p18^INK4C Is a Checkpoint For a Noncanonical Differentiation Path Of Hematopoietic Stem Cells Into Lymphoid Lineages

To address this issue, we reexamined the phenotypic markers of HSC in the p18 deficient mice (p18^-/-). Notably, we found a new stem cell like population, namely “Lin^-Sca-1⁺c-kit^low (LSK^low)” (Fig.1) in p18^-/- bone marrow (BM), which is apart from the known HSC phenotype, Lin^-Sca-1⁺c-kit⁺ (LSK⁺). LSK^low expresses low level of c-kit and is negative for CD34 and IL-7R. It is also distinct from the common lymphoid progenitors (CLPs), a canonical differentiation stage of HSC toward lymphoid lineage. LSK^low was observed only in p18^-/- BM 12 weeks after birth and was not present in p18^+/+ mice. Its frequency was similar with or higher than that of LSK⁺. To determine the differentiation potential of LSK^low into lymphoid vs. myeloid lineages, we cultured LSK^low cells with or without OP9/OP9-DL1 stromal cells in comparison with LSK⁺ cells and CLPs. Our results revealed that LSK^low cells could only be induced into lymphoid (including T, B and NK) cells as well as CLPs, while LSK⁺ cells could differentiate into both lymphoid and myeloid directions. In vivo transplantation further confirmed the differentiation spectrum of LSK^low cells and also indicated that LSK^low cells are derived from LSK⁺ cells. To obtain a molecular signature of LSK^low, we performed single cell qRT-PCR for the genes involved in HSC maintenance and lymphoid commitment. Our data showed similar expression profiles between LSK and LSK^low except higher levels of some but limited lymphoid genes such as Rag1, Rag2 and Foxo1 in LSK^low cells. In order to explore epigenetic mechanisms underlying the phenotypic alteration, we also examined a series of DNA methylation related genes. In comparison with LSK⁺ cells, LSK^low cells have a lower methylation level. This epigenetic alteration was consistent with down-regulation of DNA methyltransferases (including DNMT3a and DNMT3b) and elevation of a new class of the molecules involved in DNA demethylation (including Tet1, Tet2 and Tet3). We therefore speculate that these epigenetic changes may facilitate the formation of LSK^low cell population in p18^-/- mice.