(A–G) CD34⁺ cells cocultured with MSCs alone (CD34⁺ alone) (n = 4–7) or +AML cell lines (n = 3–7) or +AML primary patient samples (n = 3–7). After 4 days of coculture, CD34⁺ cells were plated for CFU or LTC-IC assays or implanted into NSG mice. (B) Cell counts of total non-leukemic hematopoietic cells. AML patient samples: AML1–4. (C) Representative FACS plots of cell cycle analysis of CD34⁺ cells based on DAPI and Ki-67 staining. (D) Quiescent (Ki-67^–DAPI^–) cells within normal progenitors (CD34⁺CD38⁺) and HSPCs (CD34⁺CD38^–). AML1, -2, -5, -8, and -9. (E) Proportions of normal HSPCs within CD34⁺ cells. AML1-5, -8, and -9. (F) Engraftment in primary NSG recipients. Three independent experiments with 1–7 mice/group per experiment. (G) Secondary recipients. AML1–3. Three independent experiments with 2–4 mice/group per experiment. (H–K) Collagen scaffolds seeded with MSCs were injected with CB CD34⁺ cells alone or +GFP⁺ AML cell lines (n = 4) and transplanted into NSG-SGM3 recipients. In the case of AML patient samples (n = 5–8), the CB CD34⁺ cells were either HLA-A2 mismatched or transduced to express GFP. Scaffold retrieval: 2–3 weeks (+AML cell lines) or 5–8 weeks (+AML patient samples). EdU i.p. injection: 16 hours prior to scaffold retrieval. (I) Non-leukemic human CD45⁺ hematopoietic cells per scaffold. AML1–5 and 11–13. Twelve to 26 scaffolds in 6–10 mice/group. (J) EdU incorporation in non-leukemic human CD45⁺ hematopoietic cells. AML1–5. Twelve to 26 scaffolds in 5–7 mice/group. (K) Proportions of CD34⁺ HSPCs within non-leukemic human CD45⁺ hematopoietic cells. Five to 11 mice/group. AML1, -3, and -4. Each AML cell line or patient sample is represented as a unique symbol. CD34⁺ cells alone were used as control and for normalization. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by Mann-Whitney U test (B and D–G), Kruskal-Wallis with Dunn’s (I and J), and Wilcoxon’s matched-pairs signed-rank test (K).