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Reprogramming of MLL-AF9 leukemia cells into pluripotent stem cells.

Liu Y, Cheng H, Gao S, Lu X, He F, Hu L, Hou D, Zou Z, Li Y, Zhang H, Xu J, Kang L, Wang Q, Yuan W, Gao S, Cheng T - Leukemia (2013)

Bottom Line: RNA-seq analysis showed reversible global gene expression patterns between these interchangeable leukemia and iPS cells on activation or reactivation of MLL-AF9, suggesting a sufficient epigenetic force in driving the leukemogenic process.This study represents an important step for further defining the potential interplay between oncogenic molecules and reprogramming factors during MLL leukemogenesis.More importantly, our reprogramming approach may be expanded to characterize a range of hematopoietic malignancies in order to develop new strategies for clinical diagnosis and treatment.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, Center for Stem Cell Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China.

ABSTRACT
The 'Yamanaka factors' (Oct4, Sox2, Klf4 and c-Myc) are able to generate induced pluripotent stem (iPS) cells from different cell types. However, to what degree primary malignant cells can be reprogrammed into a pluripotent state has not been vigorously assessed. We established an acute myeloid leukemia (AML) model by overexpressing the human mixed-lineage leukemia-AF9 (MLL-AF9) fusion gene in mouse hematopoietic cells that carry Yamanaka factors under the control of doxycycline (Dox). On addition of Dox to the culture, the transplantable leukemia cells were efficiently converted into iPS cells that could form teratomas and produce chimeras. Interestingly, most chimeric mice spontaneously developed the same type of AML. Moreover, both iPS reprogramming and leukemia reinitiation paths could descend from the same leukemia-initiating cell. RNA-seq analysis showed reversible global gene expression patterns between these interchangeable leukemia and iPS cells on activation or reactivation of MLL-AF9, suggesting a sufficient epigenetic force in driving the leukemogenic process. This study represents an important step for further defining the potential interplay between oncogenic molecules and reprogramming factors during MLL leukemogenesis. More importantly, our reprogramming approach may be expanded to characterize a range of hematopoietic malignancies in order to develop new strategies for clinical diagnosis and treatment.

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Experimental design and establishment of AML. (a) Schematic representation of the strategy used in this study for reprogramming primary AML cells toward iPS. (b) A Kaplan–Meyer curve showing the survival of leukemic mice. All the mice died within 2 months (n=9). (c and d) Representative peripheral blood smear and cytospin of BM cells (Wright–Giemsa staining) showing the accumulation of immature myeloid blast cells in AML mice. Scale bars, 10 μm. (e) FACS analysis of bone marrow cells indicating the GFP+Mac-1+Gr-1+CD3−B220− phenotype of the leukemia cells.
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fig1: Experimental design and establishment of AML. (a) Schematic representation of the strategy used in this study for reprogramming primary AML cells toward iPS. (b) A Kaplan–Meyer curve showing the survival of leukemic mice. All the mice died within 2 months (n=9). (c and d) Representative peripheral blood smear and cytospin of BM cells (Wright–Giemsa staining) showing the accumulation of immature myeloid blast cells in AML mice. Scale bars, 10 μm. (e) FACS analysis of bone marrow cells indicating the GFP+Mac-1+Gr-1+CD3−B220− phenotype of the leukemia cells.

Mentions: Lin− (lineage positive cell-depleted) BM HSPCs were isolated from all-iPS mice20 and transduced with a retrovirus encoding MLL-AF9 fusion gene as described before.27 OSKM factors in these HSPCs could be activated by adding Dox into the culture (Figure 1a). Transduced cells were then transplanted into lethally irradiated mice. The recipients developed leukemia within 2 months (Figure 1b). Moribund mice exhibited elevated white blood cell counts in peripheral blood (Figure 1c) and splenomegaly (Supplementary Figure S1A). Histological analysis of representative tissues and organs showed infiltration of leukemia cells (Supplementary Figure S1B). Cytospin analysis showed that the BM was completely replaced with leukemia cells (Figure 1d). FACS analysis demonstrated abundant cells positive for the GFP and myeloid lineage markers Mac-1 and Gr-1, indicating an AML phenotype (Figure 1e). Moreover, the primary leukemia cells were able to give rise to the same type of AML when injected into sublethally irradiated secondary recipients, showing the transplantablity of the leukemia cells induced by our system (Supplementary Figure S1C).


Reprogramming of MLL-AF9 leukemia cells into pluripotent stem cells.

Liu Y, Cheng H, Gao S, Lu X, He F, Hu L, Hou D, Zou Z, Li Y, Zhang H, Xu J, Kang L, Wang Q, Yuan W, Gao S, Cheng T - Leukemia (2013)

Experimental design and establishment of AML. (a) Schematic representation of the strategy used in this study for reprogramming primary AML cells toward iPS. (b) A Kaplan–Meyer curve showing the survival of leukemic mice. All the mice died within 2 months (n=9). (c and d) Representative peripheral blood smear and cytospin of BM cells (Wright–Giemsa staining) showing the accumulation of immature myeloid blast cells in AML mice. Scale bars, 10 μm. (e) FACS analysis of bone marrow cells indicating the GFP+Mac-1+Gr-1+CD3−B220− phenotype of the leukemia cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4017259&req=5

fig1: Experimental design and establishment of AML. (a) Schematic representation of the strategy used in this study for reprogramming primary AML cells toward iPS. (b) A Kaplan–Meyer curve showing the survival of leukemic mice. All the mice died within 2 months (n=9). (c and d) Representative peripheral blood smear and cytospin of BM cells (Wright–Giemsa staining) showing the accumulation of immature myeloid blast cells in AML mice. Scale bars, 10 μm. (e) FACS analysis of bone marrow cells indicating the GFP+Mac-1+Gr-1+CD3−B220− phenotype of the leukemia cells.
Mentions: Lin− (lineage positive cell-depleted) BM HSPCs were isolated from all-iPS mice20 and transduced with a retrovirus encoding MLL-AF9 fusion gene as described before.27 OSKM factors in these HSPCs could be activated by adding Dox into the culture (Figure 1a). Transduced cells were then transplanted into lethally irradiated mice. The recipients developed leukemia within 2 months (Figure 1b). Moribund mice exhibited elevated white blood cell counts in peripheral blood (Figure 1c) and splenomegaly (Supplementary Figure S1A). Histological analysis of representative tissues and organs showed infiltration of leukemia cells (Supplementary Figure S1B). Cytospin analysis showed that the BM was completely replaced with leukemia cells (Figure 1d). FACS analysis demonstrated abundant cells positive for the GFP and myeloid lineage markers Mac-1 and Gr-1, indicating an AML phenotype (Figure 1e). Moreover, the primary leukemia cells were able to give rise to the same type of AML when injected into sublethally irradiated secondary recipients, showing the transplantablity of the leukemia cells induced by our system (Supplementary Figure S1C).

Bottom Line: RNA-seq analysis showed reversible global gene expression patterns between these interchangeable leukemia and iPS cells on activation or reactivation of MLL-AF9, suggesting a sufficient epigenetic force in driving the leukemogenic process.This study represents an important step for further defining the potential interplay between oncogenic molecules and reprogramming factors during MLL leukemogenesis.More importantly, our reprogramming approach may be expanded to characterize a range of hematopoietic malignancies in order to develop new strategies for clinical diagnosis and treatment.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, Center for Stem Cell Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China.

ABSTRACT
The 'Yamanaka factors' (Oct4, Sox2, Klf4 and c-Myc) are able to generate induced pluripotent stem (iPS) cells from different cell types. However, to what degree primary malignant cells can be reprogrammed into a pluripotent state has not been vigorously assessed. We established an acute myeloid leukemia (AML) model by overexpressing the human mixed-lineage leukemia-AF9 (MLL-AF9) fusion gene in mouse hematopoietic cells that carry Yamanaka factors under the control of doxycycline (Dox). On addition of Dox to the culture, the transplantable leukemia cells were efficiently converted into iPS cells that could form teratomas and produce chimeras. Interestingly, most chimeric mice spontaneously developed the same type of AML. Moreover, both iPS reprogramming and leukemia reinitiation paths could descend from the same leukemia-initiating cell. RNA-seq analysis showed reversible global gene expression patterns between these interchangeable leukemia and iPS cells on activation or reactivation of MLL-AF9, suggesting a sufficient epigenetic force in driving the leukemogenic process. This study represents an important step for further defining the potential interplay between oncogenic molecules and reprogramming factors during MLL leukemogenesis. More importantly, our reprogramming approach may be expanded to characterize a range of hematopoietic malignancies in order to develop new strategies for clinical diagnosis and treatment.

Show MeSH
Related in: MedlinePlus