RESUMEN
Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) resets their identity back to an embryonic age and, thus, presents a significant hurdle for modeling late-onset disorders. In this study, we describe a strategy for inducing aging-related features in human iPSC-derived lineages and apply it to the modeling of Parkinson's disease (PD). Our approach involves expression of progerin, a truncated form of lamin A associated with premature aging. We found that expression of progerin in iPSC-derived fibroblasts and neurons induces multiple aging-related markers and characteristics, including dopamine-specific phenotypes such as neuromelanin accumulation. Induced aging in PD iPSC-derived dopamine neurons revealed disease phenotypes that require both aging and genetic susceptibility, such as pronounced dendrite degeneration, progressive loss of tyrosine hydroxylase (TH) expression, and enlarged mitochondria or Lewy-body-precursor inclusions. Thus, our study suggests that progerin-induced aging can be used to reveal late-onset age-related disease features in hiPSC-based disease models.
Asunto(s)
Envejecimiento/patología , Células Madre Pluripotentes Inducidas/metabolismo , Modelos Biológicos , Proteínas Nucleares/metabolismo , Precursores de Proteínas/metabolismo , Adulto , Edad de Inicio , Anciano , Anciano de 80 o más Años , Animales , Biomarcadores/metabolismo , Diferenciación Celular , Reprogramación Celular , Senescencia Celular , Niño , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Neuronas Dopaminérgicas/trasplante , Neuronas Dopaminérgicas/ultraestructura , Fibroblastos/metabolismo , Humanos , Lamina Tipo A , Mesencéfalo/patología , Ratones , Persona de Mediana Edad , Enfermedad de Parkinson/patología , Fenotipo , Donantes de TejidosRESUMEN
Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) offer great promise in regenerative medicine and disease modeling due to their unlimited self-renewal and broad differentiation capacity. There is evidence that the growth properties and critical signaling pathways differ between murine and human ESCs; therefore, it is essential to perform functional studies to test the putatively conserved mechanisms of pluripotent stem cell self-renewal between species. Previously, we identified the transcription factor Zfx as a key regulator of self-renewal in murine ESCs. Here we extend those findings to human ESCs. ZFX knockdown in hESCs hindered clonal growth and decreased colony size after serial replating. ZFX overexpression enhanced clone formation in the presence of Y-27632, increased colony size at low density and decreased expression of differentiation-related genes in human ESCs. ZFX-overexpressing hESCs resisted spontaneous differentiation but could be directed to differentiate into endodermal and neural cell fates when provided with the appropriate cues. Thus, ZFX acts as a molecular rheostat regulating the balance between self-renewal and differentiation in hESCs, revealing the close evolutionary conservation of the self-renewal mechanisms in murine and human ESCs.
Asunto(s)
Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Animales , Diferenciación Celular/genética , Línea Celular , Proliferación Celular , Tamaño de la Célula , Cromosomas Artificiales Bacterianos/genética , Células Clonales , Endodermo/citología , Endodermo/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Ratones , Transgenes/genéticaRESUMEN
Embryonic stem cells (ESCs) represent a promising source of midbrain dopaminergic (DA) neurons for applications in Parkinson disease. However, ESC-based transplantation paradigms carry a risk of introducing inappropriate or tumorigenic cells. Cell purification before transplantation may alleviate these concerns and enable identification of the specific DA neuron stage most suitable for cell therapy. Here, we used 3 transgenic mouse ESC reporter lines to mark DA neurons at 3 stages of differentiation (early, middle, and late) following induction of differentiation using Hes5::GFP, Nurr1::GFP, and Pitx3::YFP transgenes, respectively. Transplantation of FACS-purified cells from each line resulted in DA neuron engraftment, with the mid-stage and late-stage neuron grafts being composed almost exclusively of midbrain DA neurons. Mid-stage neuron cell grafts had the greatest amount of DA neuron survival and robustly induced recovery of motor deficits in hemiparkinsonian mice. Our data suggest that the Nurr1+ stage (middle stage) of neuronal differentiation is particularly suitable for grafting ESC-derived DA neurons. Moreover, global transcriptome analysis of progeny from each of the ESC reporter lines revealed expression of known midbrain DA neuron genes and also uncovered previously uncharacterized midbrain genes. These data demonstrate remarkable fate specificity of ESC-derived DA neurons and outline a sequential stage-specific ESC reporter line paradigm for in vivo gene discovery.
