In Vitro Corticogenesis from Embryonic Stem Cells Recapitulates the In Vivo Epigenetic Control of Imprinted Gene Expression

Abstract

In vitro corticogenesis from embryonic stem cells (ESCs) is an attractive model of cortical development and a promising tool for cortical therapy. It is unknown to which extent epigenetic mechanisms crucial for cortex development and function, such as parental genomic imprinting, are recapitulated by in vitro corticogenesis. Here, using genome-wide transcriptomic and methylation analyses on hybrid mouse tissues and cells, we find a high concordance of imprinting status between in vivo and ESC-derived cortices. Notably, in vitro corticogenesis strictly reproduced the in vivo parent-of-origin-dependent expression of 41 imprinted genes (IGs), including Mest and Cdkn1c known to control corticogenesis. Parent-of-origin-dependent DNA methylation was also conserved at 14 of 18 imprinted differentially methylated regions. The least concordant imprinted locus was Gpr1-Zdbf2, where the aberrant bi-allelic expression of Zdbf2 and Adam23 was concomitant with a gain of methylation on the maternal allele in vitro. Combined, our data argue for a broad conservation of the epigenetic mechanisms at imprinted loci in cortical cells derived from ESCs. We propose that in vitro corticogenesis helps to define the still poorly understood mechanisms that regulate imprinting in the brain and the roles of IGs in cortical development.

Mouse Parthenogenetic Embryonic Stem Cells with Biparental-Like Expression of Imprinted Genes Generate Cortical-Like Neurons That Integrate into the Injured Adult Cerebral Cortex

“…The Bp ESC lines were E14Tg2a (hereafter E14) , WT‐B1 [19], and Tau‐eGFP ; the Ag‐ESC lines were AK2 and AG‐B6 [19]; the Pg‐ESC lines were PG‐BT6 and PG‐08–21 (both from Columbus, U.S.) and PR8 (from Cambridge, U.K.). E14 was maintained on gelatin and the six other lines were propagated on mitomycin‐treated MEFs in DMEM (Dulbecco's Modified Eagle Medium, Life Technologies, Courtaboeuf, France) supplemented with 15% ESC‐certified FBS (Life Technologies), 0.1 mM nonessential amino acids (Life Technologies), 1 mM sodium pyruvate (Life Technologies), 0.1 mM β‐mercaptoethanol (Sigma, Saint‐Quentin Fallavier, France), 50 U/ml penicillin/streptomycin (Life Technologies), and 10³ U/ml LIF (Millipore, Molsheim, France) as described . All ESC lines were regularly controlled to exclude the presence of mycoplasma using the Mycoalert kit (Lonza, Levallois‐Perret, France).…”

“…Oppositely, we found that Pg‐ESC imprinting was surprisingly Bp‐like. Next, Pg‐ESC lines were differentiated according to an established protocol of in vitro corticogenesis that reproduces the main features of in vivo corticogenesis , including the parent‐of‐origin dependent expression of imprinted genes . We found that Pg‐ESCs generated cortical‐like progenitors and electrophysiologically active glutamatergic neurons.…”

“…In addition, imprinting is developmental stage‐ and tissue‐dependent , and these studies did not investigate PEGs and MEGs with ESC‐specific expression patterns. In this context, using RNA‐seq on hybrid ESC lines, we have recently established an extensive list of MEGs and PEGs specific to mouse Bp‐ESCs .…”

“…However, whether Pg‐ESC derivatives display specific features of cortical cells such as a radial orientation and expression of cortical markers is unknown. Numerous imprinted genes are expressed in a parent‐of‐origin dependent manner in the developing cortex generated in vivo, or in vitro from ESCs ; some of them, including Cdkn1c , Plagl1 , Dlk1 , and Igf2 , are required for proper corticogenesis in the mouse. Humans suffering from imprinting disorders (including Angelman syndrome, Prader‐Willi syndrome, and transient neonatal diabetes mellitus) exhibit impaired corticogenesis .…”

“…Here, we have first clarified the imprinting status of mouse Pg‐ESCs by quantifying the expression levels of 26 imprinted genes, including 7 validated MEGs and 12 validated PEGs in ESCs , in three independent Pg‐ESC lines, and in two normal Bp‐ESC and two androgenetic Ag‐ESC lines. Ag‐ESCs have two paternal genomes and form tumors in chimeras .…”