Figure 1. Isolation of Leukemic CD34+ Cells

Cryopreserved CML or AML PBMCs and BMMCs were thawed and prepared for isolation. CD34+ cells were isolated using EasySep™ Human Cord Blood CD34 Positive Selection Kit II. The percentage of CD34+ cells before (A, C) and after (B, D) CD34+ cell isolation was measured by flow cytometry. Dead cells were excluded by light scatter profile and viability staining. In this example the purity of CD34+ cells increased from 3% to 82% (CML) and from 16% to 93% (AML).

Figure 2. Expansion of CD34+ CML Cells

CD34+ CML cells were cultured in StemSpan™ SFEM II containing CD34+ Expansion Supplement (Exp) without or with UM171. After 7 and 14 days, the cultured cells were stained with fluorescently labeled antibodies against CD45, CD34, CD90, CD45RA, and with ALDEFLUOR™ (Catalog #01700) to measure ALDH activity, and analyzed by flow cytometry. Sequential gates were used to determine the percentages of viable CD45+, CD45+CD34+ and CD45+CD34+CD90+CD45RA- cells(based on “Fluorescence Minus One” (FMO) controls), and ALDHbr cells (based on DEAB control). (A) Representative flow cytometry profiles at day 7 are shown. The (B,D) frequency and (C,E) cell numbers of these subsets per initial CD34+ cell on (B,C) day 7 and (D,E) day 14 are shown. StemSpan™ SFEM II supplemented with CD34+ Expansion Supplement supports the expansion of CML cells in culture. The addition of UM171 enhances expansion of all subsets shown (~10-fold expansion of CD34+ progenitor cells at day 7 and ~20-fold at day 14 compared to cultures without UM171). Data shown are mean ± SEM (n = 6). P-values were calculated using a two-tailed paired Student’s t-test (*P < 0.05; **P < 0.01; ***P < 0.0001). All six CML samples tested were able to expand in culture.

Figure 3. Expansion of CD34+ AML Cells

CD34+ AML cells were cultured in StemSpan™ SFEM II containing CD34+ Expansion Supplement (Exp) alone, or with UM171. After 7 and 14 days, the cultured cells were stained with fluorescently labeled antibodies and with ALDEFLUOR™ Reagent as described in Figure 2. (A) Representative flow cytometry profiles at day 7 are shown. The (B, D) frequency and (C, E) cell numbers of these subsets per initial CD34+ cell on (B, C) day 7 and (D, E) day 14 are shown. SFEM II supplemented with CD34+ Expansion Supplement supports the expansion of AML cells in culture. The addition of UM171 further enhances expansion of all subsets shown (~3-fold expansion of all subsets at day 7 and ~7-fold at day 14 compared to cultures without UM171). Data shown are mean ± SEM (n = 6). P-values were calculated using a two-tailed paired Student’s t-test (*P < 0.05; **P < 0.01). Six out of ten AML samples tested were able to expand in culture.

Figure 4. Colony-Forming Potential of CD34+ CML Cells is Maintained During Culture

CML cells were assayed in colony assays using MethoCult™ H4435 Enriched medium directly after CD34+ cell isolation (day 0) or after 7 or 14 days of expansion without or with UM171 (as described in Figure 2). After 14 days of culture in StemSpan™ SFEM II with CD34+ expansion supplement (Exp) with or without UM171, colonies were (A) imaged with STEMvision™ and counted manually from digital images. (B) CFU output expressed as the total number of colonies per original input CD34+ cell. Numbers above each of the individual bars indicate the proportion of BCR-ABL positive colonies, measured by qRT-PCR on individual plucked colonies across 6 different samples (8-12 colonies were plucked for each sample per condition). SFEM II supplemented with CD34+ Expansion Supplement (Exp) supports the expansion of colony-forming progenitor cells in culture. UM171 further promotes colony forming progenitor cell output (~3.5-fold expansion at day 7 and ~8-fold at day 14). Single-colony qRT-PCR analysis revealed that colonies generated from samples on day 0, and colonies generated from cells expanded for 7 and 14 days, were predominantly BCR-ABL+ but also that normal BCR-ABL- progenitor cells were present at low frequencies. Data shown are mean ± SEM (n = 6). P-values were calculated using a two-tailed paired Student’s t-test (*P < 0.05).