Identification of a new intrinsically timed developmental checkpoint that reprograms key hematopoietic stem cell properties

Abstract

Hematopoietic stem cells (HSCs) execute self-renewal divisions throughout fetal and adult life, although some of their properties do alter. Here we analyzed the magnitude and timing of changes in the self-renewal properties and differentiated cell outputs of transplanted HSCs obtained from different sources during development. We also assessed the expression of several "stem cell" genes in corresponding populations of highly purified HSCs. Fetal and adult HSCs displayed marked differences in their self-renewal, differentiated cell output, and gene expression properties, with persistence of a fetal phenotype until 3 weeks after birth. Then, 1 week later, the HSCs became functionally indistinguishable from adult HSCs. The same schedule of changes in HSC properties occurred when HSCs from fetal or 3-week-old donors were transplanted into adult recipients. These findings point to the existence of a previously unrecognized, intrinsically regulated master switch that effects a developmental change in key HSC properties.

Fig. 1.

Rates of expansion and differentiation properties in vivo of HSCs reflect their developmental status. (A) Experimental design used to assess HSC regeneration kinetics in serially transplanted mice. In each HSC regeneration experiment, the initial transplant of unseparated cells contained ≈10 HSCs based on prior determination of the HSC content of the test cell suspension being used. These cells were then injected into sublethally irradiated Ly5-congenic W⁴¹/W⁴¹ recipients. This experimental design was used to maximize the contribution of the transplanted HSCs to the total HSC pool being regenerated while still allowing donor HSCs to be definitively identified, and also to remove any influences related to variable input HSCs doses that have been previously reported (17). The blue asterisks denote the groups of mice whose cells were analyzed 16 weeks posttransplant to demonstrate changes in the proportional contribution of mature myeloid cells to the total WBCs generated from the transplanted HSCs. (B) Comparison of HSC regeneration (mean ± SEM CRUs/mouse, four experiments each) from E14.5 fetal liver (FL, gray symbols) and adult bone marrow (ABM, black symbols) in sublethally irradiated W⁴¹/W⁴¹ mice injected initially with 10 HSCs (CRUs). (C) Similar analyses of mice transplanted with 10 HSCs (CRUs) from E18.5 fetal bone marrow (E18.5 FBM, pink symbols) and 3- and 4-week postnatal bone marrow (3-wk BM, red symbols; 4-wk BM, blue symbols). (D) HSCs (CRUs) produced from the HSC progeny of E14.5 fetal liver HSCs after 6 weeks in primary recipients (FL-derived, green symbols). (E) Relative outputs of donor-derived lymphoid and myeloid WBCs 16 weeks posttransplant in recipients of 3–6 CRUs. Each bar shows the mean ± SEM percentage of all donor-derived WBCs that expressed Ly6g and/or Mac1 (GM, n = 4–24). The information in the brackets in the labels for each bar refers to the number of weeks that were allowed to elapse before cells were harvested from primary recipients injected with the initial cells indicated and then transferred to secondary (or, in the case of the bottom bar, also into tertiary) recipients where the final lineage output assessments were made 16 weeks later. ∗, Values significantly lower (P < 0.05) than the value obtained for transplants of E14.5 fetal liver HSCs (CRUs). ∗∗, Values significantly higher (P < 0.05) than the value obtained for transplants of adult bone marrow HSCs (CRUs). (F) Representative FACS plots of the WBCs in mice 16 weeks after being transplanted with HSCs from 3-week (red) or 4-week (blue) postnatal bone marrow.

Fig. 2.

Kinetics of HSC self-renewal divisions in vitro. (A) Schema showing the general experimental design used to determine the in vitro division kinetics of purified HSCs from E14.5 fetal liver. (B) Cumulative first (2 cells, pink symbols), second (≥3 cells, red symbols), and third divisions (≥5 cells, black symbols) in 80 cultures, each initiated with single purified E14.5 fetal liver HSC and maintained in 50 ng/ml SF only (data pooled from two experiments). Shown for reference in the gray dotted lines are corresponding curves on the same scale for adult bone marrow HSCs stimulated with 300 ng/ml SF plus 20 ng/ml interleukin-11 plus 1 ng/ml Flt3-ligand to achieve similar maintenance of functional HSC activity (redrawn from ref. 5).

Fig. 3.

Evidence of a switch during ontogeny in the gene expression profile of HSCs. Relative transcript levels were determined by comparing expression values obtained for lin⁻ Rho⁻ SP cells isolated from adult bone marrow (ABM, black bars) to those obtained for lin⁻ Sca-1⁺ CD43⁺ Mac1⁺ cells isolated from E14.5 fetal liver (FL, values set = 1, gray bars) after internal normalization to Gapdh transcript levels. A similar comparison was made for extracts of the same populations isolated from 3- and 4-week postnatal bone marrow cells [3-wk BM (red bars) values set = 1, and 4-wk BM (blue bars), respectively]. Results are the mean ± SEM of data from two to three biological replicates, each measured in triplicate.

Fig. 4.

HSCs show a coordinated reprogramming of multiple key properties. Preswitch HSCs are actively cycling and demonstrate a high rate of self-renewal after transplantation into irradiated hosts, a higher output of myeloid (GM) cells, and a distinct gene expression profile. Postswitch HSCs are predominantly quiescent and display a lower rate of self-renewal after transplantation into irradiated hosts, a lower proportional output of myeloid cells, and a different gene expression profile from preswitch HSCs.