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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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Characterization of leukemia-derived iPS cells. (a) The reprogramming procedure of AML cells. Black arrow: medium change. (b) Representative L-iPS colony derived from AML cells cultured with Dox and typical morphology of L-iPS cells after propagation. Scale bars, 100 μm. (c) Immunofluorescence staining showing the expression of pluripotency markers (OCT4, NANOG, SOX2 and SSEA-1) in L-iPS cells. The data represent one of three independent experiments. Scale bars, 20 μm. (d) Representative teratoma from L-iPS cells containing all three germ layers (ectoderm, mesoderm and endoderm). H&E staining. The data represent one of four independent experiments. Scale bars, 50 μm. (e) Blastocyst injection of L-iPS cells generated chimeric mice with high chimerism. The data represent two of ten chimeras. (f) Genomic DNA PCR showing the integration of MLL-AF9 fusion gene in L-iPS cells, demonstrating that all iPS cells tested were derived from the primary AML cells. (g) A Kaplan–Meyer curve showing the survival of chimeric mice. Most chimeras died within 2 months (n=10).(h) FACS analysis of bone marrow cells isolated from diseased chimeras showing the GFP+Mac-1+Gr-1+CD3−B220− phenotype (identical to that in primary leukemia). (i) quantitative fluorescence in situ hybridization analysis showing the MLL-AF9 integrations in R1 cells (negative control), 1° leukemia cells, L-iPS cells and 2° leukemia cells. DAPI, blue; MLL 5′, green; MLL 3′, red. (j) Percent MLL-AF9 integration in 1° leukemia cells, L-iPS cells (2#, 5# and 7#) and 2° leukemia cells (2-1#, 5-1#, 6-1# and 7-3#). 300 cells/ sample were counted.
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fig2: Characterization of leukemia-derived iPS cells. (a) The reprogramming procedure of AML cells. Black arrow: medium change. (b) Representative L-iPS colony derived from AML cells cultured with Dox and typical morphology of L-iPS cells after propagation. Scale bars, 100 μm. (c) Immunofluorescence staining showing the expression of pluripotency markers (OCT4, NANOG, SOX2 and SSEA-1) in L-iPS cells. The data represent one of three independent experiments. Scale bars, 20 μm. (d) Representative teratoma from L-iPS cells containing all three germ layers (ectoderm, mesoderm and endoderm). H&E staining. The data represent one of four independent experiments. Scale bars, 50 μm. (e) Blastocyst injection of L-iPS cells generated chimeric mice with high chimerism. The data represent two of ten chimeras. (f) Genomic DNA PCR showing the integration of MLL-AF9 fusion gene in L-iPS cells, demonstrating that all iPS cells tested were derived from the primary AML cells. (g) A Kaplan–Meyer curve showing the survival of chimeric mice. Most chimeras died within 2 months (n=10).(h) FACS analysis of bone marrow cells isolated from diseased chimeras showing the GFP+Mac-1+Gr-1+CD3−B220− phenotype (identical to that in primary leukemia). (i) quantitative fluorescence in situ hybridization analysis showing the MLL-AF9 integrations in R1 cells (negative control), 1° leukemia cells, L-iPS cells and 2° leukemia cells. DAPI, blue; MLL 5′, green; MLL 3′, red. (j) Percent MLL-AF9 integration in 1° leukemia cells, L-iPS cells (2#, 5# and 7#) and 2° leukemia cells (2-1#, 5-1#, 6-1# and 7-3#). 300 cells/ sample were counted.

Mentions: To assess whether the cells carrying the leukemic gene could be reprogrammed, GFP+ leukemia cells were sorted and plated into six-well plates coated with MEF feeder cells. The leukemia cells were cultured in mouse ES medium containing stem cell factor (50 ng/ml), IL-3 (10 ng/ml), IL-6 (10 ng/ml) and Dox (2 μg/ml) to promote leukemia cell proliferation while reactivating the four Yamanaka reprogramming factors. After 1 week in culture, the leukemia cells were maintained only in the presence of Dox until ES-like colonies appeared (typically 7 days later) before the removal of Dox. ES-like colonies were individually picked up for propagation after 1–2 days (Figure 2a). After propagation, seven L-iPS cell lines that exhibited typical morphology of ES cells (Figure 2b) were randomly selected for further characterization. iPS cells (N-iPS) from non-transduced Lin− BM cells were used as the control. Immunofluorescence experiments revealed positive staining for ES cell markers, including Oct4, Sox2, Nanog and the surface marker SSEA-1, in all seven iPS cell lines (Figure 2c). Reverse transcription-polymerase chain reaction (RT-PCR) and quantitative RT-PCR (qRT-PCR) demonstrated the expression of endogenous pluripotency genes in all seven L-iPS cells, which was comparable to that of N-iPS cells or mouse ES cells (R1) (Supplementary Figures S2A and S2B). Most L-iPS cell lines were predominantly diploid with the normal (40 XY) karyotype (Supplementary Figure S2C). Bisulfite sequencing showed higher levels of demethylation of Oct4 and Nanog promoters in L-iPS cell lines compared with the parental leukemia cells, suggesting epigenetic remodeling during reprogramming (Supplementary Figure S2D).


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)

Characterization of leukemia-derived iPS cells. (a) The reprogramming procedure of AML cells. Black arrow: medium change. (b) Representative L-iPS colony derived from AML cells cultured with Dox and typical morphology of L-iPS cells after propagation. Scale bars, 100 μm. (c) Immunofluorescence staining showing the expression of pluripotency markers (OCT4, NANOG, SOX2 and SSEA-1) in L-iPS cells. The data represent one of three independent experiments. Scale bars, 20 μm. (d) Representative teratoma from L-iPS cells containing all three germ layers (ectoderm, mesoderm and endoderm). H&E staining. The data represent one of four independent experiments. Scale bars, 50 μm. (e) Blastocyst injection of L-iPS cells generated chimeric mice with high chimerism. The data represent two of ten chimeras. (f) Genomic DNA PCR showing the integration of MLL-AF9 fusion gene in L-iPS cells, demonstrating that all iPS cells tested were derived from the primary AML cells. (g) A Kaplan–Meyer curve showing the survival of chimeric mice. Most chimeras died within 2 months (n=10).(h) FACS analysis of bone marrow cells isolated from diseased chimeras showing the GFP+Mac-1+Gr-1+CD3−B220− phenotype (identical to that in primary leukemia). (i) quantitative fluorescence in situ hybridization analysis showing the MLL-AF9 integrations in R1 cells (negative control), 1° leukemia cells, L-iPS cells and 2° leukemia cells. DAPI, blue; MLL 5′, green; MLL 3′, red. (j) Percent MLL-AF9 integration in 1° leukemia cells, L-iPS cells (2#, 5# and 7#) and 2° leukemia cells (2-1#, 5-1#, 6-1# and 7-3#). 300 cells/ sample were counted.
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fig2: Characterization of leukemia-derived iPS cells. (a) The reprogramming procedure of AML cells. Black arrow: medium change. (b) Representative L-iPS colony derived from AML cells cultured with Dox and typical morphology of L-iPS cells after propagation. Scale bars, 100 μm. (c) Immunofluorescence staining showing the expression of pluripotency markers (OCT4, NANOG, SOX2 and SSEA-1) in L-iPS cells. The data represent one of three independent experiments. Scale bars, 20 μm. (d) Representative teratoma from L-iPS cells containing all three germ layers (ectoderm, mesoderm and endoderm). H&E staining. The data represent one of four independent experiments. Scale bars, 50 μm. (e) Blastocyst injection of L-iPS cells generated chimeric mice with high chimerism. The data represent two of ten chimeras. (f) Genomic DNA PCR showing the integration of MLL-AF9 fusion gene in L-iPS cells, demonstrating that all iPS cells tested were derived from the primary AML cells. (g) A Kaplan–Meyer curve showing the survival of chimeric mice. Most chimeras died within 2 months (n=10).(h) FACS analysis of bone marrow cells isolated from diseased chimeras showing the GFP+Mac-1+Gr-1+CD3−B220− phenotype (identical to that in primary leukemia). (i) quantitative fluorescence in situ hybridization analysis showing the MLL-AF9 integrations in R1 cells (negative control), 1° leukemia cells, L-iPS cells and 2° leukemia cells. DAPI, blue; MLL 5′, green; MLL 3′, red. (j) Percent MLL-AF9 integration in 1° leukemia cells, L-iPS cells (2#, 5# and 7#) and 2° leukemia cells (2-1#, 5-1#, 6-1# and 7-3#). 300 cells/ sample were counted.
Mentions: To assess whether the cells carrying the leukemic gene could be reprogrammed, GFP+ leukemia cells were sorted and plated into six-well plates coated with MEF feeder cells. The leukemia cells were cultured in mouse ES medium containing stem cell factor (50 ng/ml), IL-3 (10 ng/ml), IL-6 (10 ng/ml) and Dox (2 μg/ml) to promote leukemia cell proliferation while reactivating the four Yamanaka reprogramming factors. After 1 week in culture, the leukemia cells were maintained only in the presence of Dox until ES-like colonies appeared (typically 7 days later) before the removal of Dox. ES-like colonies were individually picked up for propagation after 1–2 days (Figure 2a). After propagation, seven L-iPS cell lines that exhibited typical morphology of ES cells (Figure 2b) were randomly selected for further characterization. iPS cells (N-iPS) from non-transduced Lin− BM cells were used as the control. Immunofluorescence experiments revealed positive staining for ES cell markers, including Oct4, Sox2, Nanog and the surface marker SSEA-1, in all seven iPS cell lines (Figure 2c). Reverse transcription-polymerase chain reaction (RT-PCR) and quantitative RT-PCR (qRT-PCR) demonstrated the expression of endogenous pluripotency genes in all seven L-iPS cells, which was comparable to that of N-iPS cells or mouse ES cells (R1) (Supplementary Figures S2A and S2B). Most L-iPS cell lines were predominantly diploid with the normal (40 XY) karyotype (Supplementary Figure S2C). Bisulfite sequencing showed higher levels of demethylation of Oct4 and Nanog promoters in L-iPS cell lines compared with the parental leukemia cells, suggesting epigenetic remodeling during reprogramming (Supplementary Figure S2D).

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