ORCID
0000-0002-9548-3441
Date of Award
5-8-2024
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
Early human embryogenesis is difficult to study due to practical and ethical limitations, but the successful culture of “naïve” human pluripotent stem cells (hPSCs) has expanded our ability to model cells akin to the pre-implantation epiblast. Naïve hPSCs are readily obtained through in vitro resetting of “primed” hPSCs, cells which model the post-implantation epiblast. Naïve hPSCs offer the advantages of modeling the pre-implantation transcriptome and epigenome, the ability to form embryonic and extraembryonic tissues, and the capacity to self-organize into integrated human embryo models. However, a persistent drawback of naïve hPSCs is the loss of parent-specific epigenetic marks, called imprints. Approximately 200 imprinted genes have been identified in humans and they have profound impacts on organismal growth, metabolism, neural development, and certain cancer risks. Many imprinted genes are misregulated in naïve hPSC due to loss of imprinting (LOI), which contrasts with the intact imprinting found in the natural blastocyst. Furthermore, once imprints are erased, they largely do not return upon re-priming or differentiation. To more accurately model the pre-implantation cell state and to promote advances in reproductive health and regenerative medicine, we must improve the status of imprinting in naïve hPSCs. To better understand the mechanisms regulating LOI during primed-to-naïve resetting, I constructed a dual-colored fluorescent reporter cell line that enables efficient tracking of LOI in real time and at single-cell resolution. I inserted a P2A-mRuby3 construct at the endogenous paternal SNRPN (demethylated, transcriptionally active) imprinted locus. In addition, I inserted a P2A-eGFP construct at the endogenous maternal SNRPN (methylated, transcriptionally repressed) locus. The vast majority of cells acquire biallelic SNRPN expression during primed-to-naïve resetting, which is linked to demethylation at the SNURF/SNRPN imprinted region. Biallelic SNRPN reporter activity is also associated with global demethylation. Once cells acquire biallelic SNRPN expression, they remain biallelic upon extended naïve culture or differentiation, confirming that erased imprinted methylation does not return. To explore the possibility of mitigating imprint erasure in naïve hPSCs, I tested chemical and genetic methods of imprint preservation. Inhibition of FGF signaling is currently required to reach the naïve pluripotent state, yet small molecule inhibitors of MEK and ERK have been shown to contribute to demethylation and imprint erasure. I hypothesized that reducing the amount of MEK/ERK inhibition would enable cells to reach an imprint-protected naïve state. Complete removal of either the MEK or ERK inhibitor precluded naïve resetting. However, titrations of both inhibitors gave rise to naïve, monoallelic SNRPN reporter cells that maintained imprinting at a subset of imprints. Re-priming of these imprint-protected cells revealed that imprinted DNA methylation can be retained upon downstream culture when not erased in the naïve state. Despite a wave of global demethylation during pre-implantation development, DNA methylation is maintained at imprints in vivo. A literature investigation of in vivo imprint-protecting factors revealed the potential protective effects of five maternally- or embryonically-expressed proteins: DPPA3, KHDC3L, NLRP2, NLRP7, and ZFP57. While these factors are highly expressed in naïve hPSCs, their upregulation occurs after the onset of imprint erasure during primed-to-naïve resetting. I hypothesized that overexpression of one or more of these factors throughout primed-to-naïve resetting could protect imprinted methylation. Exogenous expression of ZFP57, but not the other factors, caused a significant delay in biallelic SNRPN reporter activity, suggesting an imprint-protective effect. DNA methylation analysis revealed that ZFP57 overexpression during naïve resetting had a protective effect on imprint methylation under two different naïve culture conditions. Overall, this work demonstrates that reduction of MEK/ERK inhibition or overexpression of ZFP57 enables cells to reach in imprint-protected naïve state. Moreover, the biallelic SNRPN fluorescent reporter line offers a versatile tool for efficiently tracking DNA methylation dynamics during human cell state transitions at single-cell resolution. Together, the technology and biological insights generated here contribute to the advancement of pluripotent state modeling and our understanding of epigenetic regulation during human development, and underlie future studies in reproductive and regenerative medicine.
Language
English (en)
Chair and Committee
Thorold Theunissen
Recommended Citation
Fischer, Laura A., "Tracking and Mitigating Imprint Erasure During Human Pluripotent State Transitions" (2024). Arts & Sciences Electronic Theses and Dissertations. 3021.
https://openscholarship.wustl.edu/art_sci_etds/3021