Browsing by Author "Xia, Ninuo"

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A PAX5-OCT4-PRDM1 developmental switch specifies human primordial germ cells
(2018-04) Fang, Fang; Angulo, Benjamin; Xia, Ninuo; Sukhwani, Meena; Wang, Zhengyuan; Carey, Charles C.; Mazurie, Aurélien J.; Cui, Jun; Wilkinson, Royce A.; Wiedenheft, Blake A.; Irie, Naoko; Surani, M. Azim; Orwig, Kyle E.; Reijo Pera, Renee A.
Dysregulation of genetic pathways during human germ cell development leads to infertility. Here, we analysed bona fide human primordial germ cells (hPGCs) to probe the developmental genetics of human germ cell specification and differentiation. We examined the distribution of OCT4 occupancy in hPGCs relative to human embryonic stem cells (hESCs). We demonstrated that development, from pluripotent stem cells to germ cells, is driven by switching partners with OCT4 from SOX2 to PAX5 and PRDM1. Gain- and loss-of-function studies revealed that PAX5 encodes a critical regulator of hPGC development. Moreover, an epistasis analysis indicated that PAX5 acts upstream of OCT4 and PRDM1. The PAX5-OCT4-PRDM1 proteins form a core transcriptional network that activates germline and represses somatic programmes during human germ cell differentiation. These findings illustrate the power of combined genome editing, cell differentiation and engraftment for probing human developmental genetics that have historically been difficult to study.
Quantification of dopaminergic neuron differentiation and neurotoxicity via a genetic reporter
(2016-04) Cui, Jun; Rotstein, Megan; Bennett, Theo; Zhangm, Pengbo; Xia, Ninuo; Reijo Pera, Renee A.
Human pluripotent stem cells provide a powerful human-genome based system for modeling human diseases in vitro and for potentially identifying novel treatments. Directed differentiation of pluripotent stem cells produces many specific cell types including dopaminergic neurons. Here, we generated a genetic reporter assay in pluripotent stem cells using newly-developed genome editing technologies in order to monitor differentiation efficiency and compare dopaminergic neuron survival under different conditions. We show that insertion of a luciferase reporter gene into the endogenous tyrosine hydroxylase (TH) locus enables rapid and easy quantification of dopaminergic neurons in cell culture throughout the entire differentiation process. Moreover, we demonstrate that the cellular assay is effective in assessing neuron response to different cytotoxic chemicals and is able to be scaled for high throughput applications. These results suggest that stem cell-derived terminal cell types can provide an alternative to traditional immortal cell lines or primary cells as a quantitative cellular model for toxin evaluation and drug discovery.