RESUMEN
Several distinct differentiation protocols for deriving pancreatic progenitors (PPs) from human pluripotent stem cells have been described, but it remains to be shown how similar the PPs are across protocols and how well they resemble their in vivo counterparts. Here, we evaluated three differentiation protocols, performed RNA and assay for transposase-accessible chromatin using sequencing on isolated PPs derived with these, and compared them with fetal human pancreas populations. This enabled us to define a shared transcriptional and epigenomic signature of the PPs, including several genes not previously implicated in pancreas development. Furthermore, we identified a significant and previously unappreciated cross-protocol variation of the PPs through multi-omics analysis and demonstrate how such information can be applied to refine differentiation protocols for derivation of insulin-producing beta-like cells. Together, our study highlights the importance of a detailed characterization of defined cell populations derived from distinct differentiation protocols and provides a valuable resource for exploring human pancreatic development.
Asunto(s)
Diferenciación Celular , Páncreas/citología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Biomarcadores , Técnicas de Cultivo de Célula , Células Cultivadas , Ensamble y Desensamble de Cromatina/genética , Biología Computacional/métodos , Epigénesis Genética , Perfilación de la Expresión Génica , Humanos , Inmunofenotipificación , Islotes Pancreáticos/citologíaRESUMEN
While induced pluripotent stem cell (iPSC) technologies enable the study of inaccessible patient cell types, cellular heterogeneity can confound the comparison of gene expression profiles between iPSC-derived cell lines. Here, we purified iPSC-derived human dopaminergic neurons (DaNs) using the intracellular marker, tyrosine hydroxylase. Once purified, the transcriptomic profiles of iPSC-derived DaNs appear remarkably similar to profiles obtained from mature post-mortem DaNs. Comparison of the profiles of purified iPSC-derived DaNs derived from Parkinson's disease (PD) patients carrying LRRK2 G2019S variants to controls identified significant functional convergence amongst differentially-expressed (DE) genes. The PD LRRK2-G2019S associated profile was positively matched with expression changes induced by the Parkinsonian neurotoxin rotenone and opposed by those induced by clioquinol, a compound with demonstrated therapeutic efficacy in multiple PD models. No functional convergence amongst DE genes was observed following a similar comparison using non-purified iPSC-derived DaN-containing populations, with cellular heterogeneity appearing a greater confound than genotypic background.
Asunto(s)
Células Madre Pluripotentes Inducidas/efectos de los fármacos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Enfermedad de Parkinson/tratamiento farmacológico , Transcriptoma/genética , Autopsia , Células Cultivadas , Clioquinol/administración & dosificación , Dopamina/genética , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/biosíntesis , Mutación , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Rotenona/metabolismo , Rotenona/toxicidad , Transcriptoma/efectos de los fármacosRESUMEN
Current screening methods for ovarian cancer can only detect advanced disease. Earlier detection has proved difficult because the molecular precursors involved in the natural history of the disease are unknown. To identify early driver mutations in ovarian cancer cells, we used dense whole genome sequencing of micrometastases and microscopic residual disease collected at three time points over three years from a single patient during treatment for high-grade serous ovarian cancer (HGSOC). The functional and clinical significance of the identified mutations was examined using a combination of population-based whole genome sequencing, targeted deep sequencing, multi-center analysis of protein expression, loss of function experiments in an in-vivo reporter assay and mammalian models, and gain of function experiments in primary cultured fallopian tube epithelial (FTE) cells. We identified frequent mutations involving a 40kb distal repressor region for the key stem cell differentiation gene SOX2. In the apparently normal FTE, the region was also mutated. This was associated with a profound increase in SOX2 expression (p<2(-16)), which was not found in patients without cancer (n=108). Importantly, we show that SOX2 overexpression in FTE is nearly ubiquitous in patients with HGSOCs (n=100), and common in BRCA1-BRCA2 mutation carriers (n=71) who underwent prophylactic salpingo-oophorectomy. We propose that the finding of SOX2 overexpression in FTE could be exploited to develop biomarkers for detecting disease at a premalignant stage, which would reduce mortality from this devastating disease.