Highlights
- A biallelic, dual-colored fluorescent reporter at the imprinted SNRPN locus in hPSCs
- Biallelic SNRPN expression is rapidly induced during primed-to-naive resetting
- Acquisition of biallelic SNRPN expression is irreversible upon re-priming
- ZFP57 overexpression mitigates imprint erasure during primed-to-naive resetting
Summary
Naive human pluripotent stem cells (hPSCs) model the pre-implantation epiblast. However, parent-specific epigenetic marks (imprints) are eroded in naive hPSCs, which represents an important deviation from the epiblast in vivo. To track the dynamics of imprint erasure during naive resetting in real time, we established a dual-colored fluorescent reporter at both alleles of the imprinted SNRPN locus. During primed-to-naive resetting, SNRPN expression becomes biallelic in most naive cells, and biallelic SNRPN expression is irreversible upon re-priming. We utilized this live-cell reporter to evaluate chemical and genetic strategies to minimize imprint erasure. Decreasing the level of MEK/ERK inhibition or overexpressing the KRAB zinc-finger protein ZFP57 protected a subset of imprints during naive resetting. Combining these two strategies protected imprint levels to a further extent than either strategy alone. This study offers an experimental tool to track and enhance imprint stability during transitions between human pluripotent states in vitro.
Graphical abstract
Introduction
Parent-specific epigenetic marks (imprints) are crucial for normal growth and development, yet their mechanisms of establishment and maintenance are not fully understood. Landmark studies in mice demonstrated that imprinted genes are expressed from either the maternal or paternal allele and are essential for the development of embryonic and extraembryonic tissues (Barton et al., 1984; McGrath and Solter, 1984; Surani et al., 1984). In humans, approximately 200 imprinted genes have been discovered, and improper imprinting can manifest as growth restriction, obesity, intellectual disabilities, behavioral abnormalities, and an increased risk of certain cancers (Kalish et al., 2014; Monk et al., 2019; Peters, 2014; Tucci et al., 2019). In addition, aberrant expression of imprinted genes has been implicated in developmental defects in non-human primate embryos generated by somatic cell nuclear transfer (Liao et al., 2024). The evolution of genomic imprinting in placental mammals is thought to reflect the competition between maternal and paternal genomes over resources during gestation (Wilkins and Haig, 2003). Moreover, imprinting promotes the exchange of genetic information by raising barriers to uniparental reproduction (Li et al., 2018).
Advances in modeling early human development have surged from the delineation of pluripotent cell states, namely that of naive and primed pluripotency (Nichols and Smith, 2009). Naive human pluripotent stem cells (hPSCs) align closely with the in vivo pre-implantation epiblast in that they share a similar transcriptional profile (including that of transposable elements) (Takashima et al., 2014; Theunissen et al., 2014, 2016), demonstrate X chromosome dampening (Dror et al., 2024; Sahakyan et al., 2017), and possess the developmental plasticity to generate embryonic and extraembryonic tissues (Castel et al., 2020; Cinkornpumin et al., 2020; Dattani et al., 2024; Dong et al., 2020; Guo et al., 2021; Io et al., 2021; Okubo et al., 2024). However, a persistent issue hampering bona fide naive hPSCs is the erosion of imprints (Pastor et al., 2016; Theunissen et al., 2016). Current naive culture media require fibroblast growth factor (FGF) pathway inhibition to maintain naive identity. Interestingly, FGF pathway inhibition has also been suggested to cause greater loss of imprinting (LOI) (Keshet and Benvenisty, 2021). This complicates the accurate study of naive hPSCs. Furthermore, imprints and proper monoallelic gene expression do not return upon transition back to the primed state of pluripotency, a process known as “re-priming,” or subsequent differentiation (Keshet and Benvenisty, 2021; Theunissen et al., 2016). Thus, aberrant imprinting in naive hPSCs hinders developmental studies of lineage specification and the potential applications of naive hPSCs in regenerative medicine.
Several studies have surveyed the LOI found in cultured pluripotent cells by analyzing the expression of imprinted genes bearing distinguishing parental single-nucleotide polymorphisms (Bar et al., 2017; Keshet and Benvenisty, 2021; Rugg-Gunn et al., 2007). However, most studies pertaining to LOI have lacked the ability to monitor imprint integrity in live cultures. Stelzer et al. created a live-cell reporter for DNA methylation in mouse embryonic stem cells (ESCs) by utilizing a minimal promoter that is sensitive to methylation changes of adjacent sequences (Stelzer et al., 2015). However, there remains an unmet need for a reporter of imprinted gene expression in hPSCs that enables real-time visualization of LOI at single-cell resolution.
Here, we created a dual-colored fluorescent reporter at the endogenous SNRPN locus in primed hPSCs. We show that SNRPN acquires biallelic expression during primed-to-naive resetting, which is irreversible upon re-priming. Our reporter accurately reflects methylation at the SNRPN locus and is a proxy for global methylation levels. Titrating FGF pathway inhibition during naive resetting enabled us to capture a naive, imprint-protected cell population. We also demonstrate the imprint-protective effects of a KRAB zinc-finger protein, ZFP57, when ectopically expressed during the generation of naive hPSCs. When combined, these two imprint protection strategies produced an even greater imprint-protective effect. These findings provide an important step toward improving the imprint fidelity of naive hPSCs and their applications for studies of human development and regeneration.
Results
A live-cell reporter hPSC line displays allele-specific SNRPN expression
To track the stability of parent-specific imprints in real time, we set out to build a dual-colored fluorescent reporter at a representative imprinted locus. We selected the SNRPN locus because it shows stable monoallelic expression in primed hPSCs, and the associated imprint control center (ICR) undergoes demethylation during primed-to-naive resetting, resulting in biallelic expression of the downstream SNRPN transcript (Pastor et al., 2016; Rugg-Gunn et al., 2007; Theunissen et al., 2016). In addition, SNRPN is robustly expressed in both primed and naive hPSC conditions, an important prerequisite for an endogenous live-cell reporter. We inserted a P2A-mRuby3 sequence on one allele of SNRPN and a P2A-EGFP sequence on the other allele in H9 human embryonic stem cells (hESCs) using CRISPR-Cas9-mediated genome editing (Figures 1A and S1A). Several clones were generated, and sequence integration was validated by junction PCR (Figure S1B). We designate this genotype as H9-SNRPN-mRuby3-EGFP (H9-SRG). In the primed state, mRuby3 was highly expressed and EGFP was not expressed, suggesting that mRuby3 was integrated into the active, paternal SNRPN allele, while EGFP was integrated into the inactive, maternal SNRPN allele (Figures 1B and S1C). Consistent with LOI and a switch from mono- to biallelic SNRPN expression, EGFP became active in the majority of naive cells within 2–3 passages in PXGGY/A naive induction medium (Khan et al., 2021), resulting in a population of mostly double-positive mRuby3+/EGFP+ (R+G+) cells (Figures 1B and S1C). We confirmed the acquisition of naive identity in these H9-SRG cells by flow cytometry for the naive-specific cell surface marker SUSD2. Additional markers for primed and naive pluripotency were confirmed by qPCR (Figure S1D). G-banding of primed H9-SRG clones confirmed normal karyotypes (Figure S1E).
