Figure 2.

Characterization of expansion cultures by scRNA-seq

(A) Adult mPB CD34⁺ cells from n = 2 healthy donors were expanded with UM171 in bulk following transduction with a barcoded LV library (upper panel) or after marking FACS-sorted GMPs (CD34⁺CD38⁺CD45RA⁺ or CD34⁺CD38⁺CD45RA⁻BAH1⁻CD71⁻), MEPs (CD34⁺CD38⁺CD45RA⁻CD71⁺), or multipotent HSPCs (CD34⁺CD38⁻) with a color-coded LV expressing a blue (BFP), red (Cherry), or green (GFP) fluorescent protein, respectively (lower panel). Cells were harvested on day 4 and/or day 8 for scRNA-seq library preparation. (B) Colony-forming cell assay on sorted progeny of GMPs, MEPs, and CD38⁻ cells after 7 days of expansion. Absolute number of colony-forming units (CFU) with red (BFU-E) or white (CFU-G/M) morphology are shown for the three populations of origin (mean ± SEM, n = 5 technical replicates). (C) Uniform manifold approximation and projection (UMAP) encompassing the samples described in (A) with manually curated cell type annotation, as described in materials and methods. HSC/MLP, hematopoietic stem cells/multi-lymphoid progenitors; HSPC, hematopoietic stem/progenitor cells; Mye, myeloid lineage progenitors; Mono, monocyte progenitors; MCP, mast cell progenitors; Mk-Ery, megakaryocyte/erythroid lineage progenitors; Prog-Cycling, progenitors predicted to be in active phases of the cell cycle. (D) Alluvial plot showing population frequencies over time, calculated within each time point. NC, non-cultured sample (day 0). (E) Distribution of the expanded day-7 progeny of FACS-sorted cell populations across UMAP embedding according to cell population of origin and (F) relative frequency of scRNA-seq-based cell type annotations as defined in (C). (G) Expression of key cytokine receptors in the indicated cell type annotations. The color palette (from blue to red) represents the Z score of receptor expression (from low to high, respectively), while the size of the dots represents the percentage of cells expressing the indicated cytokine receptor transcript in the indicated population. (H) Pediatric mPB DPs from n = 2 MPS1 patients (Pt1, Pt4) were sorted for CD34⁺CD90⁺CD45RA⁻ cells (dp = day 1) or expanded for 4 (EX4) and 7 days (EX7) in UM171 before sorting into the CD34⁺CD90⁺CD45RA⁻ cell fractions. scRNA-seq was performed on the HSC-enriched subpopulations at the three time points. The UMAP on the left shows automated cell type annotation using scGate leveraging the adult mPB dataset described in (A)–(G) as reference (see materials and methods for details). The alluvial plots on the right show time-dependent changes in population frequencies. Note that CD34⁺CD90⁺CD45RA⁻ cells, an HSC-enriched subset of the cultures, were analyzed here. (I) Cells with LV-derived transcripts (WPRE⁺) are projected on the UMAP plot as a surrogate for estimating transduction efficiency (TE). (J) Proportion of LV transcript+ cells over time in the two patients. TE over time and across major populations. (K) Proportion of LV transcript+ cells in the DP (intensely colored bar) vs. the expanded cells (faintly colored bars; EX4 and EX7 samples were aggregated) for the entire population (All) or within the indicated, scRNA-seq-defined subpopulations. HSCs and HSPCs were aggregated to obtain a representative number of cells for all conditions. Left graph (blue bars), Pt1; right graph, orange, Pt4. (L) TE assessed by digital droplet PCR on individually plucked colony-forming units (gold-standard assay), either on the DP (bulk) or in the BM of the patients at 1 year after gene therapy, as reported in Gentner et al.³⁰