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Aging of the microenvironment influences clonality in hematopoiesis.

Vas V, Senger K, Dörr K, Niebel A, Geiger H - PLoS ONE (2012)

Bottom Line: Based on a retroviral-insertion mutagenesis approach to generate primitive hematopoietic cells with an intrinsic potential for clonal expansion, we determined clonality of transduced hematopoietic progenitor cells (HPCs) exposed to a young or aged microenvironment in vivo.While HPCs displayed primarily oligo-clonality within a young microenvironment, aged animals transplanted with identical pool of cells displayed reduced clonality within transduced HPCs.Our data show that an aged niche exerts a distinct selection pressure on dominant HPC-clones thus facilitating the transition to mono-clonality, which might be one underlying cause for the increased age-associated incidence of leukemia.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology and Allergic Diseases, University of Ulm, Ulm, Germany.

ABSTRACT
The mechanisms of the age-associated exponential increase in the incidence of leukemia are not known in detail. Leukemia as well as aging are initiated and regulated in multi-factorial fashion by cell-intrinsic and extrinsic factors. The role of aging of the microenvironment for leukemia initiation/progression has not been investigated in great detail so far. Clonality in hematopoiesis is tightly linked to the initiation of leukemia. Based on a retroviral-insertion mutagenesis approach to generate primitive hematopoietic cells with an intrinsic potential for clonal expansion, we determined clonality of transduced hematopoietic progenitor cells (HPCs) exposed to a young or aged microenvironment in vivo. While HPCs displayed primarily oligo-clonality within a young microenvironment, aged animals transplanted with identical pool of cells displayed reduced clonality within transduced HPCs. Our data show that an aged niche exerts a distinct selection pressure on dominant HPC-clones thus facilitating the transition to mono-clonality, which might be one underlying cause for the increased age-associated incidence of leukemia.

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Related in: MedlinePlus

Coexistence of balanced HSC populations is characteristic for young microenvironment whereas an aged microenvironment favors the expansion of single dominant hematopoietic clone.(A) Schematic representation of the retroviral insertional mutagenesis screen. DNA was extracted from individual CFCs (average: 7.3±1.8 GFP+CFCs/mouse were isolated) and digested with four-cutter enzymes. After LM-PCR, the recovered bands were isolated and sequenced. The sequences were aligned to the mouse genome with NCBI/BLAST program. The RISs were identified and the closest genes (within a ±100 kb window) were listed. Based on the LM-PCR band-pattern and the identified RISs, clonal analyses were performed. (B) Representative agarose gel analysis of LM-PCR performed on methylcellulose colonies isolated from BM of one representative young transplanted mouse. Clonality was identified based on the band pattern and DNA sequence information on individual bands. Distinct colors represent distinct clones. (C) Insertion sites recovered by LM-PCR from CFCs of one representative aged recipient mouse. The red arrows depict distinct GFP peaks. i.c.: internal control bands represent PCR products amplified from the viral genome. (D) Distribution of clonality within the GFP+ CFC population of young and aged recipient mice determined by the LM-PCR pattern and sequence information. (E) Flow cytometric analyses of PB of young and aged recipient mice (same mice were used in clonal analysis and RISs identification). Myeloid cells were identified based on Gr1 and Mac1 expression and B220 was used as B cell marker. (young mice n = 6, aged mice n = 5, from 3 independent experiments), * = p<0.05 (F) Proposed model for clonality of hematopoiesis in young and aged microenvironment.
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pone-0042080-g002: Coexistence of balanced HSC populations is characteristic for young microenvironment whereas an aged microenvironment favors the expansion of single dominant hematopoietic clone.(A) Schematic representation of the retroviral insertional mutagenesis screen. DNA was extracted from individual CFCs (average: 7.3±1.8 GFP+CFCs/mouse were isolated) and digested with four-cutter enzymes. After LM-PCR, the recovered bands were isolated and sequenced. The sequences were aligned to the mouse genome with NCBI/BLAST program. The RISs were identified and the closest genes (within a ±100 kb window) were listed. Based on the LM-PCR band-pattern and the identified RISs, clonal analyses were performed. (B) Representative agarose gel analysis of LM-PCR performed on methylcellulose colonies isolated from BM of one representative young transplanted mouse. Clonality was identified based on the band pattern and DNA sequence information on individual bands. Distinct colors represent distinct clones. (C) Insertion sites recovered by LM-PCR from CFCs of one representative aged recipient mouse. The red arrows depict distinct GFP peaks. i.c.: internal control bands represent PCR products amplified from the viral genome. (D) Distribution of clonality within the GFP+ CFC population of young and aged recipient mice determined by the LM-PCR pattern and sequence information. (E) Flow cytometric analyses of PB of young and aged recipient mice (same mice were used in clonal analysis and RISs identification). Myeloid cells were identified based on Gr1 and Mac1 expression and B220 was used as B cell marker. (young mice n = 6, aged mice n = 5, from 3 independent experiments), * = p<0.05 (F) Proposed model for clonality of hematopoiesis in young and aged microenvironment.

Mentions: To further investigate clonality in more primitive hematopoietic cells exposed to a young or aged microenvironment, retroviral insertion sites (RISs) from individual GFP+ colony forming cells (CFCs) from BM were amplified by ligation-mediated PCR and genomic location of RISs were determined (Figure 1A). The gel-band pattern of the LM-PCR reaction, in combination with the sequence information from distinct bands were used to determine the frequency of distinct clones in the myeloid progenitor-cell compartment (Figure 2A). As the same pool of transduced graft cells were transplanted into young and aged animals, differences in the two experimental groups in hematopoiesis result primarily from the distinct age of the microenvironment. In young mice, hematopoiesis within the GFP+CFCs remained in the majority oligoclonal as indicated by distinct banding patterns among individual CFC-clones and confirmed by sequencing (Figure 2B,D). GFP+CFCs from individual aged recipients presented under the LM-PCR protocol used without exception a monoclonal pattern (Figure 2C,D). The difference in the clonality pattern among the myeloid stem/progenitor population between young and aged animals was significant (p = 0.035). The LM-PCR method was sensitive enough to demonstrate minor clone contributing down to least 10% of the transduced population (Figure 2D), while the restriction enzyme used for LM-PCR analysis will cover at least 85% of all possible integration sites [20], implying that the observed mono-clonality in CFC colonies might represent in a good number of cases true monoclonality in vivo.


Aging of the microenvironment influences clonality in hematopoiesis.

Vas V, Senger K, Dörr K, Niebel A, Geiger H - PLoS ONE (2012)

Coexistence of balanced HSC populations is characteristic for young microenvironment whereas an aged microenvironment favors the expansion of single dominant hematopoietic clone.(A) Schematic representation of the retroviral insertional mutagenesis screen. DNA was extracted from individual CFCs (average: 7.3±1.8 GFP+CFCs/mouse were isolated) and digested with four-cutter enzymes. After LM-PCR, the recovered bands were isolated and sequenced. The sequences were aligned to the mouse genome with NCBI/BLAST program. The RISs were identified and the closest genes (within a ±100 kb window) were listed. Based on the LM-PCR band-pattern and the identified RISs, clonal analyses were performed. (B) Representative agarose gel analysis of LM-PCR performed on methylcellulose colonies isolated from BM of one representative young transplanted mouse. Clonality was identified based on the band pattern and DNA sequence information on individual bands. Distinct colors represent distinct clones. (C) Insertion sites recovered by LM-PCR from CFCs of one representative aged recipient mouse. The red arrows depict distinct GFP peaks. i.c.: internal control bands represent PCR products amplified from the viral genome. (D) Distribution of clonality within the GFP+ CFC population of young and aged recipient mice determined by the LM-PCR pattern and sequence information. (E) Flow cytometric analyses of PB of young and aged recipient mice (same mice were used in clonal analysis and RISs identification). Myeloid cells were identified based on Gr1 and Mac1 expression and B220 was used as B cell marker. (young mice n = 6, aged mice n = 5, from 3 independent experiments), * = p<0.05 (F) Proposed model for clonality of hematopoiesis in young and aged microenvironment.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3412859&req=5

pone-0042080-g002: Coexistence of balanced HSC populations is characteristic for young microenvironment whereas an aged microenvironment favors the expansion of single dominant hematopoietic clone.(A) Schematic representation of the retroviral insertional mutagenesis screen. DNA was extracted from individual CFCs (average: 7.3±1.8 GFP+CFCs/mouse were isolated) and digested with four-cutter enzymes. After LM-PCR, the recovered bands were isolated and sequenced. The sequences were aligned to the mouse genome with NCBI/BLAST program. The RISs were identified and the closest genes (within a ±100 kb window) were listed. Based on the LM-PCR band-pattern and the identified RISs, clonal analyses were performed. (B) Representative agarose gel analysis of LM-PCR performed on methylcellulose colonies isolated from BM of one representative young transplanted mouse. Clonality was identified based on the band pattern and DNA sequence information on individual bands. Distinct colors represent distinct clones. (C) Insertion sites recovered by LM-PCR from CFCs of one representative aged recipient mouse. The red arrows depict distinct GFP peaks. i.c.: internal control bands represent PCR products amplified from the viral genome. (D) Distribution of clonality within the GFP+ CFC population of young and aged recipient mice determined by the LM-PCR pattern and sequence information. (E) Flow cytometric analyses of PB of young and aged recipient mice (same mice were used in clonal analysis and RISs identification). Myeloid cells were identified based on Gr1 and Mac1 expression and B220 was used as B cell marker. (young mice n = 6, aged mice n = 5, from 3 independent experiments), * = p<0.05 (F) Proposed model for clonality of hematopoiesis in young and aged microenvironment.
Mentions: To further investigate clonality in more primitive hematopoietic cells exposed to a young or aged microenvironment, retroviral insertion sites (RISs) from individual GFP+ colony forming cells (CFCs) from BM were amplified by ligation-mediated PCR and genomic location of RISs were determined (Figure 1A). The gel-band pattern of the LM-PCR reaction, in combination with the sequence information from distinct bands were used to determine the frequency of distinct clones in the myeloid progenitor-cell compartment (Figure 2A). As the same pool of transduced graft cells were transplanted into young and aged animals, differences in the two experimental groups in hematopoiesis result primarily from the distinct age of the microenvironment. In young mice, hematopoiesis within the GFP+CFCs remained in the majority oligoclonal as indicated by distinct banding patterns among individual CFC-clones and confirmed by sequencing (Figure 2B,D). GFP+CFCs from individual aged recipients presented under the LM-PCR protocol used without exception a monoclonal pattern (Figure 2C,D). The difference in the clonality pattern among the myeloid stem/progenitor population between young and aged animals was significant (p = 0.035). The LM-PCR method was sensitive enough to demonstrate minor clone contributing down to least 10% of the transduced population (Figure 2D), while the restriction enzyme used for LM-PCR analysis will cover at least 85% of all possible integration sites [20], implying that the observed mono-clonality in CFC colonies might represent in a good number of cases true monoclonality in vivo.

Bottom Line: Based on a retroviral-insertion mutagenesis approach to generate primitive hematopoietic cells with an intrinsic potential for clonal expansion, we determined clonality of transduced hematopoietic progenitor cells (HPCs) exposed to a young or aged microenvironment in vivo.While HPCs displayed primarily oligo-clonality within a young microenvironment, aged animals transplanted with identical pool of cells displayed reduced clonality within transduced HPCs.Our data show that an aged niche exerts a distinct selection pressure on dominant HPC-clones thus facilitating the transition to mono-clonality, which might be one underlying cause for the increased age-associated incidence of leukemia.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology and Allergic Diseases, University of Ulm, Ulm, Germany.

ABSTRACT
The mechanisms of the age-associated exponential increase in the incidence of leukemia are not known in detail. Leukemia as well as aging are initiated and regulated in multi-factorial fashion by cell-intrinsic and extrinsic factors. The role of aging of the microenvironment for leukemia initiation/progression has not been investigated in great detail so far. Clonality in hematopoiesis is tightly linked to the initiation of leukemia. Based on a retroviral-insertion mutagenesis approach to generate primitive hematopoietic cells with an intrinsic potential for clonal expansion, we determined clonality of transduced hematopoietic progenitor cells (HPCs) exposed to a young or aged microenvironment in vivo. While HPCs displayed primarily oligo-clonality within a young microenvironment, aged animals transplanted with identical pool of cells displayed reduced clonality within transduced HPCs. Our data show that an aged niche exerts a distinct selection pressure on dominant HPC-clones thus facilitating the transition to mono-clonality, which might be one underlying cause for the increased age-associated incidence of leukemia.

Show MeSH
Related in: MedlinePlus