Derivation of hypermethylated pluripotent embryonic stem cells with high potency

Abstract

Naive hypomethylated embryonic pluripotent stem cells (ESCs) are developmentally closest to the preimplantation epiblast of blastocysts, with the potential to contribute to all embryonic tissues and the germline, excepting the extra-embryonic tissues in chimeric embryos. By contrast, epiblast stem cells (EpiSCs) resembling postimplantation epiblast are relatively more methylated and show a limited potential for chimerism. Here, for the first time, we reveal advanced pluripotent stem cells (ASCs), which are developmentally beyond the pluripotent cells in the inner cell mass but with higher potency than EpiSCs. Accordingly, a single ASC contributes very efficiently to the fetus, germline, yolk sac and the placental labyrinth in chimeras. Since they are developmentally more advanced, ASCs do not contribute to the trophoblast. ASCs were derived from blastocysts in two steps in a chemically defined medium supplemented with Activin A and basic fibroblast growth factor, followed by culturing in ABCL medium containing ActA, BMP4, CHIR99021 and leukemia inhibitory factor. Notably, ASCs exhibit a distinct transcriptome with the expression of both naive pluripotency genes, as well as mesodermal somatic genes; Eomes, Eras, Tdgf1, Evx1, hand1, Wnt5a and distinct repetitive elements. Conversion of established ESCs to ASCs is also achievable. Importantly, ASCs exhibit a stable hypermethylated epigenome and mostly intact imprints as compared to the hypomethylated inner cell mass of blastocysts and naive ESCs. Properties of ASCs suggest that they represent cells at an intermediate cellular state between the naive and primed states of pluripotency.

Zfp281 orchestrates interconversion of pluripotent states by engaging Ehmt1 and Zic2

“…Strikingly, the extinction of the Rex1 reporter and self‐renewal was similar in differentiating WT cells and Tet1 and Tet2 mutants, while the absence of Tet2 in Zfp281 / Tet2 KO cells did not revert resistance to differentiation caused by absence of Zfp281 alone (Figs E and EV2H). We furthermore noted only modest changes in Zfp281 mRNA or protein during ESC differentiation, and across existing RNA sequencing (RNA‐seq) datasets of EpiLC and EpiSC differentiation (Buecker et al , ; Factor et al , ; Bao et al , ) and epiblast development (Boroviak et al , ; Figs F and EV2I). Zfp281 has also been reported to repress Nanog transcription through interacting with the NuRD complex in Serum/Lif‐cultured ESCs (Fidalgo et al , ).…”

“…Average and SD of two experiments. ESelf‐renewal in RGd2 ESCs of specified genotypes after 72 h in N2B27. Average and SD of two experiments performed in duplicates. FZfp281 mRNA changes during ESC differentiation detected by quantitative PCR (left) and extracted from published RNA‐seq datasets (Buecker et al , ; Factor et al , ; Boroviak et al , ; Bao et al , ) (right). Average and SD of two technical replicates (left).…”

“… AmRNA log 2 fold changes (log 2 FC) in WT 16 h , WT 32 h , Zfp281 16 h , and Zfp281 32 h samples relative to WT 2i cells, and in EpiSCs relative to WT 2i/Lif cells (Factor et al , ; Bao et al , ). Zfp281 2i , Zfp281 16 h , and Zfp281 32 h and WT 16 h and WT 32 h samples were used for k ‐means clustering. B, CQuantification of (A) including mRNA log 2 FC in EpiLCs relative to WT 2i/Lif (Buecker et al , ) and as indicated (C).…”

“… (c)˜ time (EpiLCs or EpiSCs): EpiLCs (Buecker et al , ) or EpiSCs (Factor et al , ) compared to WT 2i/Lif (Buecker et al , ) and EpiSC compared to WT 2i/Lif (Bao et al , ; used for Fig A and B). (d)˜ genotime : Zfp281 16 h or Zfp281 32 h compared to WT 2i (used for Figs A and B, and EV3A), where genotime is the combination of genotype and time. (e)˜ genotype (cell state‐specific): KO cells in 2i compared to WT 2i , or KO cells 16 h or 32 h after 2i withdrawal compared to WT 16 h or WT 3 2 h , respectively (used for Figs C and C and D, and EV5B–E). …”

“…Published RNA-seq from ESCs cultured in 2i/Lif and EpiLC (Buecker et al, 2014) was 36-bp reads, and therefore, no spliced alignment could be performed. RNA-seq from ESCs cultured in 2i/Lif and EpiSCs (Factor et al, 2014;Bao et al, 2018), and global run-on sequencing (GRO-seq) data from 2i/Lif-cultured ESCs (Dorighi et al, 2017) were 100-bp and 50-bp paired-end reads, respectively, and therefore, paired="fr" was used. For in vivo embryo data (Boroviak et al, 2015), preexisting alignments to mouse GRCm38/mm10 genome were downloaded from ArrayExpress (E-MTAB-2958) and used.…”