Figure 1.

Net HSC maintenance in UM171-expanded mPB CD34⁺ cells

(A) Schematic representation of the experimental design. (B) Ex vivo expansion of drug products (DPs) from different MPS1 patients (Pt) for 6–7 additional days in the presence (dot; EX-U) or absence (square; EX) of UM171 (35 nM). Fold expansion is calculated as a ratio between final cell count on day7/8 with respect to seeding on day 1. (C) Fraction of HSC-enriched CD34⁺CD90⁺CD45RA⁻ cells at the end of expansion culture of the samples from (B). ∗p < 0.05, Mann-Whitney test. (D) Human CD45⁺ cell engraftment in the BM of NSG mice at 12 weeks post intravenous (i.v.) injection of the following mPB-derived HSPC products from 2 MPS1 patients: Pt1-Xeno (1) 400,000 non-cultured CD34⁺ cells (NC; n = 2), (2) 400,000 DP cells (DP; n = 5), or (3) 4,000,000 ex vivo-expanded cells (outgrowth of 400,000 DP cells; EX-U; n = 6). Pt2-Xeno (1) 150,000 non-cultured CD34⁺ cells (NC; n = 2), (2) 150,000 DP cells (DP; n = 4), or (3) 1,100,000 ex vivo-expanded cells (outgrowth of 150,000 DP cells; EX-U; n = 4). ∗p < 0.05, Mann-Whitney test. BM cells were obtained as terminal procedure or BM aspirate for the mice injected with cells from Pt1 and Pt2, respectively. See Figure S3 for the endpoint analysis of Pt2-Xeno. (E) Secondary transplantation of 2 and 1 × 10⁶ CD34⁺ cells isolated from primary NSG recipients engrafted with HSPC from Pt1-Xeno and Pt2-Xeno, respectively. BM engraftment of secondary recipients was assessed at 12 weeks post transplantation, respectively. Each dot represents an individual mouse. (F and G) Lineage composition of the human CD45⁺ cell graft from (D) and (E), respectively. CD19⁺, B cells; CD33⁺, myeloid cells; CD3⁺, T cells; CD34⁺, HSPC; neg, CD19⁻CD33⁻CD3⁻CD34⁻ cells. Shown is the mean ± SEM from the pooled individual mice from Pt1-Xeno and Pt2-Xeno. (H) Ex vivo expansion (mean ± SEM) of mPB CD34⁺ cells from n = 4 adult donors under EX-U conditions following transduction with a LV vector at increasing multiplicities of infection (MOI). UT, non-transduced cells. (I) Absolute number of CD34⁺ CD90⁺ cells on day 7 of expansion culture from (H). Each line represents a donor (∗p < 0.05, Kruskal-Wallis test with Dunn’s multiple comparison). (J) Relative abundance of unique LV integration sites (LVIS) from DP and EX-U groups from Pt1-Xeno in primary and secondary recipient mice. Bottom light gray stacks indicate the sum of integrations with <1% relative abundance, middle stacks indicate insertions >1% abundance, whereas the top abundant integrations are highlighted in red. (K) Left: box plots indicating number of integration sites normalized on transduction efficiency (VCN). Right: box plots indicating the Shannon diversity index as measure of clonal population diversity. Each dot represents an individual mouse of the treatment group: DP and EX-U in primary and secondary recipient, respectively (∗p < 0.05, Mann-Whitney test). (L) Left: schematic representation on how unique LVIS or LV vector barcodes (LVBC), introduced at transduction, can be exploited to prove symmetric HSC divisions in culture by retrieving, at the experiment endpoint, the same genetic mark in two different mouse recipients transplanted with cells from a given ex vivo culture batch. Right: boxplots showing the proportion of shared LVIS (Pt1-Xeno) or shared LVBCs (HD1) between at least two individual mice receiving the same day-0-equivalent CD34⁺ cell doses from the indicated HSPC products (intragroup sharing). Each dot represents the percentage of LVIS/LVBCs shared with at least one other mouse in the same experimental group. (∗p < 0.05, ∗∗p < 0.01, Mann-Whitney test). Further details on HD1 are presented in Figure S1. In all box plots shown in the figure, the whiskers represent the minimum and maximum values of the data.