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Establishment of a humanized APL model via the transplantation of PML-RARA-transduced human common myeloid progenitors into immunodeficient mice.

Matsushita H, Yahata T, Sheng Y, Nakamura Y, Muguruma Y, Matsuzawa H, Tanaka M, Hayashi H, Sato T, Damdinsuren A, Onizuka M, Ito M, Miyachi H, Pandolfi PP, Ando K - PLoS ONE (2014)

Bottom Line: The leukemia well recapitulated human APL, consisting of leukemic cells with abundant azurophilic abnormal granules in the cytoplasm, which expressed CD13, CD33 and CD117, but not HLA-DR and CD34, were clustered in the same category as human APL samples in the gene expression analysis, and demonstrated sensitivity to ATRA.In order to analyze the mechanisms underlying APL initiation and development, fractionated human cord blood was transduced with PML-RARA.Common myeloid progenitors (CMP) from CD34(+)/CD38(+) cells developed APL.

View Article: PubMed Central - PubMed

Affiliation: Research Center for Cancer Stem Cell, Tokai University School of Medicine, Isehara, Kanagawa, Japan; Medical Research Institute, Tokai University, Isehara, Kanagawa, Japan; Department of Laboratory Medicine, Tokai University School of Medicine, Isehara, Kanagawa, Japan.

ABSTRACT
Recent advances in cancer biology have revealed that many malignancies possess a hierarchal system, and leukemic stem cells (LSC) or leukemia-initiating cells (LIC) appear to be obligatory for disease progression. Acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia characterized by the formation of a PML-RARα fusion protein, leads to the accumulation of abnormal promyelocytes. In order to understand the precise mechanisms involved in human APL leukemogenesis, we established a humanized in vivo APL model involving retroviral transduction of PML-RARA into CD34(+) hematopoietic cells from human cord blood and transplantation of these cells into immunodeficient mice. The leukemia well recapitulated human APL, consisting of leukemic cells with abundant azurophilic abnormal granules in the cytoplasm, which expressed CD13, CD33 and CD117, but not HLA-DR and CD34, were clustered in the same category as human APL samples in the gene expression analysis, and demonstrated sensitivity to ATRA. As seen in human APL, the induced APL cells showed a low transplantation efficiency in the secondary recipients, which was also exhibited in the transplantations that were carried out using the sorted CD34- fraction. In order to analyze the mechanisms underlying APL initiation and development, fractionated human cord blood was transduced with PML-RARA. Common myeloid progenitors (CMP) from CD34(+)/CD38(+) cells developed APL. These findings demonstrate that CMP are a target fraction for PML-RARA in APL, whereas the resultant CD34(-) APL cells may share the ability to maintain the tumor.

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PML-RARA targeted human common myeloid progenitors for APL leukemogenesis.(A) The sorting strategy for CD34+/CD38−, CD34+/CD38+ and CD34−/CD33+ cells. Human cord blood was first separated into CD34+ and CD34− cells by magnetic beads, and then sorted into three fractions by a FACS vantage instrument. (B) The expression of PML-RARA mRNA in each of the fractions after retroviral transfection. B2M, beta 2 microglobulin. The PML-RARA expression vector was used as a positive control for the PML-RARA analysis. (C) A colony-forming assay using PML-RARA-transduced CD34+/CD38+ and CD34+/CD38− cells. The average of three independent experiments is shown. The data represent the means ± SD. (D) The total numbers of colonies of PML-RARA-transduced CD34+/CD38+ and CD34+/CD38− cells shown in (C) are highlighted. The data represent the means ± SD (n = 3). (E) The development of the induced APL from CD34+/CD38+ cells in NOG mice. Each sorted fraction from human cord blood, as seen in (A), was retrovirally transduced with PML-RARA and transplanted into irradiated NOG mice. The percentages were determined by the frequency of EGFP+/CD45+/CD33+ cells at 16 to 20 weeks after transplantation. Each dot represents a single mouse. The horizontal line represents the median value. (F) The sorting strategy for common myeloid progenitors (CMP), granulocyte-monocytic progenitors (GMP), and megakaryocyte-erythrocyte progenitors (MEP). Human cord blood was separated into CD34+ cells by magnetic beads, CD34+/CD38+ cells were sorted out, and were finally divided into CMP, GMP and MEP by the FACS vantage instrument. (G) The transduction efficiency of PML-RARA in CMP, GMP and MEP. Representative data are shown. (H) The development of the induced APL from the human hematopoietic progenitors in NOG mice. Each sorted progenitor fraction from human cord blood, as seen in (F), was retrovirally transduced with PML-RARA and transplanted into irradiated NOG mice. The percentages were determined by the frequency of EGFP+/CD45+/CD33+ cells at 16 to 20 weeks after transplantation. Each dot represents a single mouse. The horizontal line represents the median value.
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pone-0111082-g006: PML-RARA targeted human common myeloid progenitors for APL leukemogenesis.(A) The sorting strategy for CD34+/CD38−, CD34+/CD38+ and CD34−/CD33+ cells. Human cord blood was first separated into CD34+ and CD34− cells by magnetic beads, and then sorted into three fractions by a FACS vantage instrument. (B) The expression of PML-RARA mRNA in each of the fractions after retroviral transfection. B2M, beta 2 microglobulin. The PML-RARA expression vector was used as a positive control for the PML-RARA analysis. (C) A colony-forming assay using PML-RARA-transduced CD34+/CD38+ and CD34+/CD38− cells. The average of three independent experiments is shown. The data represent the means ± SD. (D) The total numbers of colonies of PML-RARA-transduced CD34+/CD38+ and CD34+/CD38− cells shown in (C) are highlighted. The data represent the means ± SD (n = 3). (E) The development of the induced APL from CD34+/CD38+ cells in NOG mice. Each sorted fraction from human cord blood, as seen in (A), was retrovirally transduced with PML-RARA and transplanted into irradiated NOG mice. The percentages were determined by the frequency of EGFP+/CD45+/CD33+ cells at 16 to 20 weeks after transplantation. Each dot represents a single mouse. The horizontal line represents the median value. (F) The sorting strategy for common myeloid progenitors (CMP), granulocyte-monocytic progenitors (GMP), and megakaryocyte-erythrocyte progenitors (MEP). Human cord blood was separated into CD34+ cells by magnetic beads, CD34+/CD38+ cells were sorted out, and were finally divided into CMP, GMP and MEP by the FACS vantage instrument. (G) The transduction efficiency of PML-RARA in CMP, GMP and MEP. Representative data are shown. (H) The development of the induced APL from the human hematopoietic progenitors in NOG mice. Each sorted progenitor fraction from human cord blood, as seen in (F), was retrovirally transduced with PML-RARA and transplanted into irradiated NOG mice. The percentages were determined by the frequency of EGFP+/CD45+/CD33+ cells at 16 to 20 weeks after transplantation. Each dot represents a single mouse. The horizontal line represents the median value.

Mentions: The findings that the PML-RARA transduced-CD34+ cells developed APL while the resultant CD34− APL cells exhibited transplantability indicate the possibility that the initiation and maintenance of APL arise at different steps of differentiation, which are not likely to involve the CD34+/CD38− fraction, as originally reported in human AML. Therefore, in order to identify a cellular target for PML-RARA that effectively develops APL, PML-RARA was transduced into fractionated cells: CD34+/CD38−, CD34+/CD38+ and CD34−/CD33+ cells from the cord blood (Figures 6A and 6B). The transduction efficiency, as evaluated by EGFP expression, ranged from 1.9% to 5.0% (median: 3.53%, n = 6) in CD34+/CD38− cells, 4.5% to 10.6% (median: 10.07%, n = 6) in CD34+/CD38+ cells and 19.1% to 22.1% (median: 20.63%, n = 4) in CD34−/CD33+ cells. Because the CD34+ fraction from human cord blood possessed a higher proportion of CD34+/CD38+ (74.5% to 94.2%) than that of CD34+/CD38− cells, the presumed absolute number of PML-RARA transplanted cells was higher in CD34+/CD38+ cells than in CD34+/CD38− cells (3,430 to 31,800 cells vs 140 to 450 cells per mouse; 22,900 to 27,700 CD34−/CD33+ cells). One hundred unfractionated human CD34+ cells, including both CD34+/CD38− and CD34+/CD38+ cells, were engrafted with multilineage differentiation in our previous study [10], thus suggesting that the transplanted cell numbers were adequate for engraftment in the NOG mice. The induction of PML-RARA in CD34+/CD38+ cells reduced the colony formation capacity and favored the formation of myeloid colonies, as seen in CD34+ cells (Figures 1B and 1C). On the other hand, the induction of PML-RARA in CD34+/CD38− cells generated very few colonies in comparison to the MIGR1 control vector-infected CD34+/CD38− cells (Figures 6C and 6D). Consistent with the results, the induced APL cells were detected mostly in the mice transplanted with CD34+/CD38+ cells (median, 16.4% in the whole bone marrow cells) (Figure 6E). These findings suggest that CD34+/CD38+ progenitors proliferate and survive more efficiently than CD34+/CD38− cells in vitro and trigger APL in vivo by inducing PML-RARA.


Establishment of a humanized APL model via the transplantation of PML-RARA-transduced human common myeloid progenitors into immunodeficient mice.

Matsushita H, Yahata T, Sheng Y, Nakamura Y, Muguruma Y, Matsuzawa H, Tanaka M, Hayashi H, Sato T, Damdinsuren A, Onizuka M, Ito M, Miyachi H, Pandolfi PP, Ando K - PLoS ONE (2014)

PML-RARA targeted human common myeloid progenitors for APL leukemogenesis.(A) The sorting strategy for CD34+/CD38−, CD34+/CD38+ and CD34−/CD33+ cells. Human cord blood was first separated into CD34+ and CD34− cells by magnetic beads, and then sorted into three fractions by a FACS vantage instrument. (B) The expression of PML-RARA mRNA in each of the fractions after retroviral transfection. B2M, beta 2 microglobulin. The PML-RARA expression vector was used as a positive control for the PML-RARA analysis. (C) A colony-forming assay using PML-RARA-transduced CD34+/CD38+ and CD34+/CD38− cells. The average of three independent experiments is shown. The data represent the means ± SD. (D) The total numbers of colonies of PML-RARA-transduced CD34+/CD38+ and CD34+/CD38− cells shown in (C) are highlighted. The data represent the means ± SD (n = 3). (E) The development of the induced APL from CD34+/CD38+ cells in NOG mice. Each sorted fraction from human cord blood, as seen in (A), was retrovirally transduced with PML-RARA and transplanted into irradiated NOG mice. The percentages were determined by the frequency of EGFP+/CD45+/CD33+ cells at 16 to 20 weeks after transplantation. Each dot represents a single mouse. The horizontal line represents the median value. (F) The sorting strategy for common myeloid progenitors (CMP), granulocyte-monocytic progenitors (GMP), and megakaryocyte-erythrocyte progenitors (MEP). Human cord blood was separated into CD34+ cells by magnetic beads, CD34+/CD38+ cells were sorted out, and were finally divided into CMP, GMP and MEP by the FACS vantage instrument. (G) The transduction efficiency of PML-RARA in CMP, GMP and MEP. Representative data are shown. (H) The development of the induced APL from the human hematopoietic progenitors in NOG mice. Each sorted progenitor fraction from human cord blood, as seen in (F), was retrovirally transduced with PML-RARA and transplanted into irradiated NOG mice. The percentages were determined by the frequency of EGFP+/CD45+/CD33+ cells at 16 to 20 weeks after transplantation. Each dot represents a single mouse. The horizontal line represents the median value.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4219701&req=5

pone-0111082-g006: PML-RARA targeted human common myeloid progenitors for APL leukemogenesis.(A) The sorting strategy for CD34+/CD38−, CD34+/CD38+ and CD34−/CD33+ cells. Human cord blood was first separated into CD34+ and CD34− cells by magnetic beads, and then sorted into three fractions by a FACS vantage instrument. (B) The expression of PML-RARA mRNA in each of the fractions after retroviral transfection. B2M, beta 2 microglobulin. The PML-RARA expression vector was used as a positive control for the PML-RARA analysis. (C) A colony-forming assay using PML-RARA-transduced CD34+/CD38+ and CD34+/CD38− cells. The average of three independent experiments is shown. The data represent the means ± SD. (D) The total numbers of colonies of PML-RARA-transduced CD34+/CD38+ and CD34+/CD38− cells shown in (C) are highlighted. The data represent the means ± SD (n = 3). (E) The development of the induced APL from CD34+/CD38+ cells in NOG mice. Each sorted fraction from human cord blood, as seen in (A), was retrovirally transduced with PML-RARA and transplanted into irradiated NOG mice. The percentages were determined by the frequency of EGFP+/CD45+/CD33+ cells at 16 to 20 weeks after transplantation. Each dot represents a single mouse. The horizontal line represents the median value. (F) The sorting strategy for common myeloid progenitors (CMP), granulocyte-monocytic progenitors (GMP), and megakaryocyte-erythrocyte progenitors (MEP). Human cord blood was separated into CD34+ cells by magnetic beads, CD34+/CD38+ cells were sorted out, and were finally divided into CMP, GMP and MEP by the FACS vantage instrument. (G) The transduction efficiency of PML-RARA in CMP, GMP and MEP. Representative data are shown. (H) The development of the induced APL from the human hematopoietic progenitors in NOG mice. Each sorted progenitor fraction from human cord blood, as seen in (F), was retrovirally transduced with PML-RARA and transplanted into irradiated NOG mice. The percentages were determined by the frequency of EGFP+/CD45+/CD33+ cells at 16 to 20 weeks after transplantation. Each dot represents a single mouse. The horizontal line represents the median value.
Mentions: The findings that the PML-RARA transduced-CD34+ cells developed APL while the resultant CD34− APL cells exhibited transplantability indicate the possibility that the initiation and maintenance of APL arise at different steps of differentiation, which are not likely to involve the CD34+/CD38− fraction, as originally reported in human AML. Therefore, in order to identify a cellular target for PML-RARA that effectively develops APL, PML-RARA was transduced into fractionated cells: CD34+/CD38−, CD34+/CD38+ and CD34−/CD33+ cells from the cord blood (Figures 6A and 6B). The transduction efficiency, as evaluated by EGFP expression, ranged from 1.9% to 5.0% (median: 3.53%, n = 6) in CD34+/CD38− cells, 4.5% to 10.6% (median: 10.07%, n = 6) in CD34+/CD38+ cells and 19.1% to 22.1% (median: 20.63%, n = 4) in CD34−/CD33+ cells. Because the CD34+ fraction from human cord blood possessed a higher proportion of CD34+/CD38+ (74.5% to 94.2%) than that of CD34+/CD38− cells, the presumed absolute number of PML-RARA transplanted cells was higher in CD34+/CD38+ cells than in CD34+/CD38− cells (3,430 to 31,800 cells vs 140 to 450 cells per mouse; 22,900 to 27,700 CD34−/CD33+ cells). One hundred unfractionated human CD34+ cells, including both CD34+/CD38− and CD34+/CD38+ cells, were engrafted with multilineage differentiation in our previous study [10], thus suggesting that the transplanted cell numbers were adequate for engraftment in the NOG mice. The induction of PML-RARA in CD34+/CD38+ cells reduced the colony formation capacity and favored the formation of myeloid colonies, as seen in CD34+ cells (Figures 1B and 1C). On the other hand, the induction of PML-RARA in CD34+/CD38− cells generated very few colonies in comparison to the MIGR1 control vector-infected CD34+/CD38− cells (Figures 6C and 6D). Consistent with the results, the induced APL cells were detected mostly in the mice transplanted with CD34+/CD38+ cells (median, 16.4% in the whole bone marrow cells) (Figure 6E). These findings suggest that CD34+/CD38+ progenitors proliferate and survive more efficiently than CD34+/CD38− cells in vitro and trigger APL in vivo by inducing PML-RARA.

Bottom Line: The leukemia well recapitulated human APL, consisting of leukemic cells with abundant azurophilic abnormal granules in the cytoplasm, which expressed CD13, CD33 and CD117, but not HLA-DR and CD34, were clustered in the same category as human APL samples in the gene expression analysis, and demonstrated sensitivity to ATRA.In order to analyze the mechanisms underlying APL initiation and development, fractionated human cord blood was transduced with PML-RARA.Common myeloid progenitors (CMP) from CD34(+)/CD38(+) cells developed APL.

View Article: PubMed Central - PubMed

Affiliation: Research Center for Cancer Stem Cell, Tokai University School of Medicine, Isehara, Kanagawa, Japan; Medical Research Institute, Tokai University, Isehara, Kanagawa, Japan; Department of Laboratory Medicine, Tokai University School of Medicine, Isehara, Kanagawa, Japan.

ABSTRACT
Recent advances in cancer biology have revealed that many malignancies possess a hierarchal system, and leukemic stem cells (LSC) or leukemia-initiating cells (LIC) appear to be obligatory for disease progression. Acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia characterized by the formation of a PML-RARα fusion protein, leads to the accumulation of abnormal promyelocytes. In order to understand the precise mechanisms involved in human APL leukemogenesis, we established a humanized in vivo APL model involving retroviral transduction of PML-RARA into CD34(+) hematopoietic cells from human cord blood and transplantation of these cells into immunodeficient mice. The leukemia well recapitulated human APL, consisting of leukemic cells with abundant azurophilic abnormal granules in the cytoplasm, which expressed CD13, CD33 and CD117, but not HLA-DR and CD34, were clustered in the same category as human APL samples in the gene expression analysis, and demonstrated sensitivity to ATRA. As seen in human APL, the induced APL cells showed a low transplantation efficiency in the secondary recipients, which was also exhibited in the transplantations that were carried out using the sorted CD34- fraction. In order to analyze the mechanisms underlying APL initiation and development, fractionated human cord blood was transduced with PML-RARA. Common myeloid progenitors (CMP) from CD34(+)/CD38(+) cells developed APL. These findings demonstrate that CMP are a target fraction for PML-RARA in APL, whereas the resultant CD34(-) APL cells may share the ability to maintain the tumor.

Show MeSH
Related in: MedlinePlus