Supplemental Information
Figure S1. Characterization of primary leukemia
(A) Gross appearance of the spleen showing severe splenomegaly in AML mice.
(B) Representative photomicrographs of the BM, liver, lungs, spleen, brain and
kidneys from AML mice under H&E staining. The BM was completely replaced
by blast cells with large nuclei. Infiltration of leukemia cells was evident in
other tissues and organs. Scale bars, 50 μm.
(C) A Kaplan–Meyer curve showing the survival of secondary leukemic recipient
mice that received 106 primary leukemia cells (n=11).
Figure S2. Characterization of iPS cells
(A) RT-PCR analysis of Oct4, Sox2, Nanog, Klf4 and c-Myc in L-iPS cells.
(B) Comparable mRNA expression level of OCT4, SOX2 and Nanog in L-iPS vs.
N-iPS cells and ES cells.
(C) The L-iPS cell line (L-iPS 2#) showing the normal 40, XY karyotype.
(D) Methylation of the promoter regions of Oct4 and Nanog in leukemia cells,
N-iPS cells and L-iPS cells. Black and white circles indicate methylated and
unmethylated CpGs, respectively.
Figure S3. Single cell clonal assay
(A) Schematic representation of the strategy used in cloning assay. GFP+ primary
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leukemia cells were flow sorted and plated in semisolid medium for 7 days.
Individual AML colonies were plucked from semisolid culture and re-plated
once, and then, after additional 5 days, cells from single colonies were
washed and pooled for secondary transplantation and iPS assay. When the
secondary recipients developed leukemia, GFP+ cells were isolated and
separated for leukemia development and iPS induction (the reprogramming
procedure is the same as described in Figure 2A).
(B) Bright-field images of representative AML colonies in 1st and 2nd plating. Scale
bars, 100 μm.
(C) The list of 7 AML colonies selected for iPS formation and leukemia
development. + or -, Yes or No.
(D) FACS analysis of bone marrow cells isolated from leukemia mouse (colony 5#)
showing the GFP+Mac-1+Gr-1+CD3-B220- phenotype.
(E) Representative an iPS colony derived from 5# AML colony cells cultured with
Dox and typical morphology of iPS cells after propagation. Scale bars, 100
μm.
(F) Immunofluorescence staining showing the expression of pluripotency markers
(OCT4, SOX2, NANOG and SSEA1) in L-iPS cells. The data represent 1 of 3
independent experiments. Scale bars, 20 μm.
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(G) GFP+ transformed leukemia cells from secondary leukemic mice initiated by
4# and 5# colony cells were able to give rise to iPS and leukemia. + or -, Yes
or No.
(H) A Kaplan–Meyer curve showing the survival of tertiary recipient mice (n=7,8).
Figure S4. Leukemia development in chimeric mice
(A) Representative peripheral blood smear (Wright-Giemsa staining) from a
chimeric mouse with signs of illness, showing immature myeloid blast cells in
the circulatory system.
(B) Representative photographs of the spleen in chimeric mice with leukemia vs.
normal mice.
(C) Representative histological sections of the BM, liver, lungs, spleen, kidneys
and brain in chimeric mice with leukemia. H&E staining. Scale bars, 50 μm.
(D) Cytospin of BM cells showing the presence of immature blast cells in chimeric
mice. Scale bars, 10 μm.
Figure S5. Silence of the leukemogenic gene in the iPS cells
(A) RNA-seq result showing the MLL-AF9 fusion gene expression in different
types of cells. MLL-AF9 was only expressed in leukemia cells.
(B) RT-PCR analysis of peripheral blood of chimeras showing that the MLL-AF9
fusion gene could be detected as early as one month after birth.
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Figure S6. Enrichment of MLL-AF9 signature genes in P4 and P5
(A-B) GSEA plots revealing that expression of MLL-AF9 signature genes were
enriched in the P4-down and P5-up (A), while the reverse enrichment was
observed in P4-up and P5-down (B).
Figure S7. Reprogramming of ICN1-overexpressed cells
(A) Morphology (Left panel, 40 x magnification; Right panel, 100 x magnification)
of ES-like colonies induced from T-ALL cells. The procedure is identical to
reprogramming AML cells. MSCV-ICN1-IRES-GFP was used to transform
HSPCs to leukemia cells. GFP (or ICN1) was not silenced in ES-like colonies,
suggesting that the leukemia cells were partially reprogrammed. The colonies
cannot be passaged successfully. Scale bars, 100 μm. (ICN1: intracellular
domain of Notch1 )
(B) AP staining of ES-like colonies induced from ICN1-overexpressed MEF cells.
MEF cells were infected with GFP vector or ICN1 and 4 ‘Yamanaka factors’,
and cultured in the ES medium.
(C) Colony formation of ICN1-overexpressed MEF cells, relative to the control.
The
result
indicates
that
ICN1-overexpression
reprogramming. Bars represent Mean ± S.D.
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could
suppress
the
Table S4. RT-PCR primers
Gene
Sequence (5’-3’)
Forward
Reverse
Gapdh
CGGAGTCAACGGATTTGGTCGTAT AGCCTTCTCCATGGTGGTGAAGAC
Sox2
GCGGAGTGGAAACTTTTGTCC
CGGGAAGCGTGTACTTATCCTT
Oct4
GAAGCAGAAGAGGATCACCTTG
TTCTTAAGGCTGAGCTGCAAG
Nanog
CCTCAGCCTCCAGCAGATGC
CCGCTTGCACTTCACCCTTTG
Klf4
TGATGGTGCTTGGTGAGTTG
TTGCACATCTGAAACCACAG
c-Myc
TCTCCATCCTATGTTGCGGTC
TCCAAGTAACTCGGTCATCATCT
AACCACCTCCGGTCAATAAGC
TTCACGATCTGCTGCAGAATG
MLL-AF9
Table S5. Real-time PCR primers
Gene
Sequence of primers (5’-3’)
Forward
Reverse
Oct4
ATCACTCACATCGCCAATCA
TGGGAAAGGTGTCCCTGTAG
Nanog
AAGCCACTAGGGAAAGCCAT
GGAAGAAGGAAGGAACCTGG
Sox2
AAGGGTTCTTGCTGGGTTTT
AGACCACGAAAACGGTCTTG
Gapdh
CGGAGTCAACGGATTTGGTCGTAT AGCCTTCTCCATGGTGGTGAAGAC
MLL-AF9
AACCACCTCCGGTCAATAAGC
5
TTCACGATCTGCTGCAGAATG
Table S6. Two-round nested PCR primers
Gene
Sequence (5’-3’)
Forward
Reverse
Oct4-outside
GAGGATTGGAGGTGTAATGGTTGTT
CTACTAACCCATCACCCCCACCTA
Oct4-inside
TGGGTTGAAATATTGGGTTTATTT
CTAAAACCAAATATCCAACCATA
Nanog-outside AAGTATGGATTAATTTATTAAGGTAGTT AAAAAACCCACACTCATATCAATATA
Nanog-inside
AAGTATGGATTAATTTATTAAGGTAGTT CAACCAAATAACCTATCTAAAAA
MLL-AF9
TGTAGGTTTGGTAAGTTAGTTTAAGT
CTTTTATTAAACTCRAAAAACAAA
CGGTTTAGGGTTAAGAATAGATGG
CACTCAAAAAAAACCCTCCCAA
outside
MLL-AF9
inside
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