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

Domen J, Cheshier SH, Weissman IL - J. Exp. Med. (2000)

Bottom Line: It is not clear whether apoptosis plays a direct role in regulating HSC numbers.This block in apoptosis affects their HSC compartment.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Developmental Biology, Stanford University School of Medicine, Stanford, California 94305-5428, USA. domen@stanford.edu

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.

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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-1.1FITC, LinPE, Sca-1TXR, and c-KitAPC. Boxed areas indicate the gates within which HSC are present. The two boxes in the Thy-1.1 versus Lin plots depict LT-HSC (Linneg, bottom) and ST-HSC (Linlo, top). (B) As in A, except that the plots depict populations gated for Sca-1+ and c-Kit+ (top) and Thy-1.1lo and Linneg (bottom), and thus allow assessment of the contribution of each marker to the purification of the HSC population. (C) Cytospin showing morphology of Linneg and Linlo 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 Thy1.1FITC, LinCy5, Sca-1TXR, c-KitAPC, and BCL-2PE, 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.
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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-1.1FITC, LinPE, Sca-1TXR, and c-KitAPC. Boxed areas indicate the gates within which HSC are present. The two boxes in the Thy-1.1 versus Lin plots depict LT-HSC (Linneg, bottom) and ST-HSC (Linlo, top). (B) As in A, except that the plots depict populations gated for Sca-1+ and c-Kit+ (top) and Thy-1.1lo and Linneg (bottom), and thus allow assessment of the contribution of each marker to the purification of the HSC population. (C) Cytospin showing morphology of Linneg and Linlo 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 Thy1.1FITC, LinCy5, Sca-1TXR, c-KitAPC, and BCL-2PE, 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.

Mentions: LT-HSC in this study are defined by the following combination of cell surface markers: Thy-1.1lo, Sca-1high, c-Kithigh, and Linneg. Cells with this phenotype, which form a rare population in whole bone marrow, have been found to contain a population of cells with long-term multilineage reconstitution potential. The limit dilution dose for reconstitution with these cells is ∼10 cells injected i.v. 31. Cells with short-term multilineage reconstitution potential (ST-HSC) differ from the LT-HSC population in increased, but low level, expression of certain Lin-markers (initially Mac-1 followed by CD4), and are present in larger numbers in whole bone marrow 31. When bone marrow is stained for these HSC markers (Fig. 1A and Fig. B), the profiles for wild-type and H2K-BCL-2–transgenic mice are similar, except for relatively minor differences, such as increased numbers of T cells (Thy-1.1high cells). The HSC populations as defined in wild-type mice can be easily recognized in bone marrow from transgenic mice. Morphologically, HSC from H2K-BCL-2–transgenic mice resemble those of wild-type mice (Fig. 1 C). LT-HSC are relatively small and uniform in size, while ST-HSC are more varied in size. Antibody staining for the presence of human BCL-2 protein in cells with the LT-HSC surface phenotype shows that all cells express the transgene at high levels (Fig. 1 D). This is in agreement with earlier data showing high levels of expression in cells sorted for three (Thy-1.1, Lin, Sca-1) of the four HSC markers, and with the fact that these cells are protected against radiation-induced apoptosis 30. All three founderlines used in this study (1038, 1043, and 1053) have very similar levels of expression of the transgene in HSC (Fig. 1 D). All of this is in agreement, but doesn't prove, that HSC in H2K-BCL-2–transgenic mice have the same cell surface characteristics as wild-type HSC. However, HSC cannot be defined by cell surface markers alone, since these can vary 32. Therefore LT- and ST-HSC from H2K-BCL-2–transgenic mice were tested for their ability to repopulate lethally irradiated animals under limit-dilution conditions. They do this at least as well as wild-type HSC (see below). Quantitation of cells with HSC surface phenotype in H2K-BCL-2–transgenic bone marrow reveals an increase in their numbers, compared with that present in wild-type bone marrow (Fig. 2). This is the case both for cells with the LT-HSC surface phenotype (Linneg) and for cells with the ST-HSC surface phenotype (Linlo). As tabulated in Table , the expansion of HSC is more dramatic in the LT-HSC compartment (2.4× wild-type levels) than in the ST-HSC compartment (1.7× wild-type levels). The spread in HSC numbers in H2K-BCL-2–transgenic bone marrow is larger than that encountered in wild-type littermates, as is demonstrated in Fig. 2 and the difference in SD in Table between wild-type and H2K-BCL-2. The transgenic data on HSC numbers in Fig. 2, relative data, and Table , actual percentages of bone marrow, are based on the analysis of mice from two different transgenic founderlines, 1038 and 1053. When analyzed separately, there is no significant difference in the number of stem cells between them (Fig. 2). Since there are no differences in the total bone marrow cellularity between H2K-BCL-2–transgenic mice and wild-type littermates 30, this increased frequency of HSC means increased numbers of HSC in the bone marrow.


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

Domen J, Cheshier SH, Weissman IL - J. Exp. Med. (2000)

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-1.1FITC, LinPE, Sca-1TXR, and c-KitAPC. Boxed areas indicate the gates within which HSC are present. The two boxes in the Thy-1.1 versus Lin plots depict LT-HSC (Linneg, bottom) and ST-HSC (Linlo, top). (B) As in A, except that the plots depict populations gated for Sca-1+ and c-Kit+ (top) and Thy-1.1lo and Linneg (bottom), and thus allow assessment of the contribution of each marker to the purification of the HSC population. (C) Cytospin showing morphology of Linneg and Linlo 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 Thy1.1FITC, LinCy5, Sca-1TXR, c-KitAPC, and BCL-2PE, 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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
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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-1.1FITC, LinPE, Sca-1TXR, and c-KitAPC. Boxed areas indicate the gates within which HSC are present. The two boxes in the Thy-1.1 versus Lin plots depict LT-HSC (Linneg, bottom) and ST-HSC (Linlo, top). (B) As in A, except that the plots depict populations gated for Sca-1+ and c-Kit+ (top) and Thy-1.1lo and Linneg (bottom), and thus allow assessment of the contribution of each marker to the purification of the HSC population. (C) Cytospin showing morphology of Linneg and Linlo 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 Thy1.1FITC, LinCy5, Sca-1TXR, c-KitAPC, and BCL-2PE, 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.
Mentions: LT-HSC in this study are defined by the following combination of cell surface markers: Thy-1.1lo, Sca-1high, c-Kithigh, and Linneg. Cells with this phenotype, which form a rare population in whole bone marrow, have been found to contain a population of cells with long-term multilineage reconstitution potential. The limit dilution dose for reconstitution with these cells is ∼10 cells injected i.v. 31. Cells with short-term multilineage reconstitution potential (ST-HSC) differ from the LT-HSC population in increased, but low level, expression of certain Lin-markers (initially Mac-1 followed by CD4), and are present in larger numbers in whole bone marrow 31. When bone marrow is stained for these HSC markers (Fig. 1A and Fig. B), the profiles for wild-type and H2K-BCL-2–transgenic mice are similar, except for relatively minor differences, such as increased numbers of T cells (Thy-1.1high cells). The HSC populations as defined in wild-type mice can be easily recognized in bone marrow from transgenic mice. Morphologically, HSC from H2K-BCL-2–transgenic mice resemble those of wild-type mice (Fig. 1 C). LT-HSC are relatively small and uniform in size, while ST-HSC are more varied in size. Antibody staining for the presence of human BCL-2 protein in cells with the LT-HSC surface phenotype shows that all cells express the transgene at high levels (Fig. 1 D). This is in agreement with earlier data showing high levels of expression in cells sorted for three (Thy-1.1, Lin, Sca-1) of the four HSC markers, and with the fact that these cells are protected against radiation-induced apoptosis 30. All three founderlines used in this study (1038, 1043, and 1053) have very similar levels of expression of the transgene in HSC (Fig. 1 D). All of this is in agreement, but doesn't prove, that HSC in H2K-BCL-2–transgenic mice have the same cell surface characteristics as wild-type HSC. However, HSC cannot be defined by cell surface markers alone, since these can vary 32. Therefore LT- and ST-HSC from H2K-BCL-2–transgenic mice were tested for their ability to repopulate lethally irradiated animals under limit-dilution conditions. They do this at least as well as wild-type HSC (see below). Quantitation of cells with HSC surface phenotype in H2K-BCL-2–transgenic bone marrow reveals an increase in their numbers, compared with that present in wild-type bone marrow (Fig. 2). This is the case both for cells with the LT-HSC surface phenotype (Linneg) and for cells with the ST-HSC surface phenotype (Linlo). As tabulated in Table , the expansion of HSC is more dramatic in the LT-HSC compartment (2.4× wild-type levels) than in the ST-HSC compartment (1.7× wild-type levels). The spread in HSC numbers in H2K-BCL-2–transgenic bone marrow is larger than that encountered in wild-type littermates, as is demonstrated in Fig. 2 and the difference in SD in Table between wild-type and H2K-BCL-2. The transgenic data on HSC numbers in Fig. 2, relative data, and Table , actual percentages of bone marrow, are based on the analysis of mice from two different transgenic founderlines, 1038 and 1053. When analyzed separately, there is no significant difference in the number of stem cells between them (Fig. 2). Since there are no differences in the total bone marrow cellularity between H2K-BCL-2–transgenic mice and wild-type littermates 30, this increased frequency of HSC means increased numbers of HSC in the bone marrow.

Bottom Line: It is not clear whether apoptosis plays a direct role in regulating HSC numbers.This block in apoptosis affects their HSC compartment.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Developmental Biology, Stanford University School of Medicine, Stanford, California 94305-5428, USA. domen@stanford.edu

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.

Show MeSH