Notch Repression by LRF Is Necessary for the Maintenance of Adult Hematopoietic Stem Cell Pool.
Authors: Lee Sung-UK , Li Min , Maeda Manami , Sakurai Nagisa , Ishikawa Yuichi , Radtke Freddy , Yan Minhong , Macdonald Hugh Robson , Maeda Takahiro 
Published: October 2019

Abstract
Abstract 2633
Among the different stem cells, hematopoietic stem cells (HSCs) are one of the best studied and characterized stem cells. To maintain life-long hematopoiesis in the bone marrow (BM), signals governing the balance between self-renewal and differentiation are tightly regulated in HSC compartment. Notch signals are critical regulators of the lymphoid lineage fate, but their role in adult HSC function in the BM is currently under debate. LRF (Leukemia/Lymphoma Related Factor, also known as Zbtb7a/pokemon) is a transcription factor that acts as a proto-oncogene and plays a key role in lymphoid and erythroid development. Previously we reported that the pool of LT-HSCs, CD150+CD48−Flt3−Vcam-1+/&minus;IL7Rα−LSK (Lin−Sca-1+c-Kit+), was significantly reduced, while lymphoid-biased multi-potential progenitors (LMPPs: CD150−CD48+Flt3+Vcam-1+/&minus;IL7Rα−LSK) and common lymphoid progenitors (CLPs: Lin−CD150−CD48+Flt3+Vcam-1−IL7Rα+) were barely detectable in LRF deficient mice. This was due to excessive differentiation of HSC into aberrant CD4/CD8 DP (double positive) T cell development in the BM caused by high Notch activity, implicating LRF role on HSC maintenance. Both gene expression profile (GSEA and DAVID analysis) and Q-PCR results indicated that LRF deficient LT-HSCs had loss of stem cell signature; but gain of T cell signature and up-regulated Notch-target gene, Hes-1, without affecting mRNA expression of Notch (1-4) or related (DLL1, DLL4, Jagged-1) genes. To determine LRF function in HSCs, we performed in vivo and in vitro experiments: 1) 5-FU (5-fluorouracil, the chemotherapy agent) treated LRF deficient mice were not able to compensate for their loss of LT-HSCs; 2) multi-lineage defects were shown in second recipient mice transplanted with 1 million of LRF deficient bone marrow cells in serial bone marrow transplantation assays, suggesting that LRF deficient LT-HSCs had defect in self-renewal and 3) LRF deficient FL-HSCs (CD150+CD48−LSK cells) were cultured on OP9 cells expressing delta-like ligand (DLL1, DLL4 and Jagged1), and enhanced T cell differentiation was only observed when they were co-cultured with delta-expressing OP9 cells. Among the Notch family, these phenotypes were Notch1-dependent. In fact, Notch1flox/floxLRFflox/floxMx1-Cre+ mice demonstrated normal LT-HSC numbers and restored B cell development, and prolonged survival over LRFflox/floxMx1-Cre+ mice in sequential 5-FU treatment in vivo. To explore which Notch-ligand(s) in BM niche is responsible for aberrant T-cell development in LRF deficient mice as well, we treated wild-type and LRFflox/floxMx1-Cre+ with anti-DLL4 antibody twice per week for 3 weeks. DLL4 blockage in LRF deficient mice rescued B cell development and prevented the development of aberrant DP T-cell development in LRF deficient mice. To further elucidate the relationship between LRF and Notch in adult HSC function, we analyzed Notch protein expression levels in HSCs and performed in-depth analysis of HSC/progenitor (HSC, LMPP and CLP) compartments in wild-type and LRF knockout (KO). Interestingly, Notch1 proteins were differentially expressed in LT-HSCs and ~50 % of them were positive for Notch1, while Notch2 was abundantly expressed in LT-HSCs. Notch1 expressing LT-HSCs were in more active cell-cycle (S phase) and absent in LRF conditional knockout mice. It is most likely that Notch1 expressing LT-HSCs were continually differentiating toward T cells in the absence of LRF, as CD4+CD8+ T cells were evident in the BM 10 months after pIpC injection. Taken together, our data strongly indicate that LRF is indispensable for hematopoietic homeostasis by preventing the lymphoid-primed HSCs from Notch/Delta-mediated T-instructive signal in the BM niche. Currently we're investigating the functional significances of Notch1 expressing LT-HSCs in detail. Our studies help us to better understand the underlying mechanism for HSC fate decision (self-renewal v.s. differentiation) in stem cell biology and its therapeutic approach in regenerative medicine.

Disclosures:
No relevant conflicts of interest to declare.