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Intrinsic and extrinsic control of hemopoietic stem cell numbers: mapping of a stem cell gene.

de Haan G, Van Zant G - J. Exp. Med. (1997)

Bottom Line: The population size of primitive stem cells (CAFC day 35) varied widely (up to sevenfold) among strains, unlike total CAFC day 7 numbers (cycling and quiescent), which were similar.Administration of the early acting cytokine flt-3 ligand to these strains resulted in activation of quiescent primitive stem cells exclusively in strains with high endogenous stem cell numbers (DBA and AKR), but was unrelated to strain-specific progenitor cell cycling.Linkage with this genomic interval was associated with a likelihood of odds score of 3.3, surpassing the level required for significance.

View Article: PubMed Central - PubMed

Affiliation: Blood and Marrow Transplant Program, Division of Hematology/Oncology, University of Kentucky Medical Center, Lexington, Kentucky 40536-0093, USA.

ABSTRACT
We evaluated in vivo interactions between extrinsic (growth factor induced) and intrinsic (genetically determined) effectors of mouse primitive hemopoietic stem cell proliferation and numbers. Accordingly, stem cell frequency and cell cycle kinetics were assessed in eight strains of inbred mice using the cobblestone area-forming cell (CAFC) assay. A strong inverse correlation was observed between mouse lifespan and the number of autonomously cycling progenitors (CAFC day 7) in the femur. The population size of primitive stem cells (CAFC day 35) varied widely (up to sevenfold) among strains, unlike total CAFC day 7 numbers (cycling and quiescent), which were similar. Administration of the early acting cytokine flt-3 ligand to these strains resulted in activation of quiescent primitive stem cells exclusively in strains with high endogenous stem cell numbers (DBA and AKR), but was unrelated to strain-specific progenitor cell cycling. To map loci affecting stem cell frequency, we quantified stem cells in BXD recombinant inbred mice (offspring of C57BL/6 and DBA/2). The resulting strain distribution pattern showed high concordance with a marker that mapped to chromosome 18 (19 cM). Linkage with this genomic interval was associated with a likelihood of odds score of 3.3, surpassing the level required for significance. Interestingly, this segment, containing the EGR-1 gene, shows synteny with human chromosome 5q, a region strongly associated with various hematological malignancies. Our findings indicate that a gene mapping to this region is mutated in either C57BL/6 or DBA/2 (and possibly AKR) mice. These studies in apparently healthy mice may facilitate the identification of a gene implicated in human 5q-syndromes.

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Analysis of stem cell frequency  in RI strains. (a) Strain distribution pattern  of the CAFC day 35 frequency in all BXD  strains. Parental values are indicated with  the leftmost (C57BL/6) and rightmost  (DBA) bars. Each value is based on the analysis of pooled marrow cells obtained from  three BXD mice. (b) Likelihood ratio statistic plot showing the results of the interval  mapping on chromosome 18. The threshold  LRS values for suggestive and significant  linkage were 9.9 and 14.7, respectively, and  are indicated. A sharp peak was observed  around D18NcvS22, located ∼19 cM from  the centromere. The corresponding map  position of orthologous genes in human is  shown at the top of the figure. The region  around D18NcvS22 (11–34 cM) shows synteny with human chromosome 5q21-35.
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Figure 6: Analysis of stem cell frequency in RI strains. (a) Strain distribution pattern of the CAFC day 35 frequency in all BXD strains. Parental values are indicated with the leftmost (C57BL/6) and rightmost (DBA) bars. Each value is based on the analysis of pooled marrow cells obtained from three BXD mice. (b) Likelihood ratio statistic plot showing the results of the interval mapping on chromosome 18. The threshold LRS values for suggestive and significant linkage were 9.9 and 14.7, respectively, and are indicated. A sharp peak was observed around D18NcvS22, located ∼19 cM from the centromere. The corresponding map position of orthologous genes in human is shown at the top of the figure. The region around D18NcvS22 (11–34 cM) shows synteny with human chromosome 5q21-35.

Mentions: To identify loci affecting the size of the endogenous stem cell pool, we made use of BXD recombinant inbred (RI) strains of mice derived from C57BL/6 (a group 2 strain, Fig. 4) and DBA/2 (a group 3 strain) parental founders. The genome of RI BXD mice consists of a mosaic of homozygous C57BL/6 and DBA/2 chromosomal segments. Typing these mice for a particular phenotype and comparing the obtained SDP with polymorphic markers previously mapped by others in the BXD strains may result in a map position for the trait of interest. In the field of hematology this type of approach has been, for example, successfully employed to characterize the gene involved in IL-3 nonresponsiveness of A/J mice (24). To this end, we quantified CAFC day 35 frequency in all 26 RI strains. DBA parental mice had approximately threefold higher CAFC day 35 frequencies than C57BL/6 mice, but nearly all of the RI strains had CAFC day 35 frequencies which were between C57BL/6 and DBA/2 values (Fig. 6 a). Two notable exceptions were RI strain number 11, which had an unusually high stem cell number, and RI strain 29, which had a very small stem cell compartment. Our data differ quantitatively and qualitatively from those reported recently by Muller-Sieburg et al. (7). We believe that this results from our use of more stringent criteria in the CAFC assay. As has been pointed out by Ploemacher et al. (25), the CAFC assay will only result in reproducible and accurate stem cell measurements if (phase contrast–negative) colonies growing beneath stromal cells are counted. Phase contrast–positive cells accumulating on top of the stroma reflect the mature progeny of primitive cells, and their presence does not indicate a proper cobblestone area. In addition, it is likely, given the different cell cycle status of C57BL/6 and DBA/2 cells, that the half-life of such phase contrast–positive cells differs markedly from (BXD-) strain to strain, further confounding an accurate stem cell analysis. It should be noted that we used the same stromal cell line as was tested by Muller-Sieburg, and that the frequency of C57BL/6 stem cells was very comparable in both studies.


Intrinsic and extrinsic control of hemopoietic stem cell numbers: mapping of a stem cell gene.

de Haan G, Van Zant G - J. Exp. Med. (1997)

Analysis of stem cell frequency  in RI strains. (a) Strain distribution pattern  of the CAFC day 35 frequency in all BXD  strains. Parental values are indicated with  the leftmost (C57BL/6) and rightmost  (DBA) bars. Each value is based on the analysis of pooled marrow cells obtained from  three BXD mice. (b) Likelihood ratio statistic plot showing the results of the interval  mapping on chromosome 18. The threshold  LRS values for suggestive and significant  linkage were 9.9 and 14.7, respectively, and  are indicated. A sharp peak was observed  around D18NcvS22, located ∼19 cM from  the centromere. The corresponding map  position of orthologous genes in human is  shown at the top of the figure. The region  around D18NcvS22 (11–34 cM) shows synteny with human chromosome 5q21-35.
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Related In: Results  -  Collection

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Figure 6: Analysis of stem cell frequency in RI strains. (a) Strain distribution pattern of the CAFC day 35 frequency in all BXD strains. Parental values are indicated with the leftmost (C57BL/6) and rightmost (DBA) bars. Each value is based on the analysis of pooled marrow cells obtained from three BXD mice. (b) Likelihood ratio statistic plot showing the results of the interval mapping on chromosome 18. The threshold LRS values for suggestive and significant linkage were 9.9 and 14.7, respectively, and are indicated. A sharp peak was observed around D18NcvS22, located ∼19 cM from the centromere. The corresponding map position of orthologous genes in human is shown at the top of the figure. The region around D18NcvS22 (11–34 cM) shows synteny with human chromosome 5q21-35.
Mentions: To identify loci affecting the size of the endogenous stem cell pool, we made use of BXD recombinant inbred (RI) strains of mice derived from C57BL/6 (a group 2 strain, Fig. 4) and DBA/2 (a group 3 strain) parental founders. The genome of RI BXD mice consists of a mosaic of homozygous C57BL/6 and DBA/2 chromosomal segments. Typing these mice for a particular phenotype and comparing the obtained SDP with polymorphic markers previously mapped by others in the BXD strains may result in a map position for the trait of interest. In the field of hematology this type of approach has been, for example, successfully employed to characterize the gene involved in IL-3 nonresponsiveness of A/J mice (24). To this end, we quantified CAFC day 35 frequency in all 26 RI strains. DBA parental mice had approximately threefold higher CAFC day 35 frequencies than C57BL/6 mice, but nearly all of the RI strains had CAFC day 35 frequencies which were between C57BL/6 and DBA/2 values (Fig. 6 a). Two notable exceptions were RI strain number 11, which had an unusually high stem cell number, and RI strain 29, which had a very small stem cell compartment. Our data differ quantitatively and qualitatively from those reported recently by Muller-Sieburg et al. (7). We believe that this results from our use of more stringent criteria in the CAFC assay. As has been pointed out by Ploemacher et al. (25), the CAFC assay will only result in reproducible and accurate stem cell measurements if (phase contrast–negative) colonies growing beneath stromal cells are counted. Phase contrast–positive cells accumulating on top of the stroma reflect the mature progeny of primitive cells, and their presence does not indicate a proper cobblestone area. In addition, it is likely, given the different cell cycle status of C57BL/6 and DBA/2 cells, that the half-life of such phase contrast–positive cells differs markedly from (BXD-) strain to strain, further confounding an accurate stem cell analysis. It should be noted that we used the same stromal cell line as was tested by Muller-Sieburg, and that the frequency of C57BL/6 stem cells was very comparable in both studies.

Bottom Line: The population size of primitive stem cells (CAFC day 35) varied widely (up to sevenfold) among strains, unlike total CAFC day 7 numbers (cycling and quiescent), which were similar.Administration of the early acting cytokine flt-3 ligand to these strains resulted in activation of quiescent primitive stem cells exclusively in strains with high endogenous stem cell numbers (DBA and AKR), but was unrelated to strain-specific progenitor cell cycling.Linkage with this genomic interval was associated with a likelihood of odds score of 3.3, surpassing the level required for significance.

View Article: PubMed Central - PubMed

Affiliation: Blood and Marrow Transplant Program, Division of Hematology/Oncology, University of Kentucky Medical Center, Lexington, Kentucky 40536-0093, USA.

ABSTRACT
We evaluated in vivo interactions between extrinsic (growth factor induced) and intrinsic (genetically determined) effectors of mouse primitive hemopoietic stem cell proliferation and numbers. Accordingly, stem cell frequency and cell cycle kinetics were assessed in eight strains of inbred mice using the cobblestone area-forming cell (CAFC) assay. A strong inverse correlation was observed between mouse lifespan and the number of autonomously cycling progenitors (CAFC day 7) in the femur. The population size of primitive stem cells (CAFC day 35) varied widely (up to sevenfold) among strains, unlike total CAFC day 7 numbers (cycling and quiescent), which were similar. Administration of the early acting cytokine flt-3 ligand to these strains resulted in activation of quiescent primitive stem cells exclusively in strains with high endogenous stem cell numbers (DBA and AKR), but was unrelated to strain-specific progenitor cell cycling. To map loci affecting stem cell frequency, we quantified stem cells in BXD recombinant inbred mice (offspring of C57BL/6 and DBA/2). The resulting strain distribution pattern showed high concordance with a marker that mapped to chromosome 18 (19 cM). Linkage with this genomic interval was associated with a likelihood of odds score of 3.3, surpassing the level required for significance. Interestingly, this segment, containing the EGR-1 gene, shows synteny with human chromosome 5q, a region strongly associated with various hematological malignancies. Our findings indicate that a gene mapping to this region is mutated in either C57BL/6 or DBA/2 (and possibly AKR) mice. These studies in apparently healthy mice may facilitate the identification of a gene implicated in human 5q-syndromes.

Show MeSH
Related in: MedlinePlus