Supplementary Materials and Methods
1. FACS sorting of HSC and progenitors
For the detection of HSC and progenitors from CMLCP, the mononuclear cells were
freshlyprepared within 24 hrs after bone marrow harvest. Mononuclear cellswere stained
with lineage-associated PE-Cy5.5-conjugatedantibodies including CD2, CD3, CD4, CD8,
CD14, CD19,CD20 and CD56 from Caltag (South San Francisco, CA).Flow-cytometric
analyses and cell sorting were performed aspreviously published (Abe et al., Int J Hematol
2008; 88 and Jamieson et al., New Engl J Med 2004; 351). The cells with the lineagecocktail
antibodies were further incubated either withHSC-associated antibodies consisting of
APC-conjugatedanti-CD34 (HPCA-2; BD Pharmingen, San Diego,
CA),PE-Cy7-conjugated anti-CD38 (BD Pharmingen), FITC-labeled CD47
andphycoerythrin-conjugated anti-CD90 (Thy-1) or with progenitor-associated antibodies
consisting of APC-conjugatedanti-CD34, PE-Cy7-conjugated
anti-CD38,phycoerythrin-conjugated anti-IL-3 receptor (9F5; BDPharmingen) and
FITC-conjugated anti-CD45RA (MEM56;Caltag).Unstained samples and isotype controls
were included toassess background fluorescence. After staining, cells wereanalyzed and
sorted using FACSAria (BD ImmunocytometrySystems, San Jose, CA). HSC identified
asCD34+CD38-Lin-, were separated into Thy-1+ (HSC/Thy-1+) and Thy-1- (HSC/Thy-1-)
cells. Common myeloidprogenitors (CMP) were identified based on
CD34+CD38+IL-3R+CD45RA-Lin- staining, and their progeny includingGMP were
CD34+CD38+IL-3R+CD45RA+Lin-,whereas megakaryocyte/erythroid progenitors
(MEP)were identified based on CD34+CD38+IL-3R-CD45RA-Lin- staining.From each
population, we collected at least 5,000 cells (most samples were over 20,000 cells).In the
preliminary study, we also carried out FISH analyses using the Dual color/Dual fusion
probe (Vysis, Downers Grove, IL, USA). By the analyses with suchsmall number of sorted
cells, in the situationaround clinically complete molecular response (CMR) during 2nd-TKI
treatments, BCR-ABL-positive cells assumed to behardly detectable in most populations.
The limited numberof sorting cells was one critical reason for the methodological limitation
regardingsubtle quantitative evaluation.
2. Quantification of BCR-ABL transcripts
RNA was isolated from HSC/Thy-1+, HSC/Thy-1-, CMP,GMP, or MEP using the RNA
STAT-60TM (TEL-TEST,INC. Friendswood, TX), and reversely transcribed intocDNA
using TaqMan Gold RT-PCR Kit TM with random hexamers (Applied Biosystems, Foster
City, CA). Primersand probes used in this study were described previously asBCR-ABL
(Branford S et al., Br J Haematol 1999; 107), and BCR (Roeder I et al., Nat Med 2006; 12).
Quantitative RT-PCRanalysis of the expression of BCR-ABL and BCR wasperformed with
50 cycles of two-step PCR (15 s at 95 C and 60 s at 60 C) after initial denaturation (95 C
for10 min) using the ABI Prism 7700 Sequence DetectorSystem (Applied Biosystems).
BCR was used as the controlgene, and the BCR-ABL levels for each sample wereexpressed
as a ratio of BCR-ABL to BCR.Quantification standards were prepared by cloning
PCRproducts of BCR-ABL and BCR from CML samples. EachPCR product was cloned into
pBluescript sk(-) vector bythe TA cloning method, sequenced and ligated into thesame
vector. The resulting plasmids were digested with theappropriate restriction enzymes and
used for stable standardsto keep the same copy number of BCR-ABL andBCR.In the
preliminary study, we also used ABLandGAPDHtranscripts as the internal controls, but the
ratio of BCR-ABL to the controlstended to be affected in cases with small number of cells.
Therefore, we concluded thatBCRas the controlgeneis more suitable with the situation using
limited number of cells for the quantitative RT-PCR analyses. However, even if we use
BCRas the controlgene, it is difficult to evaluate subtle differencesin the ratioaround
0.00001 using this PCR methodwith such a small number of sorted cells.