Supporting Information

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Supporting Figure 5
Supporting Table 2
Supporting Figure 6
Supporting Methods

Supporting Figure 5a
Supporting Figure 5b

Supporting Figure 6 a–d
Supporting Figure 6 e and f

NOD/SCID mice (n = 58) were transplanted with either 0.5–2.5 × 10³ day 0 (input) cord blood CD34⁺CD38^-lin^- progenitor cells or their progeny after 7-day culture with either thrombopoietin, stem cell factor, and flt-3 ligand (TSF) or TSF + diethylaminobenzaldehyde. Criteria for positive human cell engraftment was >1.0% of human CD45⁺ cells by flow cytometric analysis as described (1). Poisson statistical analysis demonstrated a 3.4- and 7.7-fold increase in SCID-repopulating cells within the diethylaminobenzaldehyde + TSF culture group as compared with day 0 and TSF-cultured populations, respectively.

1. Ueda, T., Tsuji, K., Yoshino, H., Ebihara, Y., Yagasaki, H., Hisakawa, H., Mitsui, T., Manabe, A., Tanaka, R., Kobayashi, K., et al. (2000) J. Clin. Invest. 105, 1013–1021.

The Human Progenitor Enrichment Mixture (StemCell Technologies, Vancouver) contains monoclonal antibodies (mAbs) to human CD2, CD3, CD14, CD16, CD9, CD56, CD66b, and glycophorin A and was used according to the manufacturer’s protocol. Briefly, CB or BM mononuclear cells were resuspended at 5–8 × 10⁷ cells per ml in PBS with 10% FBS and 1% penicillin/streptomycin and incubated with 100 ml/ml antibody mixture for 30 min followed by incubation with 60 ml/ml magnetic colloid for 30 min. Lin^– cells were washed twice, quantified by hemacytometer count, and cryopreserved in 90% FBS and 10% DMSO (Sigma-Aldrich) or used directly for further experiments.

Immunofluorescent staining was conducted by using anti-human CD34-FITC and anti-human CD38-phycoerythrin monoclonal antibodies (Becton Dickinson) for 30 min on ice. Analysis and sterile cell sorting was conducted by using a FACSVantage flow cytometer (Becton Dickinson) to isolate CD34⁺CD38^– and CD34⁺CD38⁺ subsets. The CD34⁺CD38^– sort gate was set to collect only those events falling in the lowest 5% of phycoerythrin fluorescence within the total CD34⁺ population to ensure acquisition of highly purified CD34⁺CD38^– cells.

A minimum of 3-6 experiments were performed per culture condition for the in vitro analyses. Immunophenotypic analysis was performed on progeny cells by using anti-human CD34 and CD38 mAbs (Becton Dickinson) and isotype control IgG mAbs and compared with day 0 (input) staining. Fourteen-day methylcellulose CFC assays [colony-forming unit-granulocyte monocyte (CFU-GM), burst-forming unit-erythroid (BFU-E), and colony-forming unit-mix (CFU-Mix)] were performed in triplicate as we have previously described (1, 2) to compare the number of lineage-committed CFCs within day 0 CD34⁺CD38^–lin^– cells and their progeny. Briefly, 5–10 × 10² cells from each condition were placed in methylcellulose media for 14 days, and total numbers of colonies (minimum of n = 50 cells per colony) were then scored and the total number of CFCs per million cells were then calculated. Morphologic analysis of day 0 CD34⁺CD38^–lin^– cells and their progeny after 7 days of culture was performed by using Wrights-Giemsa staining and microscopic analysis under oil immersion (100´). For the extended 14-day cultures of CB CD34⁺CD38^–lin^– cells with DEAB + TSF, all cultures were supplemented at day 7 with equal volumes of complete culture media containing 100 mM DEAB + TSF.

BM and CB CD34⁺CD38^–lin^– cells were also placed in culture with TSF with and without 1 mM ATRA (Sigma-Aldrich), an agonist of RAR. Total cell expansion, immunophenotype, CFC content, and morphologic analysis was performed on ATRA-treated progeny and compared with both day 0 CD34⁺CD38^–lin^– populations and the progeny of TSF alone to assess the impact of a retinoid agonist on the differentiation of human HSCs. 1 mM retinaldehyde (Sigma-Aldrich), which is a substrate for ALDH1-mediated production of retinoic acids, was added to cultures of primary CB CD34⁺CD38^–lin^– cells with and without DEAB to assess for the activity of DEAB against the ALDH1 isoform. Cultures were also performed by using 100 nM of the synthetic RAR-a agonist, TTNPB (Sigma-Aldrich), 1 mM of LGD101268, a synthetic RXR agonist (Ligand Pharmaceuticals), or 100 nM of 1-a, 25-dihydroxyvitamin D₃ (Sigma-Aldrich) coupled with TSF with or without the addition of 100 mM DEAB to determine whether any of these compounds could overcome the effects of DEAB on human HSC differentiation.

Cells were transplanted via tail-vein injection after irradiating NOD/SCID mice with 300 cGy by using an x-ray irradiator as described (1, 2). Because purified human CD34⁺CD38^–lin^– cells have been previously shown to have limited engraftment capacity in the NOD/SCID mice in the absence of either exogenous cytokine treatment of cotransplantation with nonrepopulating accessory cells (CD34⁺CD38⁺lin^– or CD34^–lin^– cells) (1, 3), both day 0 CD34⁺CD38^–lin^– HSCs and their progeny were cotransplanted with 2 × 10⁴ CD34⁺CD38⁺lin^– accessory cells to facilitate engraftment as described (3). All mice in each group were killed at week 8, and marrow samples were obtained by flushing their femurs with Iscove’s modified Dulbecco’s medium at 4°C. Flow cytometric analysis of human cell engraftment was performed as previously described by using commercially available mAbs against human leukocyte differentiation antigens to identify engrafted human leukocytes and discriminate their hematopoietic lineages (1, 2). Measurement of human cell engraftment in secondary transplanted mice was performed via isolation of genomic DNA from whole BM cells at 12 weeks posttransplantation. PCR analysis was performed on each sample (100 ng) to detect a 1,171-bp segment of human chromosome 17-specific a-satellite region by using primer pairs and amplification conditions as described (4). The level of human cell engraftment was determined by comparing the 1,171-bp product with that of a human/mouse DNA mixture control as described (4).

Comparisons of data from in vitro experiments were made by using Student’s t test. For purposes of our limiting dilution analysis, a transplanted mouse was scored as positively engrafted if ³1.0% of the marrow cells expressed human CD45 via high-resolution FACS analysis. This criterion is consistent with previously published criteria for human cell repopulation in NOD/SCID mice (5). SRC frequency in each cell source was calculated by using the maximum likelihood estimator as described by Taswell (6) for the single hit Poisson model (5, 7). We calculated confidence intervals for the frequencies using the profile likelihood method, and we used the likelihood ratio test to confirm the fit of the model.

Total RNA was reverse-transcribed to cDNA by using the iScript cDNA synthesis kit (Bio-Rad). cDNA concentrations were measured with a fluorometer (Turner Designs, Sunnyvale, CA) by using RiboGreen reagent (Invitrogen). PCR amplification reactions were performed in 13 ml and contained equal amounts of cDNAs, 6.5 ml of iQ SYBR Green Supermix (Bio-Rad), 0.2 mM of each forward and reverse gene-specific primers for genes of interest, and the normalization gene 36B4. PCR was performed on an iCycler (Bio-Rad) according to the following cycling conditions: an initial cycle of 15 min at 95°C, 45 cycles of 45 sec at 95°C, 15 sec at 55°C, and 15 sec at 72°C, followed by a melt-curve analysis cycle with steps of 10 sec each at 0.5°C increments from 60 to 95°C. Amplification rates were visualized and analyzed on ICYCLER IQ optical system software Version 3.0 (Bio-Rad). Gene-specific primers were purchased from Integrated DNA Technologies (Coralville, IA).

Cells were suspended at 1 × 10⁶ cells per ml in assay buffer and stained with 200 ng/ml activated ALDEFLUOR reagent, an aliquot was immediately transferred to 6 nM DEAB as a negative control, and the samples were incubated for 30 min at 37°C. After ALDEFLUOR staining, immunophenotype staining was conducted by adding anti-human CD38-phycoerythrin and anti-human CD34-allophycocyanin or isotype-matched control antibodies (Becton Dickinson) for 30 min on ice. Sample analysis was conducted on a FACSCalibur flow cytometer (Becton Dickinson).

1. Chute, J., Muramoto, G., Fung, J. & Oxford, C. (2005) Blood 105, 576–583.

2. Chute, J., Saini, A., Chute, D., Wells, M., Clark, W., Harlan, D., Park, J., Stull, M., Civin, C. & Davis, T. (2002) Blood 100, 4433–4439.

3. Bonnet, D., Bhatia, M., Wang, J., Kapp, U. & Dick, J. (1999) Bone Marrow Transplant. 23, 203-209.

4. Yahata, T., Ando, K., Sato, T., Miyatake, H., Nakamura, Y., Muguruma, Y., Kato, S. & Hotta, T. (2003) Blood 101, 2905–2913.

5. Ueda, T., Tsuji, K., Yoshino, H., Ebihara, Y., Yagasaki, H., Hisakawa, H., Mitsui, T., Manabe, A., Tanaka, R., Kobayashi, K., et al. (2000) J. Clin. Invest. 105, 1013–1021.

6. Taswell, C. (1981) J. Immunol. 126, 1614–1619.

7. Wang, J., Doedens, M. & Dick, J. (1997) Blood 89, 3919–3924.