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

Abstract

One strategy for stem cell-based therapy of the cerebral cortex involves the generation and transplantation of functional, histocompatible cortical-like neurons from embryonic stem cells (ESCs). Diploid parthenogenetic Pg-ESCs have recently emerged as a promising source of histocompatible ESC derivatives for organ regeneration but their utility for cerebral cortex therapy is unknown. A major concern with Pg-ESCs is genomic imprinting. In contrast with biparental Bp-ESCs derived from fertilized oocytes, Pg-ESCs harbor two maternal genomes but no sperm-derived genome. Pg-ESCs are therefore expected to have aberrant expression levels of maternally expressed (MEGs) and paternally expressed (PEGs) imprinted genes. Given the roles of imprinted genes in brain development, tissue homeostasis and cancer, their deregulation in Pg-ESCs might be incompatible with therapy. Here, we report that, unexpectedly, only one gene out of 7 MEGs and 12 PEGs was differentially expressed between Pg-ESCs and Bp-ESCs while 13 were differentially expressed between androgenetic Ag-ESCs and Bp-ESCs, indicating that Pg-ESCs but not Ag-ESCs, have a Bp-like imprinting compatible with therapy. In vitro, Pg-ESCs generated cortical-like progenitors and electrophysiologically active glutamatergic neurons that maintained the Bp-like expression levels for most imprinted genes. In vivo, Pg-ESCs participated to the cortical lineage in fetal chimeras. Finally, transplanted Pg-ESC derivatives integrated into the injured adult cortex and sent axonal projections in the host brain. In conclusion, mouse Pg-ESCs generate functional cortical-like neurons with Bp-like imprinting and their derivatives properly integrate into both the embryonic cortex and the injured adult cortex. Collectively, our data support the utility of Pg-ESCs for cortical therapy. Stem Cells 2018;36:192-205.

Derivation of Mouse Parthenogenetic Advanced Stem Cells

“…Similar to paAFSCs, paASCs were found to possess the expression of major pluripotency genes such as Oct4 and Nanog, but Sox2 had an apparently reduced expression in both cell lines. However, paASCs were found to have the capability to form teratomas and also contributed to E10.5 chimaeras, which confirmed their developmental pluripotency in vivo, consistent with previous studies [7,11,26,27]. Haploid embryonic stem cells from monogenetic gametes have been established as having a high expression of pluripotent genes and contributing to chimaera, though flow sorting every few passages for haploid cells is inevitable [28,29].…”

“…However, whether the low expression of Sox2 triggered the increased expression of Cdx2 and whether this gene expression pattern is related to imprinting genes in paAFSCs and paASCs require further exploration. Moreover, the expression of Sox2 in haploid stem cells is same as that in normal mouse ESCs [5,28,29,31], but no study of parthenogenetic diploid stem cells has shown the expression status of Sox2 [7,26,27]. Thus, whether the low expression of Sox2 is a common property of parthenogenetic diploid stem cells also needs further research.…”

“…Embryonic stem cells representative of naive (ESCs) and primed (EpiSCs) pluripotency have been established and converted to each other [23][24][25]. The parthenogenetic pluripotency ESCs (paESCs) has been derived from blastocysts stage; parthenogenetic EpiSCs (paEpiSCs) have only been generated post-implantation, though the culture conditions used for all paESCs and paEpiSCs included serum or feeder [7,9,26,27] and there have no reports on the conversion of paEpiSCs to paESCs. The findings of this study have revealed that paAFSCs in a primed state can be derived from blastocysts in a chemically defined medium, as well as that primed paAFSCs can be converted to naive paASCs.…”

CRISPR Activation/Inhibition Experiments Reveal that Expression of Intronic MicroRNA miR-335 Depends on the Promoter Activity of its Host Gene Mest

“…E14Tg2a mouse ESCs and their CRISPRa and CRISPRi derivatives were cultivated on gelatine coated dishes and maintained pluripotent in Serum/Lif media as described (Varrault et al 2018). Organoids were generated in 96-well (U-bottom) Ultra-Low Attachment plates (Sumitomo) by seeding 3000 ESCs in corticogenesis medium 1: DMEM/F-12/GlutaMAX supplemented with 10% KSR, 0.1 mM of non-essential amino acids, 1 mM of sodium pyruvate, 50U/ml penicillin/streptomycin, 0.1 mM of 2-mercaptoethanol (Sigma), 1 µM DMH1-HCl (in house synthesized, Vanderbilt University) and 240 nM IWP-2 (Tocris).…”

Prevalence of Hepatitis B, C and Tuberculosis in Under Privileged Children (Orphans) of District Bagh, Azad Jammu and Kashmir

“…This is a key step for activation of reconstructed oocytes/embryos in the animal cloning program (Kishigami et al, 2006). Previously it was demonstrated that embryonic stem cell have been derived from the parthenotes (Varrault et al, 2018). These embryonic stem cells are equally competent to fertilized embryonic stem cell in terms of pluripotency and proliferation (Ju et al, 2008).…”