Figure 4.
Increase in human long-term HSC self-renewal induced by 005A is characterized by delayed cell division and activation of Notch signaling. (A-B) Cell cycle status and percentage of viable cells were detected for cells cultured in the presence or absence of 005A (n = 3 in panel A, n = 4 in panel B). CD34+ UBCs were planted in 24-well plates, and cell populations were analyzed by flow cytometry after 7-day treatment with vehicle or 005A (20 nmol/mL). (C) Analysis of single cell division status at 48 and 72 hours after 005A or vehicle treatment. (D) The total number of wells containing cell colonies at 14 days after 005A or vehicle treatment. Cells and colonies were counted under a microscope, and the 3 categories indicated were recorded, and 60 wells were measured in each group (n = 3) in panels C and D. (E) The percentage of cells for each cell division type after 72-hour treatment with and without 005A (005A and control, respectively) are presented. One hundred cells were randomly measured. Representative pictures of Numb staining are shown on the left. Scale bar = 6 μm. Original magnification, 60×. (F) Differential expression analysis of the genes closely related to stem cell fate determination in cultured HSCs, HSPCs, MPPs, and progenitors (PROs) with or without 005A treatment by single-cell PCR. The genes exhibiting the greatest changes in gene expression compared with the control group are labeled. (G-H) Expression of Notch signaling genes, HOXB4, and CXCR4 in 005A-cultured HSCs, HSPCs, MPPs, and PROs by real-time PCR (n = 3). Data were normalized to the expression levels in control group. (I) Western blot analysis for c-MYC, HES1, NOTCH1, and Notch intracellular cytoplasmic domain (NICD) in human CD34+ cells treated with vehicle control or various concentrations of 005A for 4 days. Actin was used as a loading control. (J) Absolute numbers per well of human CD34+CD38−CD45RA−CD90+ (i) and CD34+CD38−CD45RA−CD90+CD49f+ (ii) cells were measured (n = 4); 1 × 105 CD34+ UBCs were planted in 24-well plate, and cell populations were analyzed by flow cytometry after 7-day treatment with vehicle, 005A (20 nmol/mL), or 005A (20 nmol/mL) + DBZ (10 µmol/L). (K) Venn diagram shows the numbers of genes expressed differentially between 005A and control group. (L) The gene set enrichment in vehicle or 005A treated HSCs by gene set enrichment analysis. The experiment was repeated 3 times. All data represent means ± SD. Compared with control unless specified: *P < .05, **P < .01, ***P < .001 by 2-tailed unpaired t test.

Increase in human long-term HSC self-renewal induced by 005A is characterized by delayed cell division and activation of Notch signaling. (A-B) Cell cycle status and percentage of viable cells were detected for cells cultured in the presence or absence of 005A (n = 3 in panel A, n = 4 in panel B). CD34+ UBCs were planted in 24-well plates, and cell populations were analyzed by flow cytometry after 7-day treatment with vehicle or 005A (20 nmol/mL). (C) Analysis of single cell division status at 48 and 72 hours after 005A or vehicle treatment. (D) The total number of wells containing cell colonies at 14 days after 005A or vehicle treatment. Cells and colonies were counted under a microscope, and the 3 categories indicated were recorded, and 60 wells were measured in each group (n = 3) in panels C and D. (E) The percentage of cells for each cell division type after 72-hour treatment with and without 005A (005A and control, respectively) are presented. One hundred cells were randomly measured. Representative pictures of Numb staining are shown on the left. Scale bar = 6 μm. Original magnification, 60×. (F) Differential expression analysis of the genes closely related to stem cell fate determination in cultured HSCs, HSPCs, MPPs, and progenitors (PROs) with or without 005A treatment by single-cell PCR. The genes exhibiting the greatest changes in gene expression compared with the control group are labeled. (G-H) Expression of Notch signaling genes, HOXB4, and CXCR4 in 005A-cultured HSCs, HSPCs, MPPs, and PROs by real-time PCR (n = 3). Data were normalized to the expression levels in control group. (I) Western blot analysis for c-MYC, HES1, NOTCH1, and Notch intracellular cytoplasmic domain (NICD) in human CD34+ cells treated with vehicle control or various concentrations of 005A for 4 days. Actin was used as a loading control. (J) Absolute numbers per well of human CD34+CD38−CD45RA−CD90+ (i) and CD34+CD38−CD45RA−CD90+CD49f+ (ii) cells were measured (n = 4); 1 × 105 CD34+ UBCs were planted in 24-well plate, and cell populations were analyzed by flow cytometry after 7-day treatment with vehicle, 005A (20 nmol/mL), or 005A (20 nmol/mL) + DBZ (10 µmol/L). (K) Venn diagram shows the numbers of genes expressed differentially between 005A and control group. (L) The gene set enrichment in vehicle or 005A treated HSCs by gene set enrichment analysis. The experiment was repeated 3 times. All data represent means ± SD. Compared with control unless specified: *P < .05, **P < .01, ***P < .001 by 2-tailed unpaired t test.

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