SATB1 Expression Helps in Identification of the Lymphoid-Lineage-Biased Trajectory of Functionally Fluctuating Hematopoietic Stem Cells
Authors: Doi Yukiko , Yokota Takafumi , Satoh Yusuke , Ueda Tomoaki , Shingai Yasuhiro , Ichii Michiko , Tanimura Akira , Ezoe Sachiko , Shibayama Hirohiko , Oritani Kenji , Kohwi-Shigematsu Terumi , Kanakura Yuzuru 
Published: October 2019

Abstract
Hematopoietic stem cells (HSCs) are purified well using a combination of surface markers. However, even highly enriched HSC fractions have heterogeneity in their self-renewal and differentiation potential. These seemingly contradictory roles are well regulated according to the changing demand for blood cells. Hence, interactions among lineage-related genes need to be set up, as their differentiation potential is restricted. However, how the functional diversity of HSCs reflects their intrinsic gene expression is not yet known.
We previously identified special AT-rich sequence binding protein 1 (SATB1), a global chromatin organizer, as a lymphoid-inducing gene in HSCs (Immunity 2013). SATB1 overexpression strongly enhanced lymphopoiesis from murine HSCs, whereas SATB1 deficiency caused HSC malfunctions. Furthermore, another report showed that SATB1-deficient HSCs were less quiescent and differentiated preferentially to myeloid-erythroid lineages (Nat Immunol 2013). These results suggested that SATB1 is indispensable not only for the lymphopoietic potential but also for the normal function of HSCs.
In this study, we first prepared hematological-lineage restricted SATB1 conditional knock out (cKO) mice to examine whether SATB1 is essential for normal HSC function in the adult bone marrow (BM). We crossed SATB1-flox mice with Cre-recombinase expressing mice under control of the Tie2 gene promoter, which efficiently inactivated the target gene in HSCs. Analyzing the BM in these mice, we observed a significant decrease in the number of HSCs as compared to those in their wild type (WT) littermates. Next, we collected HSCs from WT and Tie2-Cre SATB1-flox cKO mice using flow cytometry, and transplanted these CD45.2+ HSCs into CD45.1+ congenic mice. The chimerism of the transplanted cells was lower in recipients of SATB1 cKO mice-derived HSCs. Evaluation of the lymphocytic potential in a co-culture with MS5 stromal cells revealed that the output of lymphocytes from SATB1-cKO HSCs was lower than that of WT HSCs.
Secondly, we generated SATB1 reporter mice in which SATB1 expression can be precisely monitored in vivo, and examined the early differentiation of HSCs. We found that the HSC fraction of adult BM consists of SATB1− and SATB1+ cells. We sorted the two types of HSCs with high purity and compared their growth and differentiation potential in vitro and in vivo. In methylcellulose colony assays, SATB1+ HSCs were less potent for producing myeloid-erythroid lineage colonies. In the co-culture with MS5 stromal cells, the output of lymphocytes from SATB1+ HSCs was more robust than that from SATB1− HSCs. RNA-sequencing data showed that the expression of many lymphocyte-related genes was upregulated in the SATB1+ HSCs compared to that in the SATB1− HSCs; however, there were no significant differences between the expression of stem cell-related genes in the two HSC types. In serial transplantation experiments, the SATB1+ HSCs produced more lymphocytic cells and fewer myeloid cells in the first recipients. Moreover, both types of HSCs could equally reconstitute the complete HSC fraction that contained SATB1− and SATB1+ cells, and successfully reconstituted lympho-hematopoiesis in the secondary recipients.
In a study with SATB1-cKO mice, we found that SATB1 is indispensable for the preservation of the HSC potential for self-renewing proliferation and lymphocyte-differentiation. These results suggest that SATB1 plays a critical role for HSC integrity. With the newly generated SATB1 reporter mice, we confirmed the heterogeneity of HSCs. While the SATB1− and SATB1+ HSCs significantly differed in lineage-differentiation potential, both showed high long-term self-renewing capacity and reciprocal reconstitution in the serial transplantation. The cell dividing flow of the two HSC fractions settled in the same trajectory in the primary recipients, and then demonstrated equal ability for self-renewal and differentiation in the secondary recipients.
Thus, we successfully isolated authentic lymphoid lineage-biased HSCs using SATB1 expression levels, and our results shed light on the oscillating nature of HSCs. Therefore, we conclude that the SATB1 expression demonstrates the fluctuation of HSCs with respect to the lineage-differentiation potential, and that SATB1 probably contributes to generation of chromatin loop formation, which endows HSCs with robust lymphopoietic potential.

Disclosures
Doi: Yakult Honsha Co.,Ltd.: Speakers Bureau. Yokota:SHIONOGI & CO., LTD.: Research Funding. Shibayama:Novartis Pharma: Honoraria, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding, Speakers Bureau; Takeda: Speakers Bureau; Chugai Pharmaceutical: Speakers Bureau; Ono Pharmaceutical: Speakers Bureau. Kanakura:Chugai Pharmaceutical: Research Funding; Eisai: Research Funding; Astellas: Research Funding; Toyama Chemical: Research Funding; Fujimotoseiyaku: Research Funding; Kyowa Hakko Kirin: Research Funding; Shionogi: Research Funding; Alexionpharma: Research Funding; Pfizer: Research Funding; Bristol Myers: Research Funding; Nippon Shinyaku: Research Funding.