Abstract

Hematopoietic stem cell transplantation (HSCT) is a well-established treatment for various hematological disorders. Umbilical cord blood (UCB) has emerged as an alternative source of hematopoietic stem and progenitor cells (HSPCs). However, the limited number of functional repopulating cells in UCB units has encouraged researchers to develop methods to expand them ex vivo. Hematopoiesis is strictly regulated by epigenetic modifiers. JARID2 is a co-factor of the Polycomb Repressive Complex 2 (PRC2), which recruits the complex to specific genomic loci and promotes the deposition of Histone 3 lysine 27 trimethylation (H3K27me3), a repressive epigenetic mark that mediates condensed chromatin packing to silence key developmental genes. In our previous studies, we found that mouse multipotent progenitor (MPP) cells with genetic loss of Jarid2 regained self-renewal capacity, unlike typical transient MPPs. These reprogrammed MPPs displayed transcriptional and epigenetic profiles more similar to hematopoietic stem cells (HSCs) than control MPPs, suggesting a role for Jarid2 in the developmental transition from HSCs to MPPs. Based on this, we hypothesized that human HSPCs may follow a similar mechanism and could be expanded ex vivo through JARID2 inhibition to improve outcomes in bone marrow transplantation. To investigate whether inhibition of JARID2 can enhance the functional potential of human HSPCs, we designed lentiviral vectors to achieve JARID2 knockdown using shRNAs in both constitutive and inducible systems. Our results showed that constitutive loss of JARID2 in human CD34+ HSPCs led to improved overall engraftment outcomes and an increased number of HSPCs in a xenograft NSG mouse model. JARID2-knockdown (JARID2-KD) HSCs also demonstrated enhanced functionality, reflected by increased self-renewal and multilineage reconstitution capacity in both primary and, more prominently, secondary transplantation experiments. We further found that transient JARID2 inhibition ex vivo exerted long-term positive effects on transplantation outcomes, suggesting the clinical potential of short-term JARID2 inhibition to improve bone marrow transplantation. Notably, one experiment showed that engraftment was enhanced only after in vivo doxycycline administration to induce JARID2 knockdown, indicating that JARID2-KD-mediated HSPC expansion and transplantation benefits can be controlled in vivo—further supporting its clinical relevance. Safety is a critical concern when considering the transplantation of expanded human HSPCs into patients. To assess the safety of JARID2 loss of function in the context of improved bone marrow transplantation, we conducted a long-term study in NSG mice. Human HSPCs with permanent JARID2 knockout via CRISPR-Cas9 were transplanted into NSG mice and monitored for one year. Histological and pathological analyses revealed no signs of abnormal development or fibrosis in the blood, spleen, or bone marrow. Additionally, we performed single-cell RNA sequencing and flow cytometry-based clustering on hCD34+ cells isolated from 20-week post-transplant bone marrow. UMAP and t-SNE analyses showed comparable cell population compositions between control and JARID2-KD groups. These results indicate that long-term JARID2 loss of function does not induce pathological changes in human HSPCs, further supporting its clinical applicability. To elucidate the cellular mechanisms underlying JARID2-mediated enhancement of bone marrow transplantation, we sorted HSCs, MPPs, and MLPs and cultured them in MethoCult assays. As expected, JARID2-KD HSCs exhibited increased colony-forming capacity compared to control HSCs. Interestingly, JARID2-KD MPPs also demonstrated enhanced colony-forming ability, with this effect persisting through two additional rounds of replating—indicating a gain in self-renewal capacity comparable to that of HSCs. These findings suggest that JARID2 knockdown enhances the functional potential of both HSCs and MPPs. Since ex vivo culture significantly alters cell surface marker expression, we aimed to identify the appropriate cell population for downstream sequencing and epigenetic analyses. Using CITE-seq, we identified CD34⁺CD90⁺EPCR⁺CD49f⁻CD71⁻ cells as the functional repopulating population after 8 days of ex vivo culture. Transplantation of 1,000 cells with this marker profile into NSG mice resulted in robust engraftment and full multilineage differentiation in JARID2-KD groups. These results suggest that JARID2 knockdown not only enhances functional capacity but also helps preserve HSC identity during ex vivo culture. While JARID2 functions as a co-factor of PRC2 by guiding the complex to specific genomic loci, EZH2 serves as the core enzymatic subunit responsible for catalyzing H3K27 trimethylation. To further explore the molecular mechanism underlying JARID2-mediated enhancement of HSPC function—and to determine whether its effects are dependent on PRC2—we conducted parallel experiments using EZH2 knockdown (KD). EZH2 shRNAs were cloned into the same lentiviral vectors used for JARID2 inhibition. In contrast to JARID2-tKD UCBs, EZH2-tKD significantly impaired transplantation outcomes, as evidenced by reduced peripheral blood engraftment and decreased HSPC numbers in the bone marrow at 20 weeks post-transplant. Notably, EZH2-KD engraftment started to decline only after in vivo doxycycline-induced shRNA expression, indicating a direct detrimental effect. Moreover, EZH2-KD HSPCs exhibited abnormal lineage distribution in xenograft models, suggesting potential pathogenic alterations. At the epigenetic level, EZH2-KD led to a marked reduction in global H3K27me3 levels, while JARID2-KD had minimal impact, highlighting distinct and non-redundant roles for JARID2 and EZH2 within the PRC2 complex. In conclusion, our findings suggest that JARID2, rather than EZH2, is a promising target for enhancing hematopoietic stem cell expansion and improving bone marrow transplantation outcomes. In the future, the development of small-molecule inhibitors targeting JARID2 may offer a novel strategy to optimize current ex vivo expansion and transplantation protocols for clinical applications.

Committee Chair

Grant Challen

Committee Members

Daniel Link; Matthew Walter; Samantha Morris; Timothy Ley; Zhongsheng You

Degree

Doctor of Philosophy (PhD)

Author's Department

Biology & Biomedical Sciences (Molecular Cell Biology)

Author's School

Graduate School of Arts and Sciences

Document Type

Dissertation

Date of Award

5-6-2025

Language

English (en)

Author's ORCID

https://orcid.org/0000-0003-4395-9441

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