Purification of Human CD34+CD90+ HSCs Reduces Target Cell Population and Improves Lentiviral Transduction for Gene Therapy

Abstract

Hematopoietic stem cell (HSC) gene therapy has the potential to cure many genetic, malignant, and infectious diseases. We have shown in a nonhuman primate gene therapy and transplantation model that the CD34⁺CD90⁺ cell fraction was exclusively responsible for multilineage engraftment and hematopoietic reconstitution. In this study, we show the translational potential of this HSC-enriched CD34 subset for lentivirus-mediated gene therapy. Alternative HSC enrichment strategies include the purification of CD133⁺ cells or CD38^low/- subsets of CD34⁺ cells from human blood products. We directly compared these strategies to the isolation of CD90⁺ cells using a good manufacturing practice (GMP) grade flow-sorting protocol with clinical applicability. We show that CD90⁺ cell selection results in about 30-fold fewer target cells in comparison to CD133⁺ or CD38^low/- CD34⁺ hematopoietic stem and progenitor cell (HSPC) subsets without compromising the engraftment potential in vivo. Single-cell RNA sequencing confirmed nearly complete depletion of lineage-committed progenitor cells in CD90⁺ fractions compared to alternative selections. Importantly, lentiviral transduction efficiency in purified CD90⁺ cells resulted in up to 3-fold higher levels of engrafted gene-modified blood cells. These studies should have important implications for the manufacturing of patient-specific HSC gene therapy and gene-engineered cell products.

Keywords: CD90 CD34; Closed System Application; Gene Therapy; Hematopoietic Stem Cells; Lentivirus Transduction; Mouse xenograft transplants; Stem Cell Enrichment / Cell Sorting; single cell RNA sequencing.

Graphical abstract

Figure 1

Quantitative Comparison of Phenotype-Defined Target CD34 Subsets (A) Average frequency of CD133⁺, CD38^low/–, and CD90⁺ HSPCs within bulk ssBM-derived CD34⁺ cells (mean ± SD, n ≥ 9 independent healthy donors). (B) Schematic of the quantitative and hierarchical relationship of phenotypic CD34 subsets. (C) Frequency of phenotype-defined subsets in bulk CD34⁺ cells before FACS (top row) and cross-contamination of phenotypic subsets within FACS-purified CD133⁺ (first column), CD38^low/– (second column), and CD90⁺ (third column) HSPCs from a representative ssBM donor used for scRNA-seq analysis. (See also Figure S1.)

Figure 2

scRNA-Seq of ssBM-Derived CD34 HSPCs and FACS-Purified CD34 Subsets (A) Dimensional reduction (tSNE) and graph-based clustering (PCA) of scRNA-seq data from ssBM-derived CD34⁺ cells and representative feature plots showing the expression of genes associated with lymphoid-primed, myeloid-primed, erythroid-primed, and immature HSPCs. Level of expression is color coded as shown in the legend in (C). (B) PCA-based transformation with clusters defined in (A) projected onto the PCA analysis and expression of representative genes as shown in (A). (C) Expression of CD34, CD133, CD38, and CD90 in ssBM-derived CD34⁺ cells. Level of expression is color coded as shown in the legend. (D) Overlay of scRNA-seq data from CD34⁺ cells (black, first plot) with FACS-purified CD133⁺ (green, second plot), CD38^low/– (purple, third plot), and CD90⁺ (pink, fourth plot) HSPCs. (See also Figures S2, S3, S4 and S5 as well as Tables S1 and S2.)

Figure 3

Bulk RNA-Seq of ssBM-Derived CD34 Subsets (A) Gating strategy for phenotypic human CD34 subpopulations. (B) Heatmap of the top 200 differentially expressed genes in phenotypic CD34 subsets listed in (A) from two independent human donors. (C) Overlay of bulk RNA-seq data from FACS-purified ssBM subsets (populations a–g are color coded as defined in A) on the ssBM scRNA-seq PCA reference map (black dots). (D) Overlay of bulk RNA-seq data from FACS-purified G-CSF-mobilized CD34 subsets (populations a–d are color coded as defined in A) on the ssBM scRNA-seq PCA reference map (black dots). (See also Figures S6 and S7 as well as Tables S3 and S4.)

Figure 4

Multilineage Engraftment Potential of Human CD34 Subpopulations (A and B) Flow cytometric assessment of the frequency of human chimerism in the (A) PB and (B) BM, spleen, and thymus after transplantation of bulk CD34⁺ HSPCs as well as FACS-purified CD34 subpopulations (1 × 10⁵ cells per mouse) from a single G-CSF-mobilized human donor. Engraftment data from a second donor can be found in Figure S9. (C) Frequency of engrafted human CD34⁺ and CD90⁺ HSPCs. CD34⁺ frequency, left y axis; CD90⁺ frequency, right y axis. (D) Human CD34⁺ cells from the murine BM were flow-sorted into CFC assays and erythroid, myeloid, and erythro-myeloid CFC potentials were quantified after 12–14 days. Horizontal line at 0.1% in A and B indicates threshold for the detection of human chimerism. Horizontal bars in (B) and (C) indicate the mean for each population. CFU, colony-forming unit; CFU-M, CFU macrophages; CFU-G, CFU granulocytes; CFU-GM, CFU, granulocytes/macrophages; BFU-E, burst forming unit erythroids; CFU-MIX, CFU erythro-myeloid colonies. (See also Figure S8, S9, and S10.)

Figure 5

FACS Purification and Quality Control of CD90⁺ HSPCs (A) Schematic of the experimental design. (B) Flow cytometric assessment of cells before FACS (CD34⁺, first plot) and purified CD90⁺ cells after sorting on the Sony (second plot) and Tyto (plot). (C) Comparison of the purity, yield, and fold enrichment of CD90⁺ HSPCs on the Sony and Tyto sorters. (D) CFC potential of CD90⁺ cells within bulk CD34⁺ HSPCs and FACS-purified CD90⁺ subsets. CD90⁺ cells from all three conditions were sorted into CFC assays to exclude contaminating CD90^– cells. (E) Flow cytometric quantification of GFP-expressing CD90⁺ cells within bulk CD34⁺ cell and FACS-purified CD90⁺ subsets (left) and the delta-MFI of GFP expression in gene-modified cells (right). (F) Erythroid, myeloid, and erythro-myeloid CFC potential of gene-modified CD90⁺ cells from bulk CD34⁺ cells and FACS-purified CD90⁺ subsets. CD90⁺ cells from all three conditions were sorted into CFC assays. (G and H) Individual colonies from all three conditions in (F) were picked, and (G) the gene-modification efficiency in CFCs was determined by PCR as well as (H) the VCN in modified CFCs quantified by qPCR. (See also Table S9.) Statistics, means ± SEM; in C, third graph, median and range; significance values, two-tailed paired t test.

Figure 6

Transplantation of FACS-Purified and Gene-Modified CD90⁺ Cells into NSG Mice (A) Frequency of human chimerism in the PB over time. (B) Human chimerism in the BM, spleen, and thymus at 20 weeks post-transplant. (C and D) Frequency of GFP⁺ human cells in (C) the PB over time and (D) tissues at 20 weeks post-transplant. (E) Engraftment of human CD34⁺, CD90⁺, and CD90⁺GFP⁺ HSPCs in the BM at necropsy. (F) Human CD34⁺ cells from the murine BM were flow-sorted into CFC assays and erythroid, myeloid, and erythro-myeloid CFC potentials were determined. Horizontal line at 0.1% in A and B indicates the threshold for the detection of human chimerism. Shapes indicate different human donors (n = 3). (See also Figure S11.) Statistics, means ± SEM.