Bone marrow niches orchestrate stem cell hierarchy and immune tolerance

Stem cells reside in specialized microenvironments, termed niches, at several different locations within tissues1-3. The differential functions of heterogeneous stem cells and niches are important given increasing clinical applications of stem cell transplantation and immunotherapy. It remains unknown whether hierarchical structures amongst stem cells at distinct niches exist, and further control aspects of immune tolerance. Here, we propose novel hierarchical arrangements within hematopoietic stem cells (HSCs) and bone marrow (BM) niches, that dictate both regenerative potential and immune privilege. High level nitric oxide-generating (NOHi) HSCs are refractory to immune attack and exhibit "delayed" albeit robust long-term reconstitution. Such highly immune privileged, primitive NOHi HSCs colocalize with distinctive capillaries, characterized by primary ciliated endothelium and high levels of the immune checkpoint molecule CD200. These capillaries regulate the regenerative functions of NOHi HSCs through ciliary protein IFT20/CD200/eNOS/autophagy signals, further mediating immune protection. Notably, previously described niche constituents, sinusoidal cells and Type H vessels2-10, co-localize with less immune privileged and less potent NOLow HSCs. Therefore, we have identified highly immune privileged, "late-rising" primitive HSCs and characterized their immunoprotective niches constituted of specialized vascular domains. Our studies suggest the niche may orchestrate hierarchy in stem cells and immune tolerance, delineating future immunotherapeutic targets. Overall design: CD200Hi capillaries (CD200HiSca1+CD31+EMCN-CD45-), Type H vessels (EMCN+Sca1+CD31+CD45-), other arterial cells (CD200-Sca1+CD31+CD45-), and sinusoidal cells (CD31+Sca1+CD45-) were sorted from BM cells obtained from crushed bone (tibia, femur, and iliac tissues). RNA was isolated from sorted cells lysed in TRIzol. Biological triplicates were prepared using six B6 mice. RNA quality was assessed using Agilent Bioanalyzer RNA-Pico kit (Agilent Technologies, USA). Libraries were prepared using the Clontech Ultra Low v4 kit (Takara Bio) for cDNA synthesis, followed by NexteraXT (Illumina, USA), and sequenced on an Illumina NovaSeq6000 (Illumina, USA), resulting in libraries with read lengths of 150bp.