In Vivo and Ex Vivo Expansion of Functional Human Repopulating Cells through JARID2 Inhibition
Authors: Han Wentao , Bjeije Hassan , Celik Hamza , Rettig Michael P. , Li Yanan , Young Andrew L , Zhang Christine RC , Xavier Raj Infencia , Dunuwille Saranajith , Challen Grant A 
Published: December 2024

Hematopoietic stem cell transplantation is a common treatment for many blood diseases. Umbilical cord blood (UCB) has become a popular 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. JARID2 is a co-factor of the polycomb repressive complex 2 (PRC2). Previous studies in our lab showed that genetic loss of Jarid2 in mouse multipotent progenitor (MPP) cells endowed ectopic self-renewal capacity to these normally transient cells, and they displayed similar transcriptional and epigenetic profiles to normal HSCs. In this study, we postulated that human HSPCs can be expanded in vivo or ex vivo through JARID2 inhibition.

To investigate whether inhibition of JARID2 can enhance functional potential of human HSPCs, we leveraged a Tet-ON Doxycycline (Dox) inducible shRNA lentivirus system to transiently inhibit the expression of JARID2 or EZH2 (PRC2 enzymatic component) in UCB CD34+ cells. We transplanted the 8-day ex vivo culture progeny from 40,000 CD34+ cells expressing shRNAs against JARID2, EZH2 or Renilla (control) into NSG mice. Analysis of monthly peripheral blood and 20-week bone marrow showed enhanced engraftment capacity and a 3-4-fold increase in HSPCs post-transplant in JARID2-transient knockdown (tKD) groups compared to control groups, a phenotype not seen with EZH2 shRNA. In another experiment, NSG mice transplanted with UCBs carrying inducible vectors were treated with Dox in vivo. JARID2-KD HSPCs also showed higher engraftment capacity and increased HSPC numbers compared to control or EZH2 groups only after exposure to Dox, suggesting JARID2 inhibition enhances function of human repopulating cells in vivo.

Single-cell RNA sequencing and flow cytometry-based clustering for hCD34+ cells from 20-week NSG mouse bone marrow indicated similar differentiation spectra between control and JARID2-tKD groups. Single cell RNA sequencing data revealed upregulation of a series of HSC identity genes (HLF, CRHBP, MYCT1, HIF1A, MLLT3 and MEIS1) in JARID-KD HSCs. Long-term pathological observation (&gt;1-year) facilitated by transplanting CD34+ cells with CRISPR knockout of JARID2 into NSG mice unveiled no significant hematologic pathologies such as reticulin fibrosis or transformation, suggesting potential safety for clinical applications. In contrast to JARID2-tKD UCBs, EZH2-tKD negatively impacted the differentiation capacity of HSPCs, which correlated with contrasting levels of the H3K27me3 repressive chromatin marker between the genotypes, highlighting a discrepancy in the roles of JARID2 and EZH2.

The pinpoint the cell types affected by JARID2-KD, we sorted HSCs (CD34+CD38-CD90+CD45RA-), MPPs (CD34+CD38-CD90-CD45RA-), and MLPs (CD34+CD38-CD90-CD45RA+) from xenografted mice and conducted serial CFU assays. Both JARID2-KD HSCs and MPPs exhibited a significantly higher number of colonies compared to the control counterparts. Notably, only JARID2-KD MPPs exhibited ectopic self-renewal capacity akin to HSCs. However, the conventional human HSC cell surface marker combination was not reliable for isolating functional repopulating cells from ex vivo cultures. We conducted CITE-seq to identify reliable markers for functional repopulating cells from cultures, identifying a CD34+CD90+EPCR+CD49f-CD71- immunophenotype. Transplanting 1,500 cells sorted by these markers into NSG mice demonstrated enhanced engraftment and complete lineage differentiation capacity from JARID2-KD.

Cumulatively, our data demonstrate that transient inhibition of the PRC2 co-factor JARID2 can reprogram human HSPCs with a durable enhancement of functional potential. Development of small molecules targeting JARID2 could hold promise for ex vivo expansion of UCB HSPCs for clinical applications.