Preferential Expansion of Human CD34 + CD133 + CD90 + Hematopoietic Stem Cells Enhances Gene-Modified Cell Frequency for Gene Therapy

Abstract

CD34⁺CD133⁺CD90⁺ hematopoietic stem cells (HSCs) are responsible for long-term multilineage hematopoiesis, and the high frequency of gene-modified HSCs is crucial for the success of hematopoietic stem and progenitor cell (HSPC) gene therapy. However, the ex vivo culture and gene manipulation steps of HSPC graft preparation significantly reduce the frequency of HSCs, thus necessitating large doses of HSPCs and reagents for the manipulation. In this study, we identified a combination of small molecules, Resveratrol, UM729, and SR1 that preferentially expands CD34⁺CD133⁺CD90⁺ HSCs over other subpopulations of adult HSPCs in ex vivo culture. The preferential expansion enriches the HSCs in ex vivo culture, enhances the adhesion, and results in a sixfold increase in the long-term engraftment in NSG mice. Further, the culture-enriched HSCs are more responsive to gene modification by lentiviral transduction and gene editing, increasing the frequency of gene-modified HSCs up to 10-fold in vivo. The yield of gene-modified HSCs obtained by the culture enrichment is similar to the sort-purification of HSCs and superior to Cyclosporin-H treatment. Our study addresses a critical challenge of low frequency of gene modified HSCs in HSPC graft by developing and demonstrating a facile HSPC culture condition that increases the frequency of gene-modified cells in vivo. This strategy will improve the outcome of HSPC gene therapy and also simplify the gene manipulation process.

INTRODUCTION

Gene modification of hematopoietic stem and progenitor cells (HSPCs) has opened new possibilities for autologous HSPC-based therapy compared to allogeneic HSPC therapy, which has several limitations.¹ The promising progress of lentiviral gene modification studies for β-hemoglobinopathies, primary immunodeficiency disorders, and lysosomal storage diseases^2

–5 as well as gene-editing studies for β-hemoglobinopathies and HIV^6,7 highlight the therapeutic benefits of autologous HSPC gene therapy.

HSPC graft for gene manipulation is composed of lineage-committed progenitors marked by CD34⁺CD133⁻CD90⁻, early progenitors (CD34⁺CD133⁺CD90⁻), hematopoietic stem cells (HSCs) (CD34⁺CD133⁺CD90⁺), and differentiated cells (CD34⁻).⁸ Lentiviral transduction requires a maximum of 48 h for prestimulation with cytokines followed by 24–48 h for viral transduction and 24 h for viral washout.^3,9
–11 Similarly, gene editing requires 48 h for prestimulation and up to 24 h for relief from electroporation stress.^6,8,12 These steps demand 3–5 days of ex vivo culturing, compromising the stemness of gene-modified HSCs.

The loss of stemness associated with ex vivo culturing and the toxicity associated with the manipulation procedure particularly by the high doses of viral vectors and the gene-editing reagents reduce the number of gene-modified stem cells for transplantation.^1,13

–16 In addition, the committed progenitors are more susceptible to gene manipulation and compete with the HSCs during the gene manipulation process.⁸ Soon after transplantation, gene-modified committed progenitor cells gradually diminish and only the gene-modified HSCs repopulate.¹⁷ Thus, there is a significant decline in the frequency of gene-modified cells in vivo, rendering gene manipulation of committed progenitor cells less beneficial.

The insufficient quantity of gene-modified HSCs transplanted and retained in vivo demands the retrieval and manipulation of large doses of HSPCs from the patient, resulting in increased production cost.^17,18 For aforementioned reasons, effective genetic manipulation of CD34⁺CD133⁺CD90⁺ enriched HSPCs could reduce the requirement for a large quantity of cells for manipulation and the need for high doses of gene manipulating reagents, thus simplifying the process and improving the efficacy.^18

–21 In this study, we tested 11 small molecules that are known to expand umbilical cord blood (UCB)-HSPCs and identified a novel combination of small molecules which enriches the fraction of gene-modified CD34⁺CD133⁺CD90⁺ HSCs in the mobilized peripheral blood (mPB) HSPC graft, resulting in an increased frequency of gene-modified cells in vivo.

METHODS

Purification of adult HSPCs

The leftover granulocyte colony-stimulating factor (GCSF)-mPB product after allogeneic stem cell transplantation was collected from the hematology department, Christian Medical College, Vellore, after IRB approval. CD34⁺ cells were purified using CD34-positive selection kit (STEMCELL Technologies) and expanded with StemSpan SFEM II containing appropriate cytokines (stem cell factor [SCF] 240 ng/mL, FMS-like tyrosine kinase 3-ligand [Flt3-L] 240 ng/mL, thrombopoietin [TPO] 80 ng/mL, interleukin [IL]-6 40 ng/mL). The HSPCs were analyzed for cell surface markers, both after purification and on day 5 or 6 of the culture using BD Aria III Flow cytometer.

The study is approved by IRB and IBSC of Christian Medical College, Vellore, India.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 8.0 (GraphPad Software). Error bars are presented as the mean ± SEM. Unpaired t-test values, number of independent replicates (n), and donors are indicated in the figure legend. p score of <0.05 is considered as statistically significant. Detailed description of methods and materials used in this study are depicted in Supplementary File.

RESULTS

Preferential expansion of HSCs results in culture enrichment of HSCs

The HSPC manipulation procedure for gene therapy requires up to 5 days of ex vivo culture.^4,10 To identify a culture condition that preserves the stemness of mPB-HSPCs during the 5-day culture, we cultured HSPCs in a stem cell culture media containing SCF, Flt3-L, TPO, and IL-6 cytokines and tested the supplementation of small molecules targeting chromatin modifiers (Trichostatin A, Nicotinamide, Resveratrol, and UM729), Wnt signaling (Prostaglandin E2), Notch signaling (3,5-difluorophenylacetamide [DAPT]), p38-mitogen-activated protein kinases (SB203580), cell cycle (P18IN003), glucocorticoid receptor (Fluticasone), Aryl hydrocarbon receptor (SR1), and sphingolipid (4-hydroxyphenyl retinamide) that were previously shown to support UCB-HSPC expansion^{22

–32} (Fig. 1A).

Figure 1.

The RUS cocktail preferentially enriches the HSCs. (A) Experimental scheme: The mPB-HSPCs were cultured with the listed small molecules in the stem cell culture media containing cytokines for 5 days and analyzed by FACS for the markers for HSPCs. (B) Percentage of CD34⁺ and CD34⁻ cells. n = 6, donors = 3. (C) Percentage of cells positive for both CD34 and CD133. S+U = SR1+UM729, S+R = SR1+Resveratrol, R+U = Resveratrol+UM729, R+U+S = Resveratrol+UM729+SR1. n = 4, donors = 2. (D) The composition of subpopulations of HSPCs in day 0 (uncultured) and day 5 of the culture with vehicle (dimethylsulfoxide) or RUS (RUS = Resveratrol+UM729+SR1). The statistical comparison is between vehicle and RUS. n = 16, donors = 7. (E) Absolute number of subpopulations of HSPCs in day 0 and day 5 of the culture with vehicle or RUS. n = 12, donors = 6. (F) Absolute number of TNCs in day 0 and day 5 of culture with vehicle or RUS. n = 21, donors = 7. (G) Ratio of fold expansion of HSCs (CD34⁺CD133⁺CD90⁺ cells) and committed progenitors (CD34⁺CD133⁻CD90⁻ cells) in the HSPCs cultured with individual small molecules or the RUS cocktail. Fold expansions were calculated based on the increase in the absolute numbers from the input (day 0) numbers. n = 5, donors = 2. (H) Absolute number of CD34⁺CD133⁺CD90⁺CD38⁻CD45RA⁻CD49f⁺ HSCs in day 0 and day 5 of culture with vehicle or RUS. n = 6, donors = 2. (I) Absolute number of CD34⁺CD133⁺EPCR⁺ HSCs after 5 days of culture. n = 3, donors = 2. Error bars represent mean ± SEM, ns. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 (unpaired t-test, two-tailed). FACS, fluorescence-activated single cell sorting; HSC, hematopoietic stem cell; HSPC, hematopoietic stem and progenitor cell; mPB, mobilized peripheral blood; ns, nonsignificant; TNCs, total nucleated cells. Color images are available online.

Among the tested candidates, SR1, UM729, and Resveratrol showed significantly higher percentage of CD34⁺ cells (Fig. 1B), and on testing these three compounds in different combinations, Resveratrol+UM729+SR1 (R+U+S/RUS) combination generated higher percentage of CD34⁺CD133⁺ cells compared to other treatment conditions (Fig. 1C and Supplementary Fig. S1A). On examination of the composition of the cell product, the RUS-treated HSPCs had an increased percentage of HSCs (CD34⁺CD133⁺CD90⁺ cells) and decreased percentage of committed progenitors (CD34⁺CD133⁻ cells) and differentiated (CD34⁻) cells than the individual compounds (Supplementary Fig. S2A). This suggests that RUS cocktail supplementation enrich the HSCs additively and thus provides an ideal ex vivo culture system for HSPCs.

To further strengthen the finding that HSCs are enriched on RUS treatment and to test whether HSC enrichment is associated with the proliferation or the maintenance of HSCs, we tested HSPCs from multiple healthy donors for their immunophenotype and proliferation rates. The RUS treatment consistently showed an increased percentage of HSCs, decreased committed progenitors, and differentiated cells over the vehicle. Of note, the proportion of HSCs, early progenitors and differentiated cells in RUS-treated cell products remained similar to that of uncultured HSPCs (day 0) (Fig. 1D and Supplementary Fig. S1B).

The growth kinetics and the carboxyfluorescein succinimidyl ester dye dilution analysis showed a slower proliferation of RUS-treated HSPCs (Supplementary Fig. S2B–D). Furthermore, the proliferation of total cells as well as the differentiated and committed progenitor cells was significantly reduced on RUS treatment. On the contrary, HSC proliferation was twofold higher suggesting preferential expansion of HSCs on RUS treatment (Fig. 1E and F). While committed progenitors are known to proliferate robustly compared to HSCs, the ratio of fold expansion was 1.2 (vs. 0.2 in control) and this phenomenon was specific to the RUS cocktail (Fig. 1G). The mean fluorescence intensity of CD34, CD133, and CD90 in the total cell population remained unaltered confirming that the changes are due to the increased number of HSCs (Supplementary Fig. S2E–G).

RUS treatment increased the absolute number and enriched the CD34⁺CD133⁺CD90⁺CD38⁻CD45RA⁻CD49f⁺ cells (Fig. 1H and Supplementary Figs. S1C and S2H) which is considered to be a stringent marker for the HSCs³³ and also CD34⁺CD133⁺EPCR⁺ cells (Fig. 1I and Supplementary Fig. S2I), the marker for expanded HSCs.^34
–36 These findings support the observation that RUS treatment enriches HSCs by inhibiting proliferation of differentiated and committed progenitor cells and preferentially allowing the proliferation of HSCs.

The dose reduction of RUS caused a decrease in the percentage of HSCs (Supplementary Fig. S3A), and its withdrawal resulted in the reduction of HSCs (Supplementary Fig. S3B), confirming that the effect is associated with the RUS treatment, and is reversible. RUS-mediated increase in HSCs was reproducible in different tested parameters, like with UM171, an analog of UM729²⁵ (Supplementary Fig. S3C), cytokine conditions (Supplementary Fig. S3D and E), stem cell culture media (Supplementary Fig. S3F), oxygen levels (Supplementary Fig. S3G), and cell culture plates (Supplementary Fig. S3H). Media change on day 3 (Supplementary Fig. S3I) and a low culture density (≤2 × 10⁵/mL) (Supplementary Fig. S3J) were found to promote HSC expansion on RUS treatment.

Culture-enriched HSCs resemble functional HSCs in gene sets and stemness characteristics

Gene expression analysis showed that 305 genes were upregulated, and 100 genes were downregulated on RUS treatment (Supplementary Fig. S4A–C). The gene set enrichment analysis (GSEA) with the gene lists for the in vivo repopulating HSCs^34,37,38 showed a significantly high normalized enrichment score (NES) (Fig. 2A–C), confirming the upregulation of stem cell signatures. KEGG2019 pathway analysis showed an upregulation of genes associated with cell adhesion (Supplementary Fig. S4D), which is crucial for long-term engraftment³⁹ and a downregulation of heme metabolism, janus kinase/signal transducers and activators of transcription - 3 (JAK/STAT3) pathway involved in cell division, and transforming growth factor-β-signaling pathways (Supplementary Fig. S4E) that are associated with robust proliferation and exhaustion of HSCs.^40,41

Figure 2.

RUS-treated HSPCs showing gene sets of HSCs and enhanced engraftment in vivo. (A) GSEA of differentially expressed genes in HSPCs cultured for 5 days with vehicle or RUS. Gene list obtained from Fares et al. ³⁴ NES = 2.16, n = 4, donors = 2. (B) GSEA of differentially expressed genes in HSPCs cultured for 5 days with vehicle or RUS. Gene list obtained from Radtke et al.,³⁷ NES = 2.19, n = 4, donors = 2. (C) GSEA of differentially expressed genes in HSPCs cultured for 5 days with vehicle or RUS. Gene list obtained from Velten et al.,³⁸ NES = 2.46, n = 4, donors = 2. (D) The mice were infused with HSPCs that were cultured for 5 days with vehicle or RUS. The human chimerism in the BM was analyzed 4 weeks post transplantation. Donors = 2. (E) The mice were infused with HSPCs that were cultured for 5 days with vehicle or RUS. The human chimerism in the mouse BM was analyzed 16 weeks posttransplantation. Donors = 3. (F) The human chimerism in mouse PB at different weeks postinfusion, n = 4. (G) The multilineage reconstitution of human cells in the mouse BM 16 weeks posttransplantation. Lymphoid cells (CD19 and CD3), myeloid cells (CD13 and CD33), and HSPCs (CD34). n = 10, donors = 3. (H) Limiting dilution analysis. The mice were infused with different doses (50,000, 100,000, 200,000, 400,000, 800,000) of HSPCs that were cultured for 5 days with vehicle or RUS. The human chimerism in the BM was analyzed 16 weeks posttransplantation. Long-term reconstitution frequency was estimated using the ELDA software.⁶² Each dot depicts one mouse. Error bars represent mean ± SEM, ns. *p ≤ 0.05, **p ≤ 0.01 (unpaired t-test, two-tailed). BM, bone marrow; GSEA, gene set enrichment analysis; NES, normalized enrichment score; PB, peripheral blood. Color images are available online.

The RUS-treated HSPCs retained their differentiation potential in the colony formation assay (Supplementary Fig. S5A) and differentiated into megakaryocytes, macrophages, and erythroid cells in suspension culture (Supplementary Fig. S5B–D). In addition, they showed twofold increase in transwell migration in response to stromal cell-derived factor (SDF)-1α stimuli (Supplementary Fig. S5E), twofold decrease in the reactive oxygen species (ROS) levels (Supplementary Fig. S5F), and an increase in the live cells when cultured for 12 days (Supplementary Fig. S5G). The differentiation potential, SDF-1α response, reduced ROS,⁴² and apoptosis indicates that the RUS-treated cells retain the stem cell characteristics in vitro.

HSCs-enriched graft exhibits enhanced engraftment and repopulation in vivo

To evaluate whether RUS treatment preserves functional HSCs, we cultured HSPCs for 5 days and transplanted into irradiated NSG mice. The early phase of HSPC engraftment is critical for gene therapy and the RUS group showed a 2.6-fold higher bone marrow (BM) engraftment of hCD45⁺ cells, 4 weeks posttransplantation (Fig. 2D), suggesting rapid engraftment. Similarly, the RUS group showed a 5.6-fold increase in the long-term engraftment (16 weeks posttransplantation) (Fig. 2E). Peripheral blood (PB) chimerism analysis also showed early and persistent engraftment (Fig. 2F).

All animals of RUS group showed PB engraftment till 16 weeks of infusion (vs. 16.6% in control) (Supplementary Table S1) and retained multilineage repopulation potential, forming human T-lymphoid, B-lymphoid, and myeloid cells with no bias toward specific lineage (Fig. 2G). The limiting dilution analysis indicated 1 in 193,826 RUS-treated cells as long-term-HSCs (vs. 1/1074091 in vehicle), confirming a sixfold increase in stem cell frequency (Fig. 2H). All these data indicate that RUS-treated graft consists of increased number of functional HSCs compared to the graft maintained in regular culture conditions.

HSC culture enrichment is independent of mobilization regimens and disease characteristics

The patient HSPCs are the target cells for gene manipulation in the HSPC gene therapy. To understand whether different mobilization regimens and disease characteristics influence the culture enrichment of HSCs, we first tested the effect of RUS treatment in plerixafor mPB-HSPCs from a sickle cell disease (SCD) patient. The composition of HSPCs in SCD patient was observed to be different from healthy individuals.⁴³ However, consistent with the GCSF mobilized healthy donor cells, RUS treatment inhibited the proliferation of differentiated and committed progenitor cells and supported the proliferation of the HSCs (Fig. 3A). The HSPCs of the β-thalassemia patient that are derived from the stressed BM microenvironment responded to the RUS treatment with an increase in the absolute number of HSCs by twofold (Fig. 3B).

Figure 3.

RUS treatment enriches the HSCs in patient HSPCs and supports the culture of sort enriched HSCs. (A) Absolute number of subpopulations of HSPCs from SCD patient, in day 0 and day 5 of culture with vehicle or RUS. n = 1. (B) Absolute number of subpopulations of HSPCs from β-thalassemia patient, in day 0 and day 5 of culture with vehicle or RUS. n = 1. (C) Absolute number of subpopulations of HSPCs from hemophilia patient, in day 0 and day 5 of culture with vehicle or RUS. n = 2. (D) Number of GEMM colonies obtained in MethoCult analysis after seeding 500 SCD-HSPCs that are cultured with vehicle or RUS for 5 days n = 3. (E) Transwell migration assay; percentage of SCD-HSPCs responded to SDF-1α in the lower chamber. The HSPCs were cultured with vehicle or RUS for 5 days before the assay. n = 2. (F) Transwell migration assay; percentage of β-thalassemia-HSPCs responded to SDF-1α in the lower chamber. The HSPCs were cultured with vehicle or RUS for 5 days before the assay. n = 4. (G) Composition of subpopulations of HSPCs. The unsorted and CD34⁺CD90⁺ sorted cells were cultured with vehicle or RUS for 5 days. The CD34⁺CD90⁺ sorting was performed on day 0 and immediately seeded for culture. n = 3, donors = 2. (H) FACS analysis of CD34⁺CD90⁺ cells in the unsorted and CD34⁺CD90⁺-sorted cells that are cultured for 5 days with vehicle or RUS. n = 3, donors = 2. (I) Number of GEMM colonies obtained in MethoCult analysis after seeding unsorted and CD34⁺CD90⁺-sorted HSPCs that are cultured for 5 days with vehicle or RUS. n = 3, donors = 2. (J) FACS analysis of CD34⁺CD90⁺ cells in the CD34⁺CD90⁻-sorted cells that are cultured for 12 and 120 h. n = 3, donors = 2. (K) Absolute number of TNCs in CD34⁺CD90⁻-sorted HSPCs after 5 days of culture with vehicle or RUS. n = 3, donors = 2. Error bars represent mean ± SEM, ns. *p ≤ 0.05, **p ≤ 0.01 (unpaired t-test, two-tailed). GEMM, granulocyte, erythrocyte, monocyte, megakaryocyte; SCD, sickle cell disease; SDF, stromal cell-derived factor. Color images are available online.

In line with this, the GCSF mPB-HSPCs from a hemophilia patient also showed an increase in the absolute numbers of HSCs (Fig. 3C). The RUS treatment generated a high frequency of granulocyte, erythrocyte, monocyte, megakaryocyte (GEMM) colonies (Fig. 3D), the most primitive progenitor colonies in the colony forming unit (CFU) assay and improved the transwell migration potential of both the SCD and β-thalassemia patient HSPCs (Fig. 3E, F). All these observations support the application of RUS treatment for HSPC gene therapy.

Culture enrichment procedure supports the sort enriched HSCs

The existing approach for HSC enrichment is the fluorescence-activated single cell sorting based sort purification of CD34⁺CD90⁺ cells followed by culturing, to execute the gene manipulation.²¹ We compared the HSC yield obtained on day 5 by sort enrichment and culture enrichment. The sort enriched CD34⁺CD90⁺ HSCs, reduced in their frequency on culture, and the frequency was similar to the HSCs found in the unsorted HSPCs cultured with RUS (Fig. 3G, H and Supplementary Fig. S6A). This effect was also mirrored in the CFU analysis (Fig. 3I). On combining, that is, culturing the sort-enriched CD34⁺CD90⁺ cells with RUS, a higher frequency of HSCs and GEMM colonies were generated (Fig. 3G–I). All these assays suggest the benefit of supplementing RUS for the culture of both the unsorted and sorted cells.

The sorted CD34⁺CD90⁻ cells had a basal frequency of CD34⁺CD90⁺ cells after 12 h of culture with vehicle or RUS. This rules out any treatment mediated conversion of CD34⁺CD90⁻ cells into CD34⁺CD90⁺ cells.⁴⁴ On further analysis, after 120 h, RUS-treated cells showed an increase in the percentage of CD34⁺CD90⁺ cells from 4% to 15%, with a twofold reduction in the proliferation of total cells (Fig. 3J, K). This strengthens the observation of preferential proliferation and enrichment of HSCs with RUS treatment.

Culture enrichment of HSCs increases the frequency of transduced HSCs

As the HSCs resist gene manipulation,^8,45 increasing the HSC population alone will not be beneficial unless these cells are amenable for gene manipulation. To test whether culture enriched HSCs can be manipulated, we transduced the HSPCs with a lenti-green fluorescent protein (GFP) vector and observed that RUS treatment retained the HSC enrichment even after the transduction stress (Fig. 4A and Supplementary Fig. S7A), unaltered overall transduction efficiency (Supplementary Fig. S7B), generated a 4.2-fold increase in the frequency and in the absolute number of GFP⁺CD34⁺CD90⁺ HSCs (Fig. 4B and Supplementary Fig. S7C), and had the ratio of GFP⁺HSCs to GFP⁺ progenitors as 2.9 (vs. 0.4 in control) (Fig. 4C).

Figure 4.

The RUS treatment increases the frequency of lentiviral transduced CD34⁺CD90⁺ HSCs. Top: Scheme of the experiment. Vehicle and RUS-treated HSPCs were transduced with Lenti-GFP vector on day 3 and analyzed by FACS 48 h posttransduction. The treatment was maintained throughout the culture period. Results are presented as mean ± SEM, ns. *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001 (unpaired t-test, two-tailed), n = 8, donors = 3. (A) Percentage of HSPC subpopulations 48 h posttransduction. (B) Percentage of GFP⁺ and GFP⁻ cells in the CD34⁺CD90⁺ and CD34⁺CD90⁻ subpopulations. (C) The ratio of GFP⁺CD34⁺CD90⁺:GFP⁺CD34⁺CD90⁻ cells. (D) Percentage of GFP⁺ and GFP⁻ cells in the CD34⁺CD90⁺ and CD34⁺CD90⁻ subpopulations of SCD-HSPCs. (E) Percentage of GFP⁺CD34⁺CD90⁺ and GFP⁻CD34⁺CD90⁺cells in the HSPCs that were treated with individual small molecules or with the RUS cocktail. (F) Percentage of GFP⁺CD34⁺CD90⁺ and GFP⁻CD34⁺CD90⁺cells. The HSPCs were cultured with vehicle or RUS for 3 days and sorted for CD34⁺CD90⁺ cells. The unsorted and sorted cells were transduced with Lenti-GFP virus and analyzed 48 h posttransduction. Posttransduction, the medium was not supplemented with RUS. n = 2, donor = 1. (G) Percentage of transduction in CD34⁺CD90⁺ cells. The data were analyzed from Fig. 4F. (H) Percentage of GFP⁺CD34⁺CD90⁺ and GFP⁻CD34⁺CD90⁺ cells. HSPCs were cultured with vehicle or RUS and treated with or without CsH, transduced with Lenti-GFP and analyzed 48 h posttransduction. The RUS was maintained posttransduction. n = 6 (donors = 2) (I). Percentage of total GFP⁺ cells from experiment (H). CsH, cyclosporin H; GFP, green fluorescent protein. Color images are available online.

The RUS-treated SCD-HSPCs also showed a high frequency and the absolute number of GFP⁺HSCs and reduced frequency and absolute number of GFP⁺ progenitors over the control (Fig. 4D and Supplementary Fig. S7E and F). Similar results were obtained with the healthy donor HSPCs that were cultured for 5 days (Supplementary Fig. S8A–E).

The ratio of transduction between the CD34⁺CD90⁺ fraction and CD34⁺CD90⁻ fraction in RUS-treated cells was 1.1 (vs. 0.8 in control), suggesting the equal transduction in HSCs and progenitors (Supplementary Fig. S8D). Thus, the availability of more HSCs has resulted in the increase in the frequency of GFP⁺HSCs. This observation was further supported by varying degree of HSC enrichment by individual small molecules and proportional increase in of GFP⁺HSCs (Fig. 4E).

To test whether RUS treatment primes the HSC fraction for transduction, cultured HSPCs were sort-purified for CD34⁺CD90⁺ HSCs and transduced. The RUS treatment showed a higher frequency of GFP⁺HSCs (Fig. 4F) and transduction in HSCs (Fig. 4G and Supplementary Figs. S6B, S7D and S8E). This suggests that the HSC fraction of RUS is more responsive to transduction than that of vehicle.

The RUS-cultured HSPCs and the sorted HSCs had a similar percentage of GFP⁺HSCs, indicating that the treatment generates gene-modified HSCs at a frequency similar to the protocol that involves sort purification and transduction. Culturing the sorted HSCs with RUS further improved the yield of GFP⁺HSCs. RUS treatment also produced fourfold more GFP⁺HSCs than the best-known transduction enhancer Cyclosporin H (CsH).⁴⁶ Combining both had a little effect on the GFP⁺HSCs but increased the overall percentage of GFP⁺ cells (Fig. 4H, I and Supplementary Fig. S6C).

Collectively, the RUS-treated cell product contains a higher frequency of gene-modified HSCs compared to control and this fraction of gene-modified HSCs occupy an equal or a greater portion in the RUS-treated cell product than the gene-modified progenitors. This effect is distinct from conventional gene therapy products that have greater number of gene-modified progenitors.¹

Culture enrichment of HSCs increases the frequency of gene-edited HSCs

To investigate the impact of culture enrichment of HSCs on gene editing, we gene-edited three different loci with Cas9 ribonuleoprotein (RNP) and observed that the RUS treatment retained the HSC enrichment even after the electroporation stress (Fig. 5A), modestly improved indel rates with 72 h of treatment (pre- and postediting) (Fig. 5B and Supplementary Fig. S9A) and showed no alterations in the pattern of indels (Supplementary Fig. S9B).

Figure 5.

The RUS treatment increases the frequency of gene-edited CD34⁺CD90⁺ HSCs. (A) HSPCs were cultured for 3 days with vehicle or RUS and edited with Cas9-RNP. The edited cells were collected 72 h postediting. Percentage of HSPC subpopulations postelectroporation. RUS treatment was maintained throughout the culture period. n = 2, donors = 2. (B) Percentage of InDels. Each dot depicts individual experiments. The regions targeted by Cas9-RNP are indicated in x-axis. (C) Percentage of GFP⁺CD34⁺CD90⁺ and GFP⁻CD34⁺CD90⁺ cells; HSPCs were cultured for 3 days and edited with Cas9-GFP RNP targeting pseudo β-globin locus. The CD34⁺CD90⁺ fraction was analyzed for GFP 3 and 48 h postelectroporation. n = 2, donors = 1. (D) The percentage of GFP within CD34⁺CD90⁺ fraction. (E) The ratio of GFP⁺CD34⁺CD90⁺:GFP⁺CD34⁺CD90⁻ cells in the HSPCs that were cultured with vehicle or RUS. (F) HSPCs were cultured for 3 days and edited with Cas9-RNP targeting AAVS1 and β-globin loci. The cell numbers were calculated 72 h postediting using trypan blue dye exclusion. Donors = 2. (G) MethoCult analysis of HSPCs, edited at different timepoints of culture (24, 48, 120 h) and plated 48 h postelectroporation. n = 1. Results are presented as mean ± SEM, ns.*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 (unpaired t-test, two-tailed). RNP, ribonucleoprotein. Color images are available online.

To test the gene editing in the HSC fraction of HSPCs, we edited with Cas9-GFP RNP, which showed a threefold increase in the frequency of GFP⁺HSCs in the RUS-treated cells at 3 h postelectroporation, which doubled in 48 h (Fig. 5C) pointing that the initial increase in GFP⁺ HSCs is further boosted by preferential proliferation of HSCs. The HSC fraction of RUS-treated cells had twofold more GFP suggesting that the HSC fraction is more permissive for editing compared to the HSC fraction of vehicle (Fig. 5D). These observations were consistent in the HSPCs that were cultured for 5 days before nucleofection with different doses of Cas9-GFP RNP (Supplementary Fig. S9C, D). The ratio analysis of GFP⁺HSCs and GFP⁺ progenitors showed that the RUS-treated cell product had an equal proportion of gene-edited HSCs and progenitors after 48 h of culture, whereas the vehicle had more gene-edited progenitor cells (Fig. 5E). In addition, RUS-treated cells recovered well from electroporation stress (Fig. 5F) and generated a higher number of CFU counts in all the tested time points (Fig. 5G).

Collectively, higher frequency of HSCs (available during gene editing), their enhanced permissiveness for gene-editing, improved viability, and preferential proliferation of HSCs (post-gene editing) contribute to the increased frequency of gene-edited HSCs.

Culture enrichment increases the frequency of gene-modified cells in vivo

Supplementing RUS to the HSPC culture generated an increased frequency of gene-modified HSCs in vitro. To know whether this advantage is retained in vivo, the HSPCs were transduced with the Lenti-GFP vector on day 3 of the culture and infused into NSG mice on day 4 (Supplementary Fig. S10A, B). The RUS-cultured cells displayed up to 11-fold increased human cell engraftment in the mouse BM at 16 weeks posttransplantation (Fig. 6A, B). The frequency of GFP⁺ human cells were also up to 10-fold higher in the BM (Fig. 6C and Supplementary Fig. S10C–G). While no mice showed PB engraftment of gene-modified cells in the control, 50% of mice had PB engraftment in RUS-treated group (Supplementary Fig. S10E), confirming that the RUS treatment enhances the generation of functional gene-modified cells.

Figure 6.

The RUS treatment increases the frequency of gene modified cells in vivo. Top: The outline of the experimental scheme. HSPCs were cultured for 3 days with vehicle or RUS and transduced with Lenti-GFP vector at multiplicity of infection 40 on day 3 and transplanted on day 4. The animals were sacrificed on 16 weeks posttransplantation for mice BM analysis. Error bars represent mean ± SEM, ns. *p ≤ 0.05 (unpaired t-test, two-tailed). (A) Representative FACS plot showing the hCD45⁺GFP⁺ cells in the mouse BM. The inset refers to human cells (left) and GFP percentage in human cells (right). (B) The engraftment of human cells in mouse BM at 16 weeks post infusion. n = 4. (C) The gene-modified human cell (hCD45⁺GFP⁺ cells) chimerism in mouse BM at 16 weeks postinfusion. n = 4. (D).Top: experimental scheme. The HSPCs were cultured for 3 days, gene edited with CCR5 targeting sgRNA on day 3 and transplanted immediately postelectroporation. The animals were sacrificed at 16 weeks for mice BM analysis. Bottom: The engraftment of human cells in mouse PB at 16 weeks postinfusion (mean ± SEM, n = 4). (E) The human cells engraftment in mouse spleen at 16 weeks postinfusion. (F) The engraftment of human cells in mouse BM at 16 weeks postinfusion. (G) The human gene-modified cell (hCD45+gene-edited cells) chimerism in mouse BM at 16 weeks postinfusion. Color images are available online.

The experimental scheme for transplantation of gene-edited cells was specifically designed to negate the positive effects of RUS on the preferential proliferation of HSCs post-gene editing, thus focusing on the availability and amenability of HSC fraction for editing.

The vehicle and RUS-treated HSPCs were gene-edited for the CCR5 locus on day 3 and transplanted immediately postelectroporation. Sixteen weeks posttransplantation, there was 1.5-, 2.2-, and 2.7-fold increase in the engraftment, respectively, in PB, spleen, and BM (Fig. 6D–F). Sanger-sequencing and Inference of CRISPR Edits analysis of BM cells revealed a twofold increase in the frequency of CCR5 gene-edited human cells (Fig. 6G). This confirms that the RUS-mediated improved HSC frequency and permissiveness (pre-gene editing in vitro) resulted in an increase in the frequency of gene-edited cells in vivo. Culturing the cells with RUS post-gene editing will allow the preferential proliferation, further improving the frequency of gene-edited cells in vivo.

DISCUSSION

Accelerated engraftment and high frequency of gene-modified cells in vivo are crucial for the success of the HSPC gene therapy.^47,48 In this regard, we have developed a refined culture system for the HSPCs and made the following important observations; ex vivo culture of mPB-HSPCs with the small molecule cocktail RUS strongly restricts the proliferation of differentiated and progenitor cells and preferentially allows the proliferation of HSCs, resulting in enriched HSCs in the cultured graft and robust engraftment in vivo. These HSCs are permissive for gene manipulation at an enhanced frequency. The preferential expansion of HSCs and their enhanced gene manipulation increases the frequency of in vivo repopulating gene-modified cells up to 10-fold.

In contrast to the strategies applied for expanding UCB-HSPCs, which necessitate several hundred-fold expansions for clinical utility, a single BM harvest can provide sufficient number of cells for manipulation, thus a robust total cell expansion is not crucial for adult HSPCs. Instead, the harvested cells should retain the engraftment potential after ex vivo culture and gene manipulation. The in vivo clonal tracking of lentiviral integration sites demonstrated that the gene-modified cells in transplanted patients are derived from 2,000–50,000 gene-modified HSC clones, despite the infusion of a minimum 6 × 10⁶ cells/kg of patient.^49,50

Studies have documented that poor engraftment of ex vivo cultured HSPCs is due to disruption of adhesion molecules.^16,51,52 The upregulation of adhesion molecules in RUS-treated cells and the enhanced engraftment in the NSG mice demonstrate that the adhesion properties are preserved in RUS-treated cells, thus mediating long-term engraftment in vivo. The individual small molecules of our cocktail expand both the progenitor and HSC population. However, the cocktail usage preferentially expands the HSCs over the progenitors or the differentiated cells. SR1, UM171/729, and Resveratrol increases the UCB-HSPC numbers by inhibiting aryl hydrocarbon receptor, Lysine-specific demethylase 1A, and regulating cell cycle, respectively.^24,25,53,54 These findings suggest that different pathways are being targeted, and the synergized effect of the RUS works in nonoverlapping pathways to inhibit the proliferation of differentiated and progenitor cells during expansion.

Phase I/II clinical trials with SR1 and UM171 have shown that the small molecule expanded UCB-HSPCs engrafted and reconstituted the blood stream with no adverse events^55,56 supporting their application in culturing adult HSPCs. A recent study has shown that on sphingolipid modulation, autophagy is activated in the HSCs, but not in the progenitors, restricting the proliferation of progenitors in the ex vivo culture.³² This supports the notion that the progenitor cells and HSCs respond differentially to culture conditions. The mobilization regimes such as GCSF and plerixafor were shown to mobilize different subpopulations of HSPCs^57,58 and the disease characteristics were also shown to alter the composition of HSPCs.⁴³ Despite these variations, we consistently observed a culture enrichment of HSCs by RUS treatment strengthening its usage in the HSPC gene therapy.

The mechanism by which RUS makes HSC fraction more responsive for gene manipulation is yet to be deciphered. Few small molecules such as CsH reduce the innate immune response during the transduction process, thus enhancing the viral transduction.^46,59 Supplementing CsH with RUS has not additively increased the frequency of GFP⁺ HSCs (Fig. 4H and I). Also, Caraphenol A, a resveratrol trimer, was shown to increase the transduction by suppressing the innate immune response of the HSPCs.⁵⁹ The RUS-treated HSPCs show significant downregulation of JAK STAT3 signaling, which mediates the immune response (Fig. S4E). All these suggest that RUS treatment may have partly reduced the activation of the immune response pathway to make HSCs more permissive to genetic manipulation.

UM171, also have been reported to improve the viral transduction in HSPCs.⁶⁰ Similar to HSC enrichment, we observe an additive effect in the increase of gene manipulated HSCs, upon the cocktail usage. Recent reports linked the gene-editing stress with viability of cells.⁶¹ RUS-treated cells have downregulation of apoptosis pathway, less ROS levels and apoptosis (Supplementary Fig. S5F, G). This could be a potential reason for increased viability in these cells post-gene editing.

Currently, there are efforts to sort the HSCs for gene manipulation procedures.^17,18,21 These protocols first sort the immunophenotypic HSCs, manipulate them in ex vivo culture, and infuse them along with the nonmanipulated progenitor cells to support immediate granulopoiesis after the transplantation. The RUS treatment-mediated enrichment approach is devoid of flow sorting, and the RUS-treated cell products also have gene modified progenitor cells at a lesser frequency. Thus, with this approach, the need for “add-back” of progenitor cells is not required. Interestingly, the yield of gene modified HSCs on RUS treatment matches to the yield obtained by the sorting procedure (Fig. 3H and 4F). The combination of sort-purification and culture enrichment approaches described (Fig. 4F) could be a potential way forward to reap the benefits of both the sort-purification and culture enrichment approaches.

CONCLUSIONS

Culturing HSPCs with the identified small molecule cocktail of Resveratrol, UM729, and SR1 significantly enhances the generation of gene-modified HSCs, a key step in HSPC gene therapy. The high frequency of gene-modified cells is achieved by the preferential proliferation of HSCs and by the enhanced susceptibility of HSCs for gene modification. This procedure should reduce the doses of HSPCs required for gene manipulation and also the usage of viral vectors and gene editing reagents without compromising the rapid and long-term engraftment potential. This will decrease the manufacturing cost of the gene-modified cells and increase the accessibility to HSPC gene therapy.

Footnotes

AUTHORS' CONTRIBUTIONS

Collection and/or assembly of data, data analysis, and interpretation, A.C.C., V.V, K.V.K., S.S., P.B., M.K.K.A., K.C., A.B., N.S.R, and V.R.; administrative support, S.K. and S.K.M.; provision of study material, A.S., S.R.V, and K.M.M; conception and design, financial support, data analysis and interpretation, article writing, and final approval of article, S.T.

ACKNOWLEDGMENTS

The authors thank Dr. Somadutta Dhir for her help in analyzing GSEA; Dr. Sandya Rani for her help with flow cytometry sample acquisition; Dr. Vigneshwar for maintaining the NSG mice.

AUTHOR DISCLOSURE

No competing financial interests exist.

FUNDING INFORMATION

This work was funded by the Department of Biotechnology, Government of India (grant no. BT/PR26901/MED/31/377/2017). A.C.C. is funded by an ICMR-SRF fellowship (2019-4018/SCR-BMS). K.V.K is funded by a DST-INSPIRE fellowship (IF180018), S.S. by a DBT-JRF fellowship, and P.B by a CSIR-JRF fellowship.

Supplementary Material

Supplementary File

Supplementary Table S1

Supplementary Figure S1

Supplementary Figure S2

Supplementary Figure S3

Supplementary Figure S4

Supplementary Figure S5

Supplementary Figure S6

Supplementary Figure S7

Supplementary Figure S8

Supplementary Figure S9

Supplementary Figure S10

References

Naldini

Genetic engineering of hematopoiesis: current stage of clinical translation and future perspectives. EMBO Mol Med;11:e9958.

Thompson

, Walters

, Kwiatkowski

, et al. Gene therapy in patients with transfusion-dependent β-thalassemia. N Engl J Med, 2018; 378:1479–1493.

Ribeil

J-A

, Hacein-Bey-Abina

, Payen

, et al. Gene therapy in a patient with sickle cell disease. N Engl J Med, 2017; 376:848–855.

Ferrua

, Cicalese

, Galimberti

, et al. Lentiviral haemopoietic stem/progenitor cell gene therapy for treatment of Wiskott-Aldrich syndrome: interim results of a non-randomised, open-label, phase 1/2 clinical study. Lancet Haematol, 2019; 6:e239–e253.

Staal

FJT

, Aiuti

, Cavazzana

. Autologous stem-cell-based gene therapy for inherited disorders: state of the art and perspectives. Front Pediatr, 2019; 7:443.

Frangoul

, Altshuler

, Cappellini

, et al. CRISPR-Cas9 gene editing for sickle cell disease and β-thalassemia. N Engl J Med, 2021; 384:252–260.

, Wang

, Liu

, et al. CRISPR-edited stem cells in a patient with HIV and acute lymphocytic leukemia. N Engl J Med, 2019; 381:1240–1247.

Genovese

, Schiroli

, Escobar

, et al. Targeted genome editing in human repopulating haematopoietic stem cells. Nature, 2014; 510:235–240.

Morris

, Fox

, Chakraverty

, et al. Gene therapy for Wiskott-Aldrich syndrome in a severely affected adult. Blood, 2017; 130:1327–1335.

10.

Kohn

, Booth

, Kang

, et al. Lentiviral gene therapy for X-linked chronic granulomatous disease. Nat Med, 2020; 26:200–206.

11.

Aiuti

, Biasco

, Scaramuzza

, et al. Lentiviral hematopoietic stem cell gene therapy in patients with wiskott-Aldrich syndrome. Science, 2013; 341:1233151.

12.

Gomez-Ospina

, Scharenberg

, Mostrel

, et al. Human genome-edited hematopoietic stem cells phenotypically correct Mucopolysaccharidosis type I. Nat Commun, 2019; 10:4045.

13.

Grez

, Reichenbach

, Schwäble

, et al. Gene therapy of chronic granulomatous disease: the engraftment dilemma. Mol Ther, 2011; 19:28–35.

14.

Mazurier

, Gan

, McKenzie

, et al. Lentivector-mediated clonal tracking reveals intrinsic heterogeneity in the human hematopoietic stem cell compartment and culture-induced stem cell impairment. Blood, 2004; 103:545–552.

15.

Piras

, Riba

, Petrillo

, et al. Lentiviral vectors escape innate sensing but trigger p53 in human hematopoietic stem and progenitor cells. EMBO Mol Med, 2017; 9:1198–1211.

16.

Szilvassy

, Meyerrose

, Ragland

, et al. Homing and engraftment defects in ex vivo expanded murine hematopoietic cells are associated with downregulation of β1 integrin. Exp Hematol, 2001; 29:1494–1502.

17.

Zonari

, Desantis

, Petrillo

, et al. Efficient ex vivo engineering and expansion of highly purified human hematopoietic stem and progenitor cell populations for gene therapy. Stem Cell Reports, 2017; 8:977–990.

18.

Radtke

, Humbert

, Kiem

. Sorting out the best: Enriching hematopoietic stem cells for gene therapy and editing. Mol Ther, 2018; 26:2328–2329.

19.

Baldwin

, Urbinati

, Romero

, et al. Enrichment of human hematopoietic stem/progenitor cells facilitates transduction for stem cell gene therapy. Stem Cells, 2015; 33:1532–1542.

20.

Masiuk

, Brown

, Laborada

, et al. Improving gene therapy efficiency through the enrichment of human hematopoietic stem cells. Mol Ther, 2017; 25:2163–2175.

21.

Radtke

, Pande

, Cui

, et al. Purification of human CD34+CD90+ HSCs reduces target cell population and improves lentiviral transduction for gene therapy. Mol Ther Methods Clin Dev, 2020; 18:679–691.

22.

Araki

, Mahmud

, Milhem

, et al. Expansion of human umbilical cord blood SCID-repopulating cells using chromatin-modifying agents. Exp Hematol, 2006; 34:140–149.

23.

Peled

, Shoham

, Aschengrau

, et al. Nicotinamide, a SIRT1 inhibitor, inhibits differentiation and facilitates expansion of hematopoietic progenitor cells with enhanced bone marrow homing and engraftment. Exp Hematol, 2012; 40:342–355.e1.

24.

Heinz

, Ehrnström

, Schambach

, et al. Comparison of different cytokine conditions reveals resveratrol as a new molecule for ex vivo cultivation of cord blood-derived hematopoietic stem cells. Stem Cells Transl Med, 2015; 4:1064–1072.

25.

Fares

, Chagraoui

, Gareau

, et al. Pyrimidoindole derivatives are agonists of human hematopoietic stem cell self-renewal. Science, 2014; 345:1509–1512.

26.

Cutler

, Multani

, Robbins

, et al. Prostaglandin-modulated Umbilical cord blood hematopoietic stem cell transplantation. Blood, 2013; 122:3074–3081.

27.

Anjos-Afonso

, Currie

, Palmer

, et al. CD34- cells at the apex of the human hematopoietic stem cell hierarchy have distinctive cellular and molecular signatures. Cell Stem Cell, 2013; 13:161–174.

28.

Zou

, Zou

, Wang

, et al. Inhibition of p38 MAPK activity promotes ex vivo expansion of human cord blood hematopoietic stem cells. Ann Hematol, 2012; 91:813–823.

29.

Gao

, Yang

, Shen

, et al. Small-molecule inhibitors targeting INK4 protein p18 INK4C enhance ex vivo expansion of haematopoietic stem cells. Nat Commun, 2015; 6:1–10.

30.

Guo

, Huang

, Cooper

, et al. Glucocorticoid hormone-induced chromatin remodeling enhances human hematopoietic stem cell homing and engraftment. Nat Med, 2017; 23:424–428.

31.

Boitano

, Wang

, Romeo

, et al. Aryl hydrocarbon receptor antagonists promote the expansion of human hematopoietic stem cells. Science, 2010; 329:1345–1348.

32.

Xie

, Garcia-Prat

, Voisin

, et al. Sphingolipid modulation activates proteostasis programs to govern human hematopoietic stem cell self-renewal. Cell Stem Cell, 2019; 25:639–653.e7.

33.

Notta

, Doulatov

, Laurenti

, et al. Isolation of single human hematopoietic stem cells capable of long-term multilineage engraftment. Science, 2011; 333:218–221.

34.

Fares

, Chagraoui

, Lehnertz

, et al. EPCR expression marks UM171-expanded CD34+ cord blood stem cells. Blood, 2017; 129:3344–3351.

35.

Radtke

, Görgens

, Kordelas

, et al. CD133 allows elaborated discrimination and quantification of haematopoietic progenitor subsets in human haematopoietic stem cell transplants. Br J Haematol, 2015; 169:868–878.

36.

Subramaniam

, Talkhoncheh

, Magnusson

, et al. Endothelial protein C receptor (EPCR) expression marks human fetal liver hematopoietic stem cells. Haematologica, 2019; 104:e47–e50.

37.

Radtke

, Adair

, Giese

, et al. A distinct hematopoietic stem cell population for rapid multilineage engraftment in nonhuman primates. Sci Transl Med, 2017; 9:eaan1145.

38.

Velten

, Haas

, Raffel

, et al. Human haematopoietic stem cell lineage commitment is a continuous process. Nat Cell Biol, 2017; 19:271–281.

39.

Tomellini

, Fares

, Lehnertz

, et al. Integrin-α3 is a functional marker of ex vivo expanded human long-term hematopoietic stem cells. Cell Rep, 2019; 28:1063–1073.e5.

40.

Kirschner

, Chandra

, Kiselev

, et al. Proliferation drives aging-related functional decline in a subpopulation of the hematopoietic stem cell compartment. Cell Rep, 2017; 19:1503–1511.

41.

Wang

, Dong

, Zhang

, et al. TGF-β1 negatively regulates the number and function of hematopoietic stem cells. Stem Cell Reports, 2018; 11:274–287.

42.

Cabezas-Wallscheid

, Buettner

, Sommerkamp

, et al. Vitamin A-retinoic acid signaling regulates hematopoietic stem cell dormancy. Cell, 2017; 169:807–823.e19.

43.

Tolu

, Wang

, Yan

, et al. Characterization of hematopoiesis in sickle cell disease by prospective isolation of stem and progenitor cells. Cells, 2020; 9:2159.

44.

Papa

, Zimran

, Djedaini

, et al. Ex vivo human HSC expansion requires coordination of cellular reprogramming with mitochondrial remodeling and p53 activation. Blood Adv, 2018; 2:2766–2779.

45.

Lomova

, Clark

, Campo-Fernandez

, et al. Improving gene editing outcomes in human hematopoietic stem and progenitor cells by temporal control of DNA repair. Stem Cells, 2019; 37:284–294.

46.

Petrillo

, Thorne

, Unali

, et al. Cyclosporine H overcomes innate immune restrictions to improve lentiviral transduction and gene editing in human hematopoietic stem cells. Cell Stem Cell, 2018; 23:820–832.e9.

47.

Ferrari

, Thrasher

, Aiuti

. Gene therapy using haematopoietic stem and progenitor cells. Nat Rev Genet, 2021; 22:216–234.

48.

Cavazzana

, Bushman

, Miccio

, et al. Gene therapy targeting haematopoietic stem cells for inherited diseases: progress and challenges. Nat Rev Drug Discov, 2019; 18:447–462.

49.

Scala

, Basso-Ricci

, Dionisio

, et al. Dynamics of genetically engineered hematopoietic stem and progenitor cells after autologous transplantation in humans. Nat Med, 2018; 24:1683–1690.

50.

Six

, Guilloux

, Denis

, et al. Clonal tracking in gene therapy patients reveals a diversity of human hematopoietic differentiation programs. Blood, 2020; 135:1219–1231.

51.

Larochelle

, Gillette

, Desmond

, et al. Bone marrow homing and engraftment of human hematopoietic stem and progenitor cells is mediated by a polarized membrane domain. Blood, 2012; 119:1848–1855.

52.

Kallinikou

, Anjos-Afonso

, Blundell

, et al. Engraftment defect of cytokine-cultured adult human mobilized CD34+ cells is related to reduced adhesion to bone marrow niche elements. Br J Haematol, 2012; 158:778–787.

53.

J C

S G

, JF

, et al. UM171 preserves epigenetic marks that are reduced in ex vivo culture of human HSCs via potentiation of the CLR3-KBTBD4 complex. Cell Stem Cell, 2021; 28:48–62.e6.

54.

A S, K Ž

T, et al. Lysine-specific demethylase 1A restricts ex vivo propagation of human HSCs and is a target of UM171. Blood, 2020; 136:2151–2161.

55.

Wagner

, Brunstein

, Boitano

, et al. Phase I/II trial of StemRegenin-1 expanded umbilical cord blood hematopoietic stem cells supports testing as a stand-alone graft. Cell Stem Cell, 2016; 18:144–155.

56.

Cohen

, Roy

, Lachance

, et al. Hematopoietic stem cell transplantation using single UM171-expanded cord blood: a single-arm, phase 1–2 safety and feasibility study. Lancet Haematol, 2020; 7:e134–e145.

57.

Donahue

, Jin

, Bonifacino

, et al. Plerixafor (AMD3100) and granulocyte colony-stimulating factor (G-CSF) mobilize different CD34+ cell populations based on global gene and microRNA expression signatures. Blood, 2009; 114:2530–2541.

58.

Teipel

, Oelschlägel

, Wetzko

, et al. Differences in cellular composition of peripheral blood stem cell grafts from healthy stem cell donors mobilized with either granulocyte colony-stimulating factor (G-CSF) alone or G-CSF and Plerixafor. Biol Blood Marrow Transplant, 2018; 24:2171–2177.

59.

Ozog

, Timberlake

, Hermann

, et al. Resveratrol trimer enhances gene delivery to hematopoietic stem cells by reducing antiviral restriction at endosomes. Blood, 2019; 134:1298–1311.

60.

Ngom

, Imren

, Maetzig

, et al. UM171 enhances lentiviral gene transfer and recovery of primitive human hematopoietic cells. Mol Ther Methods Clin Dev, 2018; 10:156–164.

61.

Schiroli

, Conti

, Ferrari

, et al. Precise gene editing preserves hematopoietic stem cell function following transient p53-mediated DNA damage response. Cell Stem Cell, 2019; 24:551–565.e8.

62.

, Smyth

. ELDA: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays. J Immunol Methods, 2009; 347:70–78.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.13 MB

0.14 MB

0.18 MB

0.23 MB

0.19 MB

0.24 MB

0.20 MB

0.19 MB

0.25 MB

0.15 MB

0.16 MB

0.09 MB