromatin regulator safeguarding the balance between pluripotency and differentiation through Tet-mediated production of 5hmC.

Asunto(s)

Diferenciación Celular , Linaje de la Célula , Citosina/análogos & derivados , Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/enzimología , Proteínas Proto-Oncogénicas/metabolismo , Sirtuinas/metabolismo , 5-Metilcitosina/análogos & derivados , Acetilación , Animales , Células Cultivadas , Ensamble y Desensamble de Cromatina , Citosina/metabolismo , Proteínas de Unión al ADN/genética , Dioxigenasas , Células Madre Embrionarias/patología , Células Madre Embrionarias/trasplante , Regulación del Desarrollo de la Expresión Génica , Genotipo , Histonas/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/enzimología , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones SCID , Proteína Homeótica Nanog , Neurogénesis , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Fenotipo , Proteínas Proto-Oncogénicas/genética , Interferencia de ARN , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Transducción de Señal , Sirtuinas/deficiencia , Sirtuinas/genética , Teratoma/enzimología , Teratoma/patología , Transfección

RESUMEN

In response to fat intake, enteroendocrine K cells release the hormone glucose-dependent insulinotropic polypeptide (GIP). GIP acts on adipocytes to increase lipid uptake and enhance adipokine secretion, promoting weight gain and insulin resistance. Modulation of intestinal GIP release could therefore represent a therapeutic strategy for the treatment and prevention of obesity and diabetes. However, the prospects of using drugs to effectively target specific enteroendocrine cell types have been tempered by the realization that these cells share similar transcriptional programs and frequently employ common mechanisms of hormone secretion. To gain novel insights into the regulation of GIP release, we generated knock-in mice expressing green fluorescent protein (GFP) under the control of the endogenous GIP promoter that enable the isolation of a purified population of small intestine K cells. Using RNA sequencing, we comprehensively characterized the transcriptomes of GIP(GFP) cells as well as the entire enteroendocrine lineage derived from Neurogenin3-expressing progenitors. Among the genes differentially expressed in GIP(GFP) cells, we identified and validated fatty acid-binding protein 5 (FABP5) as a highly expressed marker of GIP-producing cells that is absent in other enteroendocrine cell types. FABP5 promotes intracellular transport and inactivation of endocannabinoids, including anandamide, which inhibits GIP release. Remarkably, we found that circulating levels of GIP were significantly decreased in FABP5-deficient mice in the fasting state and in response to acute, oral fat diet administration. Our findings highlight the power of RNA sequencing to uncover molecular signatures of specific enteroendocrine cell types that can potentially be exploited for therapeutic purposes in the treatment of metabolic disorders.

Asunto(s)

Células Enteroendocrinas/metabolismo , Proteínas de Unión a Ácidos Grasos/genética , Proteínas de Unión a Ácidos Grasos/metabolismo , Polipéptido Inhibidor Gástrico/genética , Polipéptido Inhibidor Gástrico/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Animales , Dieta , Endocannabinoides/genética , Endocannabinoides/metabolismo , Ayuno/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Resistencia a la Insulina/genética , Intestino Delgado , Enfermedades Metabólicas/genética , Enfermedades Metabólicas/metabolismo , Ratones , Obesidad/genética , Obesidad/metabolismo , Regiones Promotoras Genéticas/genética , ARN/genética , Análisis de Secuencia de ARN/métodos

RESUMEN

Transcription factor (TF)-induced reprogramming of somatic cells into induced pluripotent stem cells (iPSC) is associated with genome-wide changes in chromatin modifications. Polycomb-mediated histone H3 lysine-27 trimethylation (H3K27me3) has been proposed as a defining mark that distinguishes the somatic from the iPSC epigenome. Here, we dissected the functional role of H3K27me3 in TF-induced reprogramming through the inactivation of the H3K27 methylase EZH2 at the onset of reprogramming. Our results demonstrate that surprisingly the establishment of functional iPSC proceeds despite global loss of H3K27me3. iPSC lacking EZH2 efficiently silenced the somatic transcriptome and differentiated into tissues derived from the three germ layers. Remarkably, the genome-wide analysis of H3K27me3 in Ezh2 mutant iPSC cells revealed the retention of this mark on a highly selected group of Polycomb targets enriched for developmental regulators controlling the expression of lineage specific genes. Erasure of H3K27me3 from these targets led to a striking impairment in TF-induced reprogramming. These results indicate that PRC2-mediated H3K27 trimethylation is required on a highly selective core of Polycomb targets whose repression enables TF-dependent cell reprogramming.

Asunto(s)

Células Madre Pluripotentes Inducidas , Factor 3 de Transcripción de Unión a Octámeros , Complejo Represivo Polycomb 2 , Proteínas del Grupo Polycomb , Animales , Diferenciación Celular , Proliferación Celular , Metilación de ADN , Proteína Potenciadora del Homólogo Zeste 2 , Fibroblastos/citología , Fibroblastos/metabolismo , Silenciador del Gen , Histonas/genética , Histonas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Ratones , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Proteínas del Grupo Polycomb/genética , Proteínas del Grupo Polycomb/metabolismo

RESUMEN

The derivation of patient-specific pluripotent cell lines through the introduction of a few transcription factors into somatic cells has opened new avenues for the study and treatment of human disorders. Induced pluripotent stem cells (iPSCs) and their derivatives offer a unique platform for disease modeling, drug discovery and toxicology, as well as an invaluable source of cells for regenerative therapies. Here, we provide an overview of the various strategies currently available for iPSC generation, highlighting recent advances and discussing some of the challenges faced in harnessing the true potential of iPSCs for biomedical research and therapeutic applications.

Asunto(s)

Ingeniería Celular/métodos , Células Madre Pluripotentes Inducidas/fisiología , Factores de Transcripción/metabolismo , Animales , Diferenciación Celular/genética , Células Cultivadas , Vectores Genéticos , Humanos , Células Madre Pluripotentes Inducidas/citología , Ratones , Modelos Biológicos , Ratas , Factores de Transcripción/genética

RESUMEN

Delivery of the transcription factors Oct4, Klf4, Sox2 and c-Myc via integrating viral vectors has been widely employed to generate induced pluripotent stem cell (iPSC) lines from both normal and disease-specific somatic tissues, providing an invaluable resource for medical research and drug development. Residual reprogramming transgene expression from integrated viruses nevertheless alters the biological properties of iPSCs and has been associated with a reduced developmental competence both in vivo and in vitro. We performed transcriptional profiling of mouse iPSC lines before and after excision of a polycistronic lentiviral reprogramming vector to systematically define the overall impact of persistent transgene expression on the molecular features of iPSCs. We demonstrate that residual expression of the Yamanaka factors prevents iPSCs from acquiring the transcriptional program exhibited by embryonic stem cells (ESCs) and that the expression profiles of iPSCs generated with and without c-Myc are indistinguishable. After vector excision, we find 36% of iPSC clones show normal methylation of the Gtl2 region, an imprinted locus that marks ESC-equivalent iPSC lines. Furthermore, we show that the reprogramming factor Klf4 binds to the promoter region of Gtl2. Regardless of Gtl2 methylation status, we find similar endodermal and hepatocyte differentiation potential comparing syngeneic Gtl2(ON) vs Gtl2(OFF) iPSC clones. Our findings provide new insights into the reprogramming process and emphasize the importance of generating iPSCs free of any residual transgene expression.

Asunto(s)

Epigénesis Genética , Perfilación de la Expresión Génica , Células Madre Pluripotentes Inducidas/metabolismo , Factores de Transcripción/genética , Transcripción Genética , Animales , Diferenciación Celular/genética , Línea Celular , Reprogramación Celular , Análisis por Conglomerados , Metilación de ADN , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Silenciador del Gen , Hepatocitos/citología , Hepatocitos/metabolismo , Células Madre Pluripotentes Inducidas/citología , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/metabolismo , Masculino , Ratones , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Factores de Transcripción/metabolismo , Transgenes

RESUMEN

The directed differentiation of iPS and ES cells into definitive endoderm (DE) would allow the derivation of otherwise inaccessible progenitors for endodermal tissues. However, a global comparison of the relative equivalency of DE derived from iPS and ES populations has not been performed. Recent reports of molecular differences between iPS and ES cells have raised uncertainty as to whether iPS cells could generate autologous endodermal lineages in vitro. Here, we show that both mouse iPS and parental ES cells exhibited highly similar in vitro capacity to undergo directed differentiation into DE progenitors. With few exceptions, both cell types displayed similar surges in gene expression of specific master transcriptional regulators and global transcriptomes that define the developmental milestones of DE differentiation. Microarray analysis showed considerable overlap between the genetic programs of DE derived from ES/iPS cells in vitro and authentic DE from mouse embryos in vivo. Intriguingly, iPS cells exhibited aberrant silencing of imprinted genes known to participate in endoderm differentiation, yet retained a robust ability to differentiate into DE. Our results show that, despite some molecular differences, iPS cells can be efficiently differentiated into DE precursors, reinforcing their potential for development of cell-based therapies for diseased endoderm-derived tissues.

Asunto(s)

Células Madre Embrionarias/citología , Endodermo/citología , Regulación del Desarrollo de la Expresión Génica , Impresión Genómica/fisiología , Células Madre Pluripotentes Inducidas/citología , Animales , Diferenciación Celular/fisiología , Línea Celular/citología , Línea Celular/metabolismo , Linaje de la Célula , Separación Celular , Quimera , Mapeo Cromosómico , Células Clonales/citología , Células Clonales/metabolismo , Metilación de ADN , Células Madre Embrionarias/metabolismo , Proteínas Fetales/biosíntesis , Proteínas Fetales/genética , Genes Reporteros , Células Madre Pluripotentes Inducidas/metabolismo , Hígado/embriología , Ratones , Ratones SCID , Células Madre Multipotentes/citología , Células Madre Multipotentes/metabolismo , Trasplante de Neoplasias , Teratoma/patología , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética

RESUMEN

Induced pluripotent stem cells (iPSCs) can differentiate into mineralizing cells and thus have a great potential in application in engineered bone substitutes with bioactive scaffolds in regeneration medicine. In the current study we characterized and demonstrated the pluripotency and osteogenic differentiation of mouse iPSCs. To enhance the osteogenic differentiation of iPSCs, we then transduced the iPSCs with the potent transcription factor, nuclear matrix protein SATB2. We observed that in SATB2-overexpressing iPSCs there were increased mineral nodule formation and elevated mRNA levels of key osteogenic genes, osterix (OSX), Runx2, bone sialoprotein (BSP) and osteocalcin (OCN). Moreover, the mRNA levels of HoxA2 was reduced after SATB2 overexpression in iPSCs. The SATB2-overexpressing iPSCs were then combined with silk scaffolds and transplanted into critical-size calvarial bone defects created in nude mice. Five weeks post-surgery, radiological and micro-CT analysis revealed enhanced new bone formation in calvarial defects in SATB2 group. Histological analysis also showed increased new bone formation and mineralization in the SATB2 group. In conclusion, the results demonstrate that SATB2 facilitates the differentiation of iPSCs towards osteoblast-lineage cells by repressing HoxA2 and augmenting the functions of the osteoblast determinants Runx2, BSP and OCN.

Asunto(s)

Regeneración Ósea/fisiología , Células Madre Pluripotentes Inducidas/fisiología , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Seda/química , Cráneo/patología , Ingeniería de Tejidos/instrumentación , Andamios del Tejido/química , Factores de Transcripción/metabolismo , Animales , Materiales Biocompatibles/química , Materiales Biocompatibles/metabolismo , Diferenciación Celular , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Células Madre Pluripotentes Inducidas/citología , Sialoproteína de Unión a Integrina/genética , Sialoproteína de Unión a Integrina/metabolismo , Ensayo de Materiales , Proteínas de Unión a la Región de Fijación a la Matriz/genética , Ratones , Ratones Desnudos , Osteocalcina/genética , Osteocalcina/metabolismo , Cráneo/citología , Cráneo/metabolismo , Factor de Transcripción Sp7 , Ingeniería de Tejidos/métodos , Factores de Transcripción/genética , Transducción Genética , Cicatrización de Heridas/fisiología

RESUMEN

TET family enzymes convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. Here, we show that Tet1 and Tet2 are Oct4-regulated enzymes that together sustain 5hmC in mouse embryonic stem cells (ESCs) and are induced concomitantly with 5hmC during reprogramming of fibroblasts to induced pluripotent stem cells. ESCs depleted of Tet1 by RNAi show diminished expression of the Nodal antagonist Lefty1 and display hyperactive Nodal signaling and skewed differentiation into the endoderm-mesoderm lineage in embryoid bodies in vitro. In Fgf4- and heparin-supplemented culture conditions, Tet1-depleted ESCs activate the trophoblast stem cell lineage determinant Elf5 and can colonize the placenta in midgestation embryo chimeras. Consistent with these findings, Tet1-depleted ESCs form aggressive hemorrhagic teratomas with increased endoderm, reduced neuroectoderm, and ectopic appearance of trophoblastic giant cells. Thus, 5hmC is an epigenetic modification associated with the pluripotent state, and Tet1 functions to regulate the lineage differentiation potential of ESCs.

Asunto(s)

Citosina/análogos & derivados , Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , 5-Metilcitosina/análogos & derivados , Animales , Sitios de Unión/genética , Sitios de Unión/fisiología , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Linaje de la Célula , Inmunoprecipitación de Cromatina , Biología Computacional , Citosina/metabolismo , Proteínas de Unión al ADN/genética , Dioxigenasas , Ratones , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Proteínas Proto-Oncogénicas/genética , Teratoma/genética , Teratoma/metabolismo

RESUMEN

Tissue engineering provides a new paradigm for periodontal tissue regeneration in which proper stem cells and effective cellular factors are very important. The objective of this study was, for the first time, to investigate the capabilities and advantages of periodontal tissue regeneration using induced pluripotent stem (iPS) cells and enamel matrix derivatives (EMD). In this study the effect of EMD gel on iPS cells in vitro was first determined, and then tissue engineering technique was performed to repair periodontal defects in three groups: silk scaffold only; silk scaffold + EMD; and silk scaffold + EMD + iPS cells. EMD greatly enhanced the mRNA expression of Runx2 but inhibited the mRNA expression of OC and mineralization nodule formation in vitro. Transplantation of iPS cells showed higher expression levels of OC, Osx, and Runx2 genes, both 12 and 24 days postsurgery. At 24 days postsurgery in the iPS cell group, histological analysis showed much more new alveolar bone and cementum formation with regenerated periodontal ligament between them. The results showed the commitment role that EMD contributes in mesenchymal progenitors to early cells in the osteogenic lineage. iPS cells combined with EMD provide a valuable tool for periodontal tissue engineering, by promoting the formation of new cementum, alveolar bone, and normal periodontal ligament.

Asunto(s)

Regeneración Tisular Guiada Periodontal/métodos , Células Madre Pluripotentes/fisiología , Animales , Regeneración Ósea/fisiología , Diferenciación Celular , Cemento Dental/metabolismo , Proteínas del Esmalte Dental/farmacología , Regulación de la Expresión Génica/fisiología , Humanos , Masculino , Ratones , Ratones Desnudos , Ligamento Periodontal/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ingeniería de Tejidos , Andamios del Tejido

RESUMEN

The development of methods to achieve efficient reprogramming of human cells while avoiding the permanent presence of reprogramming transgenes represents a critical step toward the use of induced pluripotent stem cells (iPSC) for clinical purposes, such as disease modeling or reconstituting therapies. Although several methods exist for generating iPSC free of reprogramming transgenes from mouse cells or neonatal normal human tissues, a sufficiently efficient reprogramming system is still needed to achieve the widespread derivation of disease-specific iPSC from humans with inherited or degenerative diseases. Here, we report the use of a humanized version of a single lentiviral "stem cell cassette" vector to accomplish efficient reprogramming of normal or diseased skin fibroblasts obtained from humans of virtually any age. Simultaneous transfer of either three or four reprogramming factors into human target cells using this single vector allows derivation of human iPSC containing a single excisable viral integration that on removal generates human iPSC free of integrated transgenes. As a proof of principle, here we apply this strategy to generate >100 lung disease-specific iPSC lines from individuals with a variety of diseases affecting the epithelial, endothelial, or interstitial compartments of the lung, including cystic fibrosis, α-1 antitrypsin deficiency-related emphysema, scleroderma, and sickle-cell disease. Moreover, we demonstrate that human iPSC generated with this approach have the ability to robustly differentiate into definitive endoderm in vitro, the developmental precursor tissue of lung epithelia.

Asunto(s)

Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Células Cultivadas , Reprogramación Celular/genética , Reprogramación Celular/fisiología , Endodermo/citología , Fibroblastos/citología , Fibroblastos/metabolismo , Citometría de Flujo , Vectores Genéticos/genética , Humanos , Lentivirus/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

Derivation of autologous induced pluripotent stem cells (iPSCs) through direct reprogramming of easily accessible somatic cells holds the potential to transform the field of regenerative medicine. Since Takahashi and Yamanaka's groundbreaking study describing the generation of iPSCs by retroviral-mediated delivery of defined transcription factors, substantial progress has been made to improve both the efficiency and safety of the method. These advances have provided new insights into the molecular mechanisms of reprogramming and promise to accelerate the clinical translation of iPSC technology. Here, we summarize current reprogramming methodologies with a focus on the production of transgene-free or genetically unmanipulated iPSCs and highlight important technical details that ultimately may influence the biological properties of pluripotent stem cells.

Asunto(s)

Reprogramación Celular/genética , Vectores Genéticos/genética , Células Madre Pluripotentes Inducidas/citología , Factores de Transcripción/metabolismo , Diferenciación Celular/genética , Técnicas de Transferencia de Gen , Humanos , Medicina Regenerativa

Asunto(s)

Leucocitos Mononucleares/citología , Linfocitos T/citología , Southern Blotting , Técnicas de Cultivo de Célula , Células Cultivadas , Reprogramación Celular , Humanos , Inmunohistoquímica , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Leucocitos Mononucleares/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/metabolismo