Figure 3.

Expansion protocol optimization for mPB HSPC gene-therapy applications

(A) (Top) The matrix outlines the IL-6 and IL-3 doses (ng/mL) used in the different culture combinations (identified by the letters A–H), tested on three healthy donors. All conditions contained SCF 100 ng/mL; FLT3L 100 ng/mL; TPO 50 ng/mL. (Bottom) Fold expansion over time (mean ± SEM) from three donors cultured with the different IL-3 and IL-6 concentrations. (B) The percentage (top) and absolute number (bottom) of CD34⁺ cells after 7 days of expansion. Bars represent the mean ± SEM, individual donor values are shown as dots. (C) Absolute number (#) of CD34⁺CD90⁺ cells after 7 days of expansion . Bars represent the mean ± SEM, individual donor values are shown as dots. (A–C) Linear mixed-effects models with fixed effects: IL-3, IL-6, IL-3 × IL-6 and random effect for donor; log2-scale for fold expansion and absolute output, raw scale for %CD34⁺. Type III ANOVA (Satterthwaite); post hoc Holm-adjusted contrasts. See materials and methods. (D) Percentage of human CD45⁺ cells present in the BM of mice transplanted (n = 3–6) with cells derived from the indicated culture conditions 17 weeks post transplant. The mice were transplanted using a pooled sample from the three donors for each condition, each mouse receiving a limiting dose of 35,000–39,000 day-0-equivalent cells (Kruskal-Wallis test). (E) Different commercial media were compared (A, SCGM Cell Genix; B, StemSpan StemCell Technologies; C, StemPro GIBCO). Boxplot (left) showing the in vitro fold expansion measured after 7 days of culture of mPB CD34⁺ cells from four different healthy donors. In all tested conditions, the culture media were supplemented with the same cytokine cocktail (100-ng/mL SCF, 100-ng/mL FLT3L, 50-ng/mL TPO, 50-ng/mL IL-6, 50-ng/mL IL-3). The percentage of primitive CD34⁺CD90⁺CD45RA⁻CD201⁺ITGA3⁺ cells (right) was measured by flow cytometry after 7 days of expansion in different media. Each dot represents an individual donor (Kruskal-Wallis test with Dunn’s multiple comparison, ∗p < 0.05, ∗∗p < 0.01). (F) Boxplot showing the percentage of human CD45⁺ cells engraftment in the BM of mice transplanted (n = 8) with cells cultured in medium A or medium B at 16 weeks post transplant. Each dot represents a single mouse; mice were transplanted using a pool from different donors for each condition, with 48,000–57,000 day-0-equivalent cells injected per mouse (Mann-Whitney test, ∗p < 0.05). (G) CD34⁺ cells from n = 3 adult mPB donors were expanded over 3 weeks in medium A supplemented with SFT63 cytokines, UM729 and SR1 (SFT63), or a chemically defined medium (ChemD; see materials and methods) with or without IL-3. The projected absolute number (#) of CD34^high cells (left) and more primitive CD34^hiCD90⁺CD45RA⁻ cells (right) over a 3-week culture period is shown, taking into consideration the proportion of cells that has been removed during consecutive passages. Connected dots refer to longitudinal data from an individual donor and the indicated condition. (H) Schematic representation of the experimental design. Different compound combinations were tested: UM171 (35 nM) alone (U) or in combination with SR1 (US) (750 nM). In both conditions, cells were cultured in SCGM with the optimized cytokine cocktail described above. (I) Percentage of human CD45⁺ cells (mean ± SEM) present in the peripheral blood (PB) of U (n = 8) and US (n = 10) conditions at the indicated number of weeks post transplant. ns, not significant (U vs. US, mixed-effects analysis). (J) Percentage of human CD45⁺ cells present in the BM at 17 weeks post transplant. (K) Box plots indicating the Shannon diversity index as measure of clonal population diversity. Each dot indicates an individual mouse of the U and US primary recipient groups. (L) Sharing of barcodes within the same treatment group, measured as the percentage of shared barcodes from individual mice (dots) of the same treatment group. Each dot represents the percentage of LVBCs shared with at least one other mouse in the same experimental group. (M) Percentage of human CD45⁺ cells (mean ± SEM) present in the peripheral blood (PB) of U and US (n = 4) conditions at the indicated number of weeks post-secondary transplant. The mice were transplanted using a pooled sample from BM of primary recipients depleted of murine cells. The amount of CD34⁺ cells transplanted into secondary recipients ranged between 1–1.3 × 10⁶ CD34⁺ cells. ∗p < 0.05 (U vs. US, two-way ANOVA). (N) Boxplot with percentage of human CD45⁺ cells present in the BM of mice transplanted (n = 4) with cells derived from primary recipients, 17 weeks post secondary transplant. (O) Alluvial plots showing the frequency of shared barcodes between primary and secondary transplants. Barcode frequencies are arranged from most abundant to least abundant within the primary transplant recipients' BM, only shared barcodes across primary and secondary transplant recipients are plotted. Barcode frequency is calculated as the mean abundance of the barcode across mice from the same experimental condition in which the barcode could be detected. Left and right alluvial plots show the sharing in the UM171 (U) and UM171/SR1 (US) conditions, respectively.