Abstract
Parent of origin imprints on the genome have been implicated in the regulation of neural cell type differentiation. The ability of human parthenogenetic (PG) embryonic stem cells (hpESCs) to undergo neural lineage and cell type-specific differentiation is undefined. We determined the potential of hpESCs to differentiate into various neural subtypes. Concurrently, we examined DNA methylation and expression status of imprinted genes. Under culture conditions promoting neural differentiation, hpESC-derived neural stem cells (hpNSCs) gave rise to glia and neuron-like cells that expressed subtype-specific markers and generated action potentials. Analysis of imprinting in hpESCs and in hpNSCs revealed that maternal-specific gene expression patterns and imprinting marks were generally maintained in PG cells upon differentiation. Our results demonstrate that despite the lack of a paternal genome, hpESCs generate proliferating NSCs that are capable of differentiation into physiologically functional neuron-like cells and maintain allele-specific expression of imprinted genes. Thus, hpESCs can serve as a model to study the role of maternal and paternal genomes in neural development and to better understand imprinting-associated brain diseases.
Highlights
Due to their unlimited self-renewal and multilineage differentiation potential, human pluripotent stem cells have become key cell sources for cell differentiation research, disease modeling, drug discovery, and have potential for cell replacement strategies [1]
Cell culture was performed as described [10] with slight modifications. hpESCs were maintained on mitomycin C-treated human foreskin fibroblasts (HFFs) (ATCC-LGC Standards, Wesel, Germany) at 5% CO2 in a medium containing knockout-DMEM, 20% knockout serum replacement, 1% non-essential amino acids, 2 mM Lglutamine, 0.1 mM ßmercaptoethanol (Sigma-Aldrich, Schnelldorf, Germany) and 4 ng/mL FGF2 (PeproTech, Hamburg, Germany)
The NSC identity of monolayer cells was confirmed by gene expression analysis revealing upregulation of NSC markers Sox1, Nestin, Pax6, and Musashi1 (MS1)
Summary
Due to their unlimited self-renewal and multilineage differentiation potential, human pluripotent stem cells have become key cell sources for cell differentiation research, disease modeling, drug discovery, and have potential for cell replacement strategies [1]. While asexual development of offspring from an oocyte without male genetic contribution (parthenogenesis) occurs naturally in various invertebrate and some vertebrate species [5], mammalian uniparental (PG, gynogenetic: GG, or androgenetic: AG, with only paternally derived genomes) embryos do not develop to term as a consequence of imbalanced expression of imprinted genes with parent of origin-dependent allele-specific expression patterns [6]. Despite this developmental limitation, stable ESC lines can be isolated from uniparental blastocysts of several species including human [7,8,9,10]. The in vitro differentiation capacity of murine uniparental ESC into various cell lineages, including neural and transplantable hematopoietic progenitors [11,12,13,14,15,16] indicates that these cells represent a unique model system to study the role of maternal and paternal genomes in normal development and the contribution of imprinting in disease development
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