Reverting to Quiescence Preserves Hematopoietic Stem Cells Following Genome Editing
Authors: Shiroshita Kohei , Kobayashi Hiroshi , Okamoto Shinichiro , Kataoka Keisuke , Takubo Keiyo 
Published: November 2021

Abstract
Hematopoietic stem cells (HSCs) in steady-state are quiescent in cell cycle. CRISPR-Cas9 genome editing has revolutionized the HSC research and therapeutic application of HSCs for hematological diseases. Although these methods and clinical results are promising, keeping HSC function after highly efficient genome editing is still challenging because HSCs gradually lose their repopulation capacity following cell cycle activation. Preserving the function of HSCs after genome editing is an urgent issue. In this study, we interrogated the culture method following genome editing to reverse the cell cycle status of HSCs into a quiescent state to test how cell cycle status affects the genome editing efficiency and the HSC potential.
To assess the relationship between genome editing efficiency and cell cycle status, we compared genome editing efficiency among the freshly-isolated or cultured HSCs and progenitors (HSPCs) at various time points. Ribonucleoprotein (RNP) complex was delivered into cells by electroporation. Genome-editing efficiency was evaluated by CD45 knockout rate comparing sgRNA for Rosa and CD45. All the HSPC fractions cultured displayed higher genome-editing efficiency following over-night preculture. Genome-editing efficiency of fresh HSC was lower than 20% while fresh granulocyte/monocyte progenitor (GMP) showed higher than 40%. Cell cycle analysis using EdU or Ki67 and Hoechst 33342 staining confirmed that genome-editing efficiency positively correlated with cell cycle activation. We further tested the effect of pre- and post-culture conditions for genome editing. While preculture with high cytokine concentration for a long period of time (&gt; 16 hours) is required for the efficient genome editing, post-culture condition did not compromise the genome-editing efficiency.
Given the quiescent nature of HSCs, we hypothesized that reverting activated HSCs back to quiescent state may improve the function of HSCs following genome editing. To test this, genome-edited HSCs were cultured in the quiescence-maintaining condition (SCF 1.5 ng/mL and TPO 1.0 ng/mL) under 1% O 2 atmosphere. After 7-day culture, more than 30% of cells kept the surface marker phenotype of CD150 + CD48 - LSK, and over 60% of cells were successfully underwent genome editing. Less than 10% of HSCs were EdU +, suggesting that HSCs reverted to cell cycle quiescence after genome editing. By contrast, HSCs cultured in a conventional high cytokine condition (proliferative condition) lost the surface marker phenotypes and highly incorporated EdU. To assess the long-term reconstitution potential of edited HSCs, the Evi1 expression level was evaluated using Evi1-IRES-GFP mice. The expression level of Evi1 was significantly higher in quiescent HSCs than proliferating HSCs after editing. These results suggest that, as post-electroporation culture, quiescence-maintaining condition reverts precultured HSCs back to a quiescent state in cell cycle. This protocol maintains phenotypic HSCs without compromising the genome-editing efficiency.
To further determine the function of genome-edited HSCs, single cell colony assay was performed. Clonally sorted CD45 knockout HSCs cultured in the quiescence-maintaining condition after gene editing fully maintained colony-forming capacity, but HSCs cultured in the proliferating condition lost their capacity. We then performed transplantation assays using Ubc-GFP mice. GFP + HSCs were genome-edited for Rosa and transplanted into lethally-irradiated recipient mice with competitor cells. The donor-derived chimerism of edited HSCs with quiescence-maintaining condition in peripheral blood and bone marrow was generally superior to that of edited HSCs with proliferative condition. These data demonstrates that edited HSCs cultured in quiescence-maintaining condition maintain stem cell potential in vitro and in vivo.
Altogether, we established an HSC-optimized, highly efficient genome-editing protocol. This study demonstrated that effectiveness of keeping HSC in a quiescent state even in the setting of genome editing. Our protocol is suitable for unveiling the function of genes distinguishing cycling and quiescent HSCs.

Disclosures
Kataoka: Celgene: Honoraria; Eisai: Honoraria; Astellas Pharma: Honoraria; Novartis: Honoraria; Chugai Pharmaceutical: Honoraria; AstraZeneca: Honoraria; Sumitomo Dainippon Pharma: Honoraria; Kyowa Kirin: Honoraria; Janssen Pharmaceutical: Honoraria; MSD: Honoraria; Takeda Pharmaceutical: Honoraria; Otsuka Pharmaceutical: Honoraria; Asahi Genomics: Current equity holder in publicly-traded company; Otsuka Pharmaceutical: Research Funding; Chordia Therapeutics: Research Funding; Chugai Pharmaceutical: Research Funding; Takeda Pharmaceutical: Research Funding; Bristol-Myers Squibb: Research Funding; Eisai: Other: Scholarship; Otsuka Pharmaceutical: Other: Scholarship; Ono Pharmaceutical: Other: Scholarship; Kyowa Kirin: Other: Scholarship; Shionogi: Other: Scholarship; Takeda Pharmaceutical: Other: Scholarship; Summitomo Dainippon Pharma: Other: Scholarship; Chugai Pharmaceutical: Other: Scholarship; Teijn Pharma: Other: Scholarship; Japan Blood Products Organization: Other: Scholarship; Mochida Pharmaceutical: Other: Scholarship; JCR Pharmaceuticals: Other: Scholarship; Genetic Alterations: Patents & Royalties: PD-L1 abnormalties .