Cxcl10 and Cxcr3 regulate self-renewal and differentiation of hematopoietic stem cells
Background: The function of hematopoietic stem cells (HSC) is regulated by HSC internal signaling pathways and their microenvironment. Chemokines and chemokine ligands play important roles in the regulation of HSC function. Yet, their functions in HSC are not fully understood.  Methods: We established Cxcr3 and Cxcl10 knockout mouse models (Cxcr3-/- and Cxcl10-/-) to analyze the roles of Cxcr3 or Cxcl10 in regulating HSC function. The cell cycle distribution of LT-HSC was assessed via flow cytometry. Cxcr3-/-and Cxcl10-/-stem/progenitor cells showed reduced self-renewal capacity as measured in serial transplantation assays. To study the effects of Cxcr3 or Cxcl10 deficient bone marrow microenvironment, we transplanted CD45.1 donor cells into Cxcr3-/-or Cxcl10-/- recipient mice (CD45.2) and examined donor-contributed hematopoiesis. Results: Deficiency of Cxcl10 and its receptor Cxcr3 led to decreased BM cellularity in mice, with a significantly increased proportion of LT-HSC. Cxcl10-/- stem/progenitor cells showed reduced self-renewal capacity in the secondary transplantation assay. Notably, Cxcl10-/- donor-derived cells preferentially differentiated into B lymphocytes, with skewed myeloid differentiation ability. Meanwhile, Cxcr3-deficient HSCs demonstrated a reconstitution disadvantage in secondary transplantation, but the lineage bias was not significant. Interestingly, the absence of Cxcl10 or Cxcr3 in bone marrow microenvironment did not affect HSC function.  Conclusions: The Cxcl10 and Cxcr3 regulate the function of HSC, including self-renewal and differentiation, adding to the understanding of the roles of chemokines in the regulation of HSC function. Overall design: LT-HSCs (Lin-Sca-1+c-Kit+CD135-CD150+CD48-) were isolated from bone marrow cells of three WT mice, Cxcl10-/- and Cxcr3-/- mice, respectively. 500~800 drops per mouse were sorted into 96-well plates containing 10µl buffer (2%FBS+DPBS). RNA extraction and amplification were performed with Discover-sc WTA Kit V2 following the manufacturer's instructions (Vazyme, N711-02). cDNA concentration was determined by Qubit 3.0. Sequencing libraries were prepared using TruePrep DNA Library Prep Kit V2 for Illumina according to the manufacturer's protocol (Vazyme, TD503-01) and sequenced on the Illumina sequencing platform by Genedenovo Biotechnology Co., Ltd (Guangzhou, China).