Date of Award

Spring 2021

Document Type


Degree Name

Doctor of Philosophy (PhD)



First Advisor

Park, In-Hyun


In the works described below, I investigate and describe heterogeneity in naïve human pluripotent stem cells (hPSCs)—both the heterogeneity exhibited in XCI status and the nearly unexplored transcriptional heterogeneity in these cells. Stem cells are a highly desirable model for studying XCI, as both pluripotency and XCI play out during peri-implantation. By comparing female naïve and primed stem cells, I elucidate the functional differences in their XCI state. Recent studies incorporating single-cell RNA sequencing (scRNA-seq) have demonstrated that transcript-based technologies are insufficiently developed to unequivocally resolve these questions in a human system (Kaur et al., 2020; Mandal et al., 2020; Moreira de Mello et al., 2017; Petropoulos et al., 2016a; Reinius and Sandberg, 2019; Sahakyan et al., 2017). Further studies have instead shown that 3D genomics holds immense promise for studying XCI by interrogating changes to the X chromosomes’ 3D states (Darrow et al., 2016; Giorgetti et al., 2016; Wang et al., 2016b). As such, I have collaborated with the Wang lab to characterize the 3D state of the X chromosome in naïve and primed human pluripotent stem cells. Using a combination of Hi-C and DNA FISH, we analyzed 40 TADs across the X chromosome in multiple naïve and primed hPSC lines. From this, we determined that naïve cells typically exhibit the 3D conformation of active X chromosomes. Naïve X chromosomes generally compartmentalize around the centromere and appear more dispersed than primed X chromosomes on average, which further suggests that naive X chromosomes exist in the active state. The addition of FGF does not seem to have a major effect on the X chromosome conformation in the naïve state either. In naïve hPSCs, transcriptional heterogeneity is an almost completely open question regarding its variation across cell lines, mechanism(s), cultural conditions, and even its effects. However, multiple studies using primed cells, including those published by my lab, have shown that some degree of heterogeneity is present in this culture system. Because naïve cells are a fairly recent discovery—and transgene-free naïve hPSCs are an even more recent innovation—there is ample space in this undeveloped field to leverage single-cell RNA sequencing to conduct a basal characterization of transcriptional heterogeneity in these cells. From this, we found that the cells clustered into two distinct populations, independently of the scRNA-seq method used. We also leveraged previously published scRNA-seq data from the same cell line (performed by another group in a different cultural context) in order to correlate our findings; their data also contained these two populations. Based on the research described here, my colleagues and I have made novel advancements within the field of stem cell biology, implementing novel experimental approaches. Critically, we have found that naïve X chromosomes are not explicitly heterogeneous, finding no evidence of X chromosome inactivation in female naïve hPSCs. From a transcriptional perspective, we conclude that naïve cells demonstrate discrete heterogeneity and form developmentally and functionally distinct populations. This body of work represents one of the first efforts in the field to characterize the heterogeneity of naïve hPSCs at single-cell resolution, both in a 3D genomics and transcriptional context.