RESUMEN
Human pluripotent stem cells hold potential for regenerative medicine, but available cell types have significant limitations. Although embryonic stem cells (ES cells) from in vitro fertilized embryos (IVF ES cells) represent the 'gold standard', they are allogeneic to patients. Autologous induced pluripotent stem cells (iPS cells) are prone to epigenetic and transcriptional aberrations. To determine whether such abnormalities are intrinsic to somatic cell reprogramming or secondary to the reprogramming method, genetically matched sets of human IVF ES cells, iPS cells and nuclear transfer ES cells (NT ES cells) derived by somatic cell nuclear transfer (SCNT) were subjected to genome-wide analyses. Both NT ES cells and iPS cells derived from the same somatic cells contained comparable numbers of de novo copy number variations. In contrast, DNA methylation and transcriptome profiles of NT ES cells corresponded closely to those of IVF ES cells, whereas iPS cells differed and retained residual DNA methylation patterns typical of parental somatic cells. Thus, human somatic cells can be faithfully reprogrammed to pluripotency by SCNT and are therefore ideal for cell replacement therapies.
Asunto(s)
Reprogramación Celular , Células Madre Pluripotentes/metabolismo , Animales , Línea Celular , Aberraciones Cromosómicas , Cromosomas Humanos X/genética , Cromosomas Humanos X/metabolismo , Variaciones en el Número de Copia de ADN , Metilación de ADN , Estudio de Asociación del Genoma Completo , Impresión Genómica , Humanos , Técnicas de Transferencia Nuclear/normas , Células Madre Pluripotentes/citología , TranscriptomaRESUMEN
The original version of this Article contained an error in Fig. 3. Panel b was inadvertently duplicated and the correct panel c was originally omitted. This error has been corrected in both the PDF and HTML versions of the Article.
RESUMEN
Cytokinin fulfills its diverse roles in planta through a series of transcriptional responses. We identify the in vivo DNA binding site profiles for three genetically redundant type-B ARABIDOPSIS RESPONSE REGULATORS (B-ARRs): ARR1, ARR10, and ARR12. The expression and genome-wide DNA binding locations of the three B-ARRs extensively overlap. Constructing a primary cytokinin response transcriptional network reveals a recurring theme of widespread cross-regulation between the components of the cytokinin pathway and other plant hormone pathways. The B-ARRs are found to have similar DNA binding motifs, though sequences flanking the core motif were degenerate. Cytokinin treatments amalgamate the three different B-ARRs motifs to identical DNA binding signatures (AGATHY, H(a/t/c), Y(t/c)) which suggests cytokinin may regulate binding activity of B-ARR family members. Furthermore, we find that WUSCHEL, a key gene required for apical meristem maintenance, is a cytokinin-dependent B-ARR target gene, demonstrating the importance of the cytokinin transcription factor network in shoot development.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/fisiología , Citocininas/metabolismo , Regulación de la Expresión Génica de las Plantas/fisiología , Proteínas de Homeodominio/genética , Reguladores del Crecimiento de las Plantas/genética , Proteínas de Arabidopsis/metabolismo , Sitios de Unión , Citocininas/genética , ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Redes Reguladoras de Genes/fisiología , Meristema/fisiología , Motivos de Nucleótidos/fisiología , Plantas Modificadas Genéticamente , Unión Proteica/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Oocyte defects lie at the heart of some forms of infertility and could potentially be addressed therapeutically by alternative routes for oocyte formation. Here, we describe the generation of functional human oocytes following nuclear transfer of first polar body (PB1) genomes from metaphase II (MII) oocytes into enucleated donor MII cytoplasm (PBNT). The reconstructed oocytes supported the formation of de novo meiotic spindles and, after fertilization with sperm, meiosis completion and formation of normal diploid zygotes. While PBNT zygotes developed to blastocysts less frequently (42%) than controls (75%), genome-wide genetic, epigenetic, and transcriptional analyses of PBNT and control ESCs indicated comparable numbers of structural variations and markedly similar DNA methylation and transcriptome profiles. We conclude that rescue of PB1 genetic material via introduction into donor cytoplasm may offer a source of oocytes for infertility treatment or mitochondrial replacement therapy for mtDNA disease.
Asunto(s)
Genoma Humano , Técnicas de Transferencia Nuclear , Oocitos/metabolismo , Cuerpos Polares/metabolismo , Adulto , Blastocisto/metabolismo , Metilación de ADN/genética , Desarrollo Embrionario/genética , Epigénesis Genética , Femenino , Fertilización In Vitro , Perfilación de la Expresión Génica , Inestabilidad Genómica , Células Madre Embrionarias Humanas/metabolismo , Humanos , Masculino , Metafase , Ploidias , Análisis de Secuencia de ARN , Espermatozoides/metabolismo , Huso Acromático/metabolismo , Transcripción GenéticaRESUMEN
Recent studies have aimed to convert cultured human pluripotent cells to a naive state, but it remains unclear to what extent the resulting cells recapitulate in vivo naive pluripotency. Here we propose a set of molecular criteria for evaluating the naive human pluripotent state by comparing it to the human embryo. We show that transcription of transposable elements provides a sensitive measure of the concordance between pluripotent stem cells and early human development. We also show that induction of the naive state is accompanied by genome-wide DNA hypomethylation, which is reversible except at imprinted genes, and that the X chromosome status resembles that of the human preimplantation embryo. However, we did not see efficient incorporation of naive human cells into mouse embryos. Overall, the different naive conditions we tested showed varied relationships to human embryonic states based on molecular criteria, providing a backdrop for future analysis of naive human pluripotency.