Wnt signaling in the niche enforces hematopoietic stem cell quiescence and is necessary to preserve self-renewal in vivo

Abstract

Wingless (Wnt) is a potent morphogen demonstrated in multiple cell lineages to promote the expansion and maintenance of stem and progenitor cell populations. Wnt effects are highly context dependent, and varying effects of Wnt signaling on hematopoietic stem cells (HSCs) have been reported. We explored the impact of Wnt signaling in vivo, specifically in the context of the HSC niche by using an osteoblast-specific promoter driving expression of the paninhibitor of canonical Wnt signaling, Dickkopf1 (Dkk1). Here we report that Wnt signaling was markedly inhibited in HSCs and, unexpectedly given prior reports, reduction in HSC Wnt signaling resulted in reduced p21Cip1 expression, increased cell cycling, and a progressive decline in regenerative function after transplantation. This effect was microenvironment determined, but irreversible if the cells were transferred to a normal host. Wnt pathway activation in the niche is required to limit HSC proliferation and preserve the reconstituting function of endogenous hematopoietic stem cells.

Figure 1. Seven color FACS analysis of primitive populations in wt and Dkk1-tg BM

BM from Dkk1 tg and littermates was assayed by multiparameter FACS for relative proportion of primitive HSC populations. BM was stained with antibodies against Lineage markers, cKit, Sca-1, CD34, Flk2, CD16/32 and CD127 and gated as shown in panels (A) and (C). At least 10 mice per genotype were compared, over at least three separate experiments. The proportion of BM corresponding to the HSC-containing LK+S+ fraction (A, blue gate) is shown in (B, left axis), and is sub-sectioned according to CD34 and Flk2 expression to yield phenotypic assessments of LT-HSC and ST-HSC fractions (B, right axis). More differentiated progenitors gated in the LK+S− population (A, left, green gate) were sub-sectioned based on CD16/32 and CD34 expression to compare CMP, GMP and MEP progenitors as shown in (C, left panel). Frequencies of each population, from the same samples quantified for HSC frequency in (B) are shown in (D, left axis). The CLP fraction, gated on LK^loS^lo in (A, red gate), and gated further on CD127+ cells in (C, right panel) are quantified in (D, right axis). Significance was determined by a Student’s 2-tailed T-test. Error bars indicate the SE of the mean.

Figure 2. Assessment of canonical Wnt signal activity in HSC-containing populations of Dkk1-tg mice

Dkk1-tg mice were crossbred with Topgal transgenic mice. Three pairs of topgal+ littermates, with and without the Dkk1-tg were used to isolate the HSC-containing LK+S+ primitive fraction, and more differentiated LK+S− cells. FACS-sorted populations were used to isolate mRNA, and assessed for expression of the Topgal reporter. Data are expressed as the relative Topgal mRNA expression, compared to the levels present in LK+S+ wt cells (A). Three pairs of Dkk1-tg and wt littermates were also used isolate the LK+S+ fraction of BM, from which mRNA was isolated (non-Topgal transgenics). mRNA was also isolated from the tibea (bone + marrow) of wt and Dkk1-tg mice. Quantitative PCR was performed using primers specific for the Dkk1 transgene. Data are expressed as the relative amount of Dkk1 mRNA compared to that found in wt tibea (B), and error bars indicate SE of the mean.

Figure 3. Functional assessment of HSCs isolated from Dkk1-tg mice

(A) BM from 8 pairs of wt and Dkk1-tg mice was plated in methylcellulose with growth factors (SCF, IL-3, IL-6, Epo) and scored for CFU-C (combined scoring for BFU-E, CFU-GM and CFU-GEMM colonies) after 12 days. All live colonies of more than 30 cells were counted for each of three wells plated per sample. Data are shown as mean colonies per well for each of 8 mice studied over three individual experiments. Significance was determined using a two-tailed Student’s T test. (B) Limiting dilution experiments were performed using three doses of test marrow (CD45.1) transplanted with 5×10⁵ competing cells (CD45.2) into groups of at least 9 recipients (CD45.2) per dose. Test marrow was isolated from two wt and two Dkk1-tg mice, and the Dkk1-tg donors shown here were transplanted into separate groups of irradiated recipients. Data points are plotted as the percent of recipients per group that did not exhibit at least 1% multi-lineage PB engraftment at 6 months (percent unreconstituted). LT-HSC frequency and significance were determined using Poisson statistics: wt, 1 in 63,00 (circles) vs tg, 1 in 31,500 or 1: 37,000 (squares); p<0.02. Similar results were obtained in an independent assessment of two Dkk1-tg donors. (C) Non-competitive serial transplants were initiated by transplanting 1×10⁶ whole BM pooled from three wt or Dkk1-tg donors (CD45.1) into each of 10 irradiated recipients (CD45.2). Secondary and tertiary transplants were performed after 14 weeks of engraftment by pooling BM from 3-4 reconstituted recipients to transplant 1×10⁶ whole BM into new groups of 10 irradiated CD45.2 recipients. The Kaplan-Meier survival graph depicts the survival of tertiary recipients, mice receiving BM from Dkk1-tg mice (solid line) or wt controls (dashed line). Similar results were obtained in an independent assessment of 2 wt and 2 Dkk1-tg mice. (D) Prior to transplant into tertiary recipients, BM from 5 secondary recipients of both genotypes was assayed by FACS for the frequency of LT-HSCs (LK+S+CD34loFlk2−). Error bars indicated SD of the mean, and significance was determined by a two-tailed T test.

Figure 4. Transplant analysis of HSC function following residence in a Dkk1-tg environment

Control, 1×10⁶ wt whole BM cells (CD45.2) was transplanted into each of 5 wt or Dkk1-tg irradiated recipients (CD45.1). Peripheral blood was assayed by FACS for engraftment by CD45.2 staining of wbc after 14 weeks (A). Contribution to both B220+ and Mac1+ populations was confirmed. BM was harvested from 2 engrafted recipients for each genotype and groups of 5 irradiated wt CD45.1 secondary hosts received 9×10⁵ CD45.2 cells from individual primary recipients, mixed with 3×10⁵ fresh CD45.1 wt BM. PB engraftment of secondary recipients was assayed after 6 months (B). Data shown compare the reconstitution from 10 recipients of wt-resident BM versus groups of 5 wt recipients transplanted with BM from two individual Dkk1-tg hosts. Error bars are SD of the means in each case, significance determined by two-tailed T tests. Similar results were obtained in a second experiment of two independently transplanted and re-transplanted Dkk1-tg hosts.

Figure 5. Examination of cell cycle status of primitive BM in wt and Dkk1-tg mice

BM was isolated from 3 pairs of wt and Dkk1-tg mice and subjected to FACS analysis after surface staining and treatment with Hoechst 33342 and Pyronin Y. Proportion of LK+S+ gated (A) or LK+S+CD34lo gated (B) cells present in gates corresponding to G0, G1 or S-G2-M phases of the cell cycle (see inset diagram) are depicted for a representative pair of wt (left) and Dkk1-tg (right) mice. (C) To functionally assay the preservation of a quiescent BM fraction, control recipient mice were reconstituted with wt or Dkk1 tg cells and allowed to engraft for 16 weeks. Peripheral neutrophil (NE) counts were monitored by weekly cbc analysis following i.p. injection of 5-fluorouracil at day 0 and day 42 (injections indicated by downward arrows). Data indicate mean +/− SE for groups of 5 recipients per genotype (wt, dashed line; Dkk1-tg, solid line), and are representative of two independent experiments (** p<0.005, * p<0.05 – two-tailed T test).

Figure 6. Gene expression by quantitative PCR of sorted primitive populations

BM was isolated from three independent pairs of wt and Dkk1-tg mice, and LK+S+ (white) or LK+S− (grey) populations were sorted prior to mRNA isolation. Gene expression was assayed by q-PCR for the indicated targets. Data are expressed as the delta-CT between the gene of interest and the Hprt1 levels expressed in the same Dkk1-tg sample, relative to the delta-CT observed in the paired wt sample. Means from three independent experiments are depicted with SE.