Distinct hematopoietic stem cell subtypes are differentially regulated by TGF-beta1

Abstract

The traditional view of hematopoiesis has been that all the cells of the peripheral blood are the progeny of a unitary homogeneous pool of hematopoietic stem cells (HSCs). Recent evidence suggests that the hematopoietic system is actually maintained by a consortium of HSC subtypes with distinct functional characteristics. We show here that myeloid-biased HSCs (My-HSCs) and lymphoid-biased HSCs (Ly-HSCs) can be purified according to their capacity for Hoechst dye efflux in combination with canonical HSC markers. These phenotypes are stable under natural (aging) or artificial (serial transplantation) stress and are exacerbated in the presence of competing HSCs. My- and Ly-HSCs respond differently to TGF-beta1, presenting a possible mechanism for differential regulation of HSC subtype activation. This study demonstrates definitive isolation of lineage-biased HSC subtypes and contributes to the fundamental change in view that the hematopoietic system is maintained by a continuum of HSC subtypes, rather than a functionally uniform pool.

Figure 1

Phenotypes of SP subtypes. Left column shows surface marker HSC-identification strategy from whole bone marrow based on lineage-negative, c-kit, Scal-1 expression, as well as Flk-2 and CD34. Top middle panel demonstrates delineation of SP. Top right shows regions designated as lower-SP and upper-SP and below that, c-kit and Sca-1 cells expression of cells gated on these and through a lineage-negative gate. Surface markers on these cells, designated as SP^KLS are shown below. CD150 expression is heterogeneous, with CD150⁺ cells more prevalent in the lower-SP (see also Figure S1).

Figure 2

Repopulation kinetics of transplanted SP subtypes. (A) Competitive transplant scheme for lower- and upper-SP^KLS cells individually (200 per recipient) or in combination (100 of each subtype per recipient), with 200,000 whole bone marrow competitor cells in all transplants. (B) Overall peripheral blood contribution (Engraft) and proportional contribution to myeloid, B- and T-cells at 4- and 16-weeks after transplantation of SP fractions into separate mice. (C) Peripheral blood analysis after transplantation of both upper- and lower-SP into the same recipients. (D) Scheme for re-purification of primary HSCs and transplantation into secondary recipients. (E) Contribution to the blood of SP sub-populations after secondary transplantation at 4- and 16-weeks. (F) Reconstitution of blood in secondary recipients by SP originally co-transplanted in primary transplants (see also Figure S2A).

Figure 3

Donor-derived contribution to stem and progenitor cell compartments of transplant recipients. Analysis of the bone marrow of recipient mice to determine donor cell contribution to stem and progenitor cell compartments 18-weeks post-transplant of SP subtypes transplanted separately (A) and into the same recipients (B). (C) Analysis of the donor cell contribution to SP of recipient mice. In reference to the whole bone marrow competitor cells which serve as an internal control, each SP subfraction tended to regenerate itself. Data presented are averages of three separate pooled experiments (see also Figure S2B).

Figure 4

Clonal analysis of HSC subtypes using single-cell transplantation. (A) Tracking of eight individual lower- and upper-SP^KLS clones showing overall hematopoietic chimerism in recipient mice (engraftment; top panels in grey) and lineage distribution of test cells 12-weeks post-transplant. (B) Analysis of overall engraftment and lineage contribution of lower- and upper-SP^KLS cells also fractionated with respect to CD150. (C) Distribution of lineage contribution 12-weeks after transplant from mice transplanted with single HSCs with the indicated phenotypes. (D) Average level of hematopoietic chimerism from single-cell transplantation experiments. (E) Lineage contribution measured 12 weeks after secondary transplantation of cells derived from primary mice reconstituted with a single HSC. Bone marrow from each clone was tested in four new recipients. All clones met the assigned threshold of at least 0.1% overall contribution to all 4 secondary recipients at 12-weeks post-transplant, with the exception of USP-3, in which 3/4 recipients fell just below the assigned threshold (see also Figure S3).

Figure 5

Aging SP subtypes retain lineage-differentiation biases. (A) Contour plot comparisons of young (10-week old) and old (90-week old) SPs showing an accumulation of lower-SP cells in aged mice. (B) Overall peripheral blood contribution (Engraft) and proportional contribution to myeloid, B- and T-cells at 4- and 16-weeks after transplantation of old and young SP fractions. (C) Peripheral blood contribution after secondary transplantation from recipients of aged lower- and upper-SP^KLS cells.

Figure 6

HSC subtypes show differential responses to TGFβ1. (A) Low (10 pg/μL) concentrations of TGFβ1 accelerated colony formation of My-HSCs (more colonies at day 7), but did not change the total number of colonies formed (at day 15), while TGFβ1 proved inhibitory to Ly-HSC colony formation. The lines indicate the total number of colonies per plate, while the bar graphs represent the number of CFU-GM colonies at each timepoint. Data presented are cumulative of four individual experiments each comprising two replicate plates for each condition. (B) The CFU-GEMM colonies from My-HSCs in the presence of TGFβ1 were markedly larger in size than control colonies. (C) Analysis of Pyronin Y staining of My- and Ly-HSCs following 5-hour in vitro exposure to TGFβ1. Data are averages of three separate experiments. (D) BrdU uptake after in vivo TGFβ1 exposure by progeny of transplanted My- and Ly-HSCs. In analyzing the progeny of transplanted HSCs, CD150 expression was used as a surrogate marker for myeloid- (CD150⁺) and lymphoid-biased (CD150^-) daughter HSCs. (E) Real-time PCR analysis of purified My- and Ly-HSCs following 5-hour in vitro TGFβ1 exposure. Data presented are averages for three separate experiments. Stars indicate the genes that exhibit statistically significant differences in response between My- and Ly-HSCs (see also Figure S5-7).

Figure 7

Model for HSC clonal diversity and its relation to the SP phenotype. (A) The traditional clonal succession model (left) in which all mature blood cells are the progeny of a single uniform pool of LT-HSCs, and the clonal diversity model (right), supported by our data, in which distinct HSC subtypes are capable of contributing to all lineages, but are stably programmed to do so in a highly biased fashion (B) The SP allows visual representation of the continuum of HSC subtypes encompassing the spectrum from the most myeloid-biased CD150⁺ lower-SP^KLS to the most lymphoid-biased CD150⁻ upper-SP^KLS. The HSC subtypes exhibit additional cellular, molecular, and functional distinctions. A parental unbiased HSC likely exists during development, and conceivably in the adult.