The role of apoptosis in the regulation of hematopoietic stem cells: Overexpression of Bcl-2 increases both their number and repopulation potential

Abstract

Hematopoietic stem cells (HSC) give rise to cells of all hematopoietic lineages, many of which are short lived. HSC face developmental choices: self-renewal (remain an HSC with long-term multilineage repopulating potential) or differentiation (become an HSC with short-term multilineage repopulating potential and, eventually, a mature cell). There is a large overcapacity of differentiating hematopoietic cells and apoptosis plays a role in regulating their numbers. It is not clear whether apoptosis plays a direct role in regulating HSC numbers. To address this, we have employed a transgenic mouse model that overexpresses BCL-2 in all hematopoietic cells, including HSC: H2K-BCL-2. Cells from H2K-BCL-2 mice have been shown to be protected against a wide variety of apoptosis-inducing challenges. This block in apoptosis affects their HSC compartment. H2K-BCL-2-transgenic mice have increased numbers of HSC in bone marrow (2.4x wild type), but fewer of these cells are in the S/G(2)/M phases of the cell cycle (0.6x wild type). Their HSC have an increased plating efficiency in vitro, engraft at least as well as wild-type HSC in vivo, and have an advantage following competitive reconstitution with wild-type HSC.

Figure 1

HSC FACS^®-staining profiles and expression of hBCL-2 in HSC. (A) Staining profiles on WBM of wild-type and H2K-BCL-2–transgenic mice for the four surface markers used to define HSC: Thy-1.1^FITC, Lin^PE, Sca-1^TXR, and c-Kit^APC. Boxed areas indicate the gates within which HSC are present. The two boxes in the Thy-1.1 versus Lin plots depict LT-HSC (Lin^neg, bottom) and ST-HSC (Lin^lo, top). (B) As in A, except that the plots depict populations gated for Sca-1⁺ and c-Kit⁺ (top) and Thy-1.1^lo and Lin^neg (bottom), and thus allow assessment of the contribution of each marker to the purification of the HSC population. (C) Cytospin showing morphology of Lin^neg and Lin^lo HSC sorted from WT and H2K-BCL-2–transgenic mice. May-Grünwald/Giemsa stain. (D) Expression of the H2K-BCL-2 transgene in HSC from three different founderlines. Bone marrow was simultaneously stained for Thy1.1^FITC, Lin^Cy5, Sca-1^TXR, c-Kit^APC, and BCL-2^PE, and analyzed using a modified FACS^® Vantage. The histograms depict BCL-2 staining in cells within the LT-HSC gate (top) and ST-HSC gates (bottom). Gray-filled histogram depicts staining in wild-type cells, open histograms in cells from founderlines 1038, 1043, and 1053.

Figure 4

In vitro plating of HSC from H2K-BCL-2–transgenic and wild-type mice. (A) Colony formation at 2 wk after plating of single HSC in 96-well plates with confluent, irradiated, AC11 stroma cells. Solid black bars represent H2K-BCL-2, gray bars wild-type HSC. (Left) Data for LT-HSC; (right) data for ST-HSC. Combined data from three experiments using founderlines 1038 and 1053. (B) Colony formation after plating of single sorted HSC in methylcellulose with IL-3, IL-6, SF, GM-CSF, and erythropoietin. Wells containing colonies of more than 50 cells were counted 8 d after plating. (C) Survival of growth factor-deprived HSC from wild-type (gray bars) and H2K-BCL-2 (solid bars) HSC. Single HSC were clones, sorted into Terasaki wells containing myelocult M5300 medium without factors. SF, Flt3L, IL-6, and Tpo were added after 70 h. The graph shows the surviving (proliferating) cells as a percentage of the plating efficiency of nonstarved cells, to compensate for the initial difference in plating efficiency (as illustrated in A and B). One representative experiment is shown with data on 150 plated cells per genotype for each cell type. Statistical analysis using Fisher's Exact test.

Figure 2

HSC numbers in bone marrow from H2K-BCL-2 and wild-type mice. Staining profiles showing Lin versus c-Kit staining for wild-type (A) and transgenic (B) WBM. The boxed areas indicate HSC gates for these markers for LT-HSC (left) and ST-HSC (right). (C) LT-HSC numbers in bone marrow from wild-type and H2K-BCL-2–transgenic mice. Open symbols represent wild-type mice, closed symbols H2K-BCL-2–transgenic mice. Circles represent founderline 1038, squares founderline 1053. Averages are indicated by the horizontal bars. (D) Same as in B, but showing ST-HSC numbers.

Figure 3

Cell cycle status of H2K-BCL-2–transgenic HSC. (A) Percentage of LT-HSC in bone marrow from wild-type and H2K-BCL-2–transgenic mice that is in the S/G₂/M phases of the cell cycle. Open symbols represent wild-type mice, closed symbols H2K-BCL-2–transgenic mice. Circles represent founderline 1038, triangles founderline 1043, and squares founderline 1053. Data from 12 transgenic and 13 wild-type mice, averages are indicated by the horizontal bars. At the bottom are representative plots showing Hoechst staining of bone marrow cells within the LT-HSC gates for wild-type (left) and transgenic (right) mice. (B) Same as A, but showing ST-HSC. (Bottom) Relationship between HSC numbers and cell cycle status for LT-HSC (C) and ST-HSC (D).

Figure 7

Competitive repopulation in vivo using ST-HSC from H2K-BCL-2–transgenic and wild-type mice. Markers as in Fig. 5. Lethally irradiated CD45.1×CD45.2 host animals were reconstituted with 10 transgenic ST-HSC (CD45.2), 10 wild-type ST-HSC (CD45.1), and 250,000 whole bone marrow cells from a CD45.1×CD45.2 mouse. The figure shows, as a percentage of nucleated blood cells, the myeloid cells derived from transgenic ST-HSC (left) and wild-type ST-HSC (right). Data from 10 reconstituted animals, the line shows the average reconstitution at each time point, the symbols show the data for individual animals. The gray areas indicate the background staining in nonreconstituted CD45.1×CD45.2 mice. The difference in background levels is caused by different staining characteristics of the CD45.1 and CD45.2 antibody conjugates used. One representative experiment (out of three) is shown.

Figure 5

Competitive repopulation in vivo using LT-HSC from H2K-BCL-2–transgenic and wild-type mice. (A) Experimental model that allows differential monitoring of wild-type and H2K-BCL-2–transgenic donor cells and host cells. Donor cells are either CD45.2 (H2K-BCL-2) or CD45.1 (wild-type), host cells are CD45.1×CD45.2. All three populations can easily be distinguished in the blood of reconstituted animals (right plot). (B) Ratio in blood of total donor-derived cells in animals reconstituted with two different combinations of wild-type and transgenic cells, as indicated in the figure. The t = 0 time point shows the HSC ratio used to reconstitute lethally irradiated animals. Peripheral blood data are shown for 10, 18, and 36 wk after reconstitution. (Left) Ratios for total white blood cells; (right) ratios for myeloid cells. Data points from 12 animals total. (C) Experimental set-up as in B, except that CD45.2 HSC are either wild-type or H2K-BCL-2 transgenic. (Left) Donor ratios for total white blood cells 14 wk after reconstitution with 50 CD45.2 HSC (wild-type or transgenic), 1,000 CD45.1 HSC (wild type) and 3 × 10⁵ F1 bone marrow cells. (Gray bars) Wild-type CD45.2 HSC; (black bars) H2K-BCL-2–transgenic CD45.2 HSC. (Right) Ratio of myeloid (Gr-1⁺, Mac-1⁺) donor-derived cells in the same animals. Data are from seven reconstituted animals.

Figure 6

Comparative increase in H2K-BCL-2/wild-type donor ratio in different cell populations, including HSC. Experimental set-up as in Fig. 5. The input (HSC) ratio was set as 1. (A) The overrepresentation of cells derived from H2K-BCL-2 HSC in a mouse reconstituted with 12 H2K-BCL-2–transgenic LT-HSC and 250,000 WT WBM cells 60 wk after reconstitution. (B) The same data for an animal reconstituted with 50 H2K-BCL-2–transgenic LT-HSC and 1,000 WT LT-HSC ∼14 wk after reconstitution. In both animals, the increased ratio of HSC mirrors that of myeloid cells, while the increase is much stronger for lymphoid cells.

Figure 8

Model for regulation of HSC numbers. (A) HSC homeostasis through balancing self-renewal and differentiation. (B) HSC homeostasis by balancing self-renewal, differentiation, and apoptosis.