The BM lin CD CD population significantly overlaps with LSK

The BM lin−CD201+CD27+ population significantly overlaps with LSK nrf2 keap1 in B6 mice and consists of early HSPCs. Lin−CD201+CD27+ cells have been proposed for alternative HSPC identification in mouse strains (e.g., BALB/c or NOD strains) or under stress conditions, which lack a distinct LSK immunophenotype (Vazquez et al., 2015). Our result that CD201 expression level correlates with quiescence of HSPCs similar to SCA-1 facilitates identification and isolation of quiescent subsets among lin−CD201+CD27+ HSPCs. In contrast, ESAM, which was proposed to label HSCs under stress conditions in a cell-cycle-specific manner (Sudo et al., 2012), did not seem to be useful for prospective enrichment of quiescent HSPCs at steady state, as KI67-RFP and ESAM expression showed poor correlation. We demonstrate that the properties of SCA-1hi cell populations were not a result of constitutive type I IFN signaling. The latter was surprising, as steady-state type I IFN partially accounted for SCA-1 expression (Figure S2G) and “tonic” (basal or constitutive) type I IFN signaling has been implied in HSC maintenance (Gough et al., 2012). However, our transplantation data revealed that SCA-1hi as well as SCA-1loIfnar1 HSCs outgrew wild-type (wt) competitor cells in secondary recipients, which may be explained either by a general competitive advantage of Ifnar1 HSCs over wt competitor HSCs or, due to lower overall SCA-1 expression in Ifnar1 mice, potent HSCs still reside in the SCA-1lo sorted population. The latter is very likely, as our SCA-1 sorting strategy for Ifnar1 donor cells separated only the brightest fraction of HSCs into the SCA-1hi subset, while SCA-1lo cells contained the majority of the cell population. Nevertheless, purified SCA-1hiIfnar1 donor cells repopulated primary recipients faster and more robustly. Moreover, cell-cycle analysis of Ifnar1 HSPCs revealed that the increased quiescence of the SCA-1hi population was type I IFN independent. Hematopoietic contribution, differentiation pattern, and the relationship between the HSPC subpopulations and diverse SCA-1 expression have not been addressed in situ so far. We speculate that discrepancies between recent studies reporting high (Sawai et al., 2016) or low (Busch et al., 2015; Schoedel et al., 2016; Sun et al., 2014) contribution of HSCs to steady-state hematopoiesis might reflect lineage tracing of exclusive HSC subpopulations that differ by SCA-1 expression and cell-cycle activity.
Experimental Procedures
Author Contributions M.N.F.M., A.G., K.B.S., A.H., and R.B. performed experiments and analyzed data. O.B. and H.C.C. provided Ki67-RFP mice. A.G. conceived the study and designed experiments. A.G. and A.R. wrote the manuscript.
Acknowledgments This work was supported by the German Research Foundation (DFG) through grant SFB 655 B11 to A.R., by the Excellence Initiative of the German Federal and State Governments “Support the best” (ZUK64, to A.R.), and TU Dresden Medical Faculty “MeDDrive 2015” grant to A.G. The authors thank Livia Schulze, Christa Haase, Tobias Häring, and Christina Hiller for expert technical assistance.
Introduction Intestinal epithelia are known to require nearly constant self-renewal, supported by small populations of intestinal stem cells (ISCs). Homeostasis in the mammalian intestine is maintained by ISCs that reside in crypts. Two distinct types of stem cells have been reported to maintain ISC pools in the mouse small intestine: the LGR5+ crypt basal columnar cells (CBCs) (Barker, 2014), and the Bmi1+ “+4” cells (Yan et al., 2012), that are positioned just above CBCs. The stem cell population is maintained mainly by asymmetric ISC divisions that generate one new ISC and one cell destined to differentiate. However, it has been reported that LGR5+ crypt cells also divide symmetrically at a high frequency, to generate either two stem cells or two transient amplifying (TA) cells (Barker, 2014; Lopez-Garcia et al., 2010; Snippert et al., 2010). This condition gives rise to “neutral drift” whereby individual ISC lineages are regularly extinguished and replaced, and homeostasis in the stem cell pool size is maintained by an even balance between ISC losses and duplications. Importantly, it was also found that “reserve” +4 stem cells can revert to stem cells following injury, and that this is important in maintaining the stem cell pool (Buczacki et al., 2013; Tian et al., 2011). Moreover, recent work has also shown that even more differentiated cells, including LGR5- TA cells and Paneth cells, could de-differentiate into LGR5+ cells when they were exposed to WNT3A or following irradiation injury, respectively (Sato et al., 2011; Roth et al., 2012).