Abstract
Given the difficulties of obtaining diseased cells, differentiation of neurons from patient-specific human induced pluripotent stem cells (iPSCs) with neural progenitor cells (NPCs) as intermediate precursors is of great interest. While cellular and transcriptomic changes during the differentiation process have been tracked, little attention has been given to examining spatial re-organization, which has been revealed to control gene regulation in various cells. To address the regulatory mechanism by 3D chromatin structure during neuronal differentiation, we examined the changes that take place during differentiation process using two cell types that are highly valued in the study of neurodegenerative disease - iPSCs and NPCs. In our study, we used Hi-C, a derivative of chromosome conformation capture that enables unbiased, genome-wide analysis of interaction frequencies in chromatin. We showed that while topologically associated domains remained mostly the same during differentiation, the presence of differential interacting regions in both cell types suggested that spatial organization affects gene regulation of both pluripotency maintenance and neuroectodermal differentiation. Moreover, closer analysis of promoter–promoter pairs suggested that cell fate specification is under the control of cis-regulatory elements. Our results are thus a resourceful addition in benchmarking differentiation protocols and also provide a greater appreciation of NPCs, the common precursors from which required neurons for applications in neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease, schizophrenia and spinal cord injuries are utilized.
Highlights
The use of patient-specific human induced pluripotent stem cells and their differentiated derivatives in disease-modelling and cell therapy development has been gaining traction in the studyGenes 2020, 11, 1176; doi:10.3390/genes11101176 www.mdpi.com/journal/genesGenes 2020, 11, 1176 of neurodegenerative diseases as the cells involved are difficult to isolate and scarce in number [1].Of special interest are neural progenitor cells (NPCs)—precursor cells from which neurons are generated for applications in diseases such as Parkinson’s disease [2], Alzheimer’s disease [3,4], schizophrenia [5]and spinal injury [6]
Chromatin interaction changes can be observed in defined regions called topologically associated domains (TAD) and these changes affect the activity of genes related in differentially interacted regions (DIR) [10]
We examined chromatin changes in the human induced pluripotent stem cell-neural progenitor cell (NPC) differentiation model using Hi-C, a variation of the chromosome conformation capture (3C) method first published by Dekker et al [9] and is distinguished from other adaptations such as 4C, 5C, ChIA-PET [16,17] by its unbiased genome-wide analysis of chromatin interaction frequencies [18]
Summary
The use of patient-specific human induced pluripotent stem cells (iPSCs) and their differentiated derivatives in disease-modelling and cell therapy development has been gaining traction in the studyGenes 2020, 11, 1176; doi:10.3390/genes11101176 www.mdpi.com/journal/genesGenes 2020, 11, 1176 of neurodegenerative diseases as the cells involved are difficult to isolate and scarce in number [1].Of special interest are neural progenitor cells (NPCs)—precursor cells from which neurons are generated for applications in diseases such as Parkinson’s disease [2], Alzheimer’s disease [3,4], schizophrenia [5]and spinal injury [6]. The use of patient-specific human induced pluripotent stem cells (iPSCs) and their differentiated derivatives in disease-modelling and cell therapy development has been gaining traction in the study. Comprehensive understanding of the iPSC-NPC model is crucial and other groups have tracked changes during the differentiation process through cellular [7] and transcriptomic studies [8]. Genome-wide 3D chromosomal construct mapping technologies have unearthed crucial findings on chromosome folding [12,13], and it has been reported that spatial conformation of chromatin is highly related with gene regulation such as promoter–promoter interaction and promoter–enhancer interaction [14,15]. Chromatin interaction changes can be observed in defined regions called topologically associated domains (TAD) and these changes affect the activity of genes related in differentially interacted regions (DIR) [10]
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