Expanding human hematopoietic stem cells by blocking ferroptosis

Abstract

Disclosed herein are methods of expanding hematopoietic stem cells (HSC) ex vivo and their uses thereof, the methods comprising administrating a radical trapping antioxidant (RTA) to the HSC isolated from a subject, wherein the RTA blocks ferroptosis in the HSC and increases HSC expansion. Also disclosed herein are methods of treating blood disorders, such as, for example, sickle cell disease or β-thalassemia. Further disclosed is a kit for use of HSC expansion.

Description

[0001] PCT / US25 / 47496 23 September 2025 (23.09.2025)

[0002] ATTORNEY DOCKET NO. 11624-021WO1

[0003] EXPANDING HUMAN HEMATOPOIETIC STEM CELLS BY BLOCKING FERROPTOSIS

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS

[0005] This application claims the benefit of priority to U.S. Provisional Application No.

[0006] 5 63 / 698,315, filed September 24, 2024, the disclosure of which is incorporated herein by reference in its entirety.

[0007] STATEMENT OF GOVERNMENT INTEREST

[0008] This invention was made with government support under Grant Nos. DK103794, CA265726, CA292941, CA278393 and HL146500 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0009] BACKGROUND

[0010] Tremendous advances in allogeneic hematopoietic transplantation from diverse cell sources and genome engineering of autologous hematopoietic stem cells (HSCs) have resulted in curative treatments for hundreds of thousands of patients. However, the application of these

[0011] 15 approaches has been limited by inadequate cell doses in many instances. Current approaches to collect and manipulate human HSCs are associated with limitations, such as, the ability to maintain, expand, and genetically modify HSCs ex vivo without compromising their functional properties for effective transplantation. Even in successful trials, such as the recently reported and now approved use of CRISPR / Cas9 genome editing of the BCL11A +58 enhancer to cure

[0012] 20 sickle cell disease, all patients required prolonged waiting periods for apheresis between 3-18 days after HSC mobilization to collect sufficient numbers of cells.

[0013] Thus, there is a need for methods or systems to expand hematopoietic stem cells used in the treatment of blood disorders, such as, for example, sickle cell disease.

[0014] SUMMARY

[0015] The present invention relates to methods of expanding hematopoietic stem cells (HSC) ex vivo, in vivo, or in vitro and their uses thereof, the methods comprising administrating a radical trapping antioxidant (RTA) such as liproxstatin-1 (Lip-1), ferrostatin-1 (Fer-1), or a functional derivative thereof, to the HSC isolated from a subject, wherein the RTA blocks ferroptosis in the

[0016] 30 HSC and increases HSC expansion. In some aspects, the subject is a human.

[0017] In one aspect, as disclosed herein is a method of reducing ferroptosis by at least 10% in a hematopoietic stem cell (HSC) population treated with an RTA compared to a control in which PCT / US25 / 47496 23 September 2025 (23.09.2025)

[0018] ATTORNEY DOCKET NO. 11624-021WO1

[0019] RTA is not administered. In an additional aspect, cells are expanded by at least 10% compared to a control in which an RTA is not administered.

[0020] In one aspect, the HSC is supplemented with an RTA and at least one additional composition. In some aspects, the additional composition is 7-dehydrocholesterol.

[0021] In one aspect, as disclosed herein, is a method of treating a blood disorder in a subject, comprising, isolating a hematopoietic stem cell (HSC) from a sample from a subject; administering at least one radical trapping antioxidant, such as Lip-1, Fer-1, or a functional derivative thereof to the HSC in culture, wherein the RTA blocks ferroptosis in the HSC; genetically engineering the HSC, and administering to a subject the engineered hematopoietic stem cell. In some embodiments, engineering the HSC comprises introducing a mutation in an adeno-associated virus integration site 1 (AAVS1) locus, a CD33 locus or a BCL11A +58 enhancer locus using ex vivo gene editing, wherein the mutation increases expansion of HSCs compared to a control. In some aspects, administration of an RTA protects HSCs from loss-post editing and preserves edited cells. In further embodiments, the genetic engineering of the HSC improves the function of blood cells. In some embodiments, the blood disorder is severe acquired immune deficiency, metabolic diseases, muscular dystrophy, myotonic dystrophy, genetic blood disorders, congenital neutropenia, Diamond-Blackfan anemia, Shwachman- Diamond syndrome, telomerase disorders, acquired aplastic anemia, bone marrow failure syndromes, immunodeficiency syndromes, lysosomal storage diseases, hemoglobinopathies including sickle cell disease and P-thalassemia, Fanconi anemia, leukodystrophies, congenital dyserythropoietic anemia, severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome, chronic granulomatous disease, osteopetrosis, mucopolysaccharidoses, Gaucher disease, Niemann-Pick disease, Hurler syndrome, Krabbe disease, metachromatic leukodystrophy, pyruvate kinase deficiency, and X-linked adrenoleukodystrophy. In some embodiments, the ex vivo gene-editing comprises administrating an endonuclease and a synthetic base-modified single-guide RNAs (sgRNAs) to the HSC. In some embodiments, the endonuclease is Cas9. In some embodiments, the treatment is autologous. In other embodiments the treatment is allogenic.

[0022] In any of the preceding aspects, the HSC is isolated from cord blood or peripheral blood or bone marrow of a subject. In some embodiments, administrating an RTA to the HSC increases expression of ferroptosis protective gene, GPX4, 7-dehydrocholesterol levels, ribosome biogenesis and / or cholesterol biosynthesis in the HSC compared to a control. In some embodiments, 7-dehydrocholesterol is an inhibitor of ferroptosis and is administered in addition to an RTA. In some embodiments, administrating an RTA to the HSC decreases cell PCT / US25 / 47496 23 September 2025 (23.09.2025)

[0023] ATTORNEY DOCKET NO. 11624-021WO1 proliferation and / or lipid peroxidation in the HSC compared to a control.

[0024] In one aspect, as disclosed herein, is a kit for use in the expansion of hematopoietic stem cells, comprising a medium appropriate for culturing stem cells and a radical trapping antioxidant (RTA). In some embodiments, the medium appropriate for culturing stem cells is a standard serum-free medium. In some embodiments, the medium appropriate for culturing stem cells is a chemically-defined cytokine-free medium. In some embodiments the kit consists of at least one additional composition. In some embodiments, the additional composition is 7- dehydrocholesterol .

[0025] Additional aspects and advantages of the disclosure will be set forth, in part, in the detailed description and any claims which follow, and in part will be derived from the detailed description or can be learned by practice of the various aspects of the disclosure. The advantages described below will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosure.

[0026] BRIEF DESCRIPTION OF THE FIGURES

[0027] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain examples of the present disclosure and together with the description, serve to explain, without limitation, the principles of the disclosure. Like numbers represent the same elements throughout the figures.

[0028] Figs. 1A-1G show ferroptosis prevention boosts the expansion of Long-term (LT) -HSC regardless of HSPC source or culture condition. Fig. 1A shows the experimental workflow. mPB- or CB -derived HSPCs were cultured for up to 14 days in a serum-free expansion medium in the presence or absence of ferroptosis inhibitors (Lip-1, SS, and -ME). Fig. IB shows cell viability analysis of mPB-HSPCs on day 14 conducted using flow cytometry. The Apotracker probe allowed the detection of dead cells (n=9,3,3,3,3,9,4). Fig. 1C shows the percentage of LT- HSCs in mPB cells at day 14. LT-HSC population was defined as CD34+CD45RA" CD133+CD90+EPCR+ITGA3+(n=3). Student’s t-test. Fig. ID shows the fold change of LT-HSC at day 14 calculated as the ratio of Lip-1 group %LT-HSC / untreated group %LT-HSC (n=9). One sample t- and Wilcoxon test. Fig. IE shows cell viability analysis of CB-HSPCs on day 14 conducted using flow cytometry. The Apotracker probe allowed the detection of dead cells (n=6,4,4,6,4). Fig. IF shows percentage of LT-HSCs (CD34+CD45RA_ PCT / US25 / 47496 23 September 2025 (23.09.2025)

[0029] ATTORNEY DOCKET NO. 11624-021WO1

[0030] CD133+CD90+EPCR+ITGA+) in CB cells at day 14 (n=6,4,4,6,4). Student’s t-test. Fig. 1G shows fold change of LT-HSC at day 14 calculated as the ratio of Lip-1 group %LT-HSC / untreated group %LT-HSC (n=6). One sample t-test. Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean ± SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0031] Figs. 2A-2I show ferroptosis inhibition enhances LT-HSC expansion and enriches molecularly-defined HSCs in chemically-defined clusters. Fig. 2A shows experimental workflow. CB-derived HSPCs were cultured for up to 3 weeks in a cytokine-free expansion medium in the presence or absence of ferroptosis inhibitor (Lip-1). Fig. 2B shows percentage of primitive HSPCs (defined as CD34+CD45RA CD90+) in CB cells at day 21 (n=4 per condition). Student’s t-test. Fig. 2C shows percentage of ST-HSCs (CD34+CD45RA CD90+EPCR+) in CB cells at day 21 (n=4). Student’s t-test. Fig. 2D shows percentage of LT-HSC (CD34+CD45RA_CD90+EPCR+ITGA3+) in CB cells at day 21 (n=4). Student’s t-test. Fig. 2E shows fold change of LT-HSC at day 21 calculated as the ratio of Lip- 1 group %LT-HSC / untreated group %LT- HSC (n=4). One sample t- test. Fig. 2F shows UMAP of 29,096 scRNA-seq cells sorted for HSPCs (CD34+CD45RA-CD90+) based on annotated cell population, comprising two conditions with or without Lip-1 treatment for 10 days (n=2 per condition). Fig. 2G shows stacked bar plot showing the proportion of cell types assigned to individual samples. Fig. 2H shows UMAPs illustrating the cell state density between Lipl-treated and untreated control cells. The grey-dotted eclipse indicates the HSC compartment. Fig. 21 shows box plot of z-score normalized HSC signature expression of all the cells of the untreated and Lip-1 groups (n=2 per condition). The significance of differences in the two conditions was calculated based on a two- sided Wilcoxon rank-sum test. Each dot represents an independent donor unless otherwise indicated and all the data are represented as Mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0032] Figs. 3A-3I show Lip-1 supplementation improves cord blood HSPC in vivo repopulation capacity. Fig. 3A shows CB-derived HSPCs from a single donor were split into two groups and cultured for 7 days in a cytokine-free expansion medium either with or without Lip-1. Each condition was then transplanted into four separate NBSGW recipient mice (n=4 per group). Fig. 3B shows the percentage of human CD45+cells measured at the indicated time points in the PB of mice transplanted with HSPCs cultured as indicated (n=4). Student’s t-test (calculated at the last time point for PB). Fig. 3C shows the percentage of CD45+cells measured at 15w in the BM of mice transplanted with HSPCs cultured as indicated (n=4). Student’s t-test (calculated at the last time point for PB). Fig. 3D shows percentage of human CD34+cells measured at 15w in PCT / US25 / 47496 23 September 2025 (23.09.2025)

[0033] ATTORNEY DOCKET NO. 11624-021WO1 the BM of mice transplanted with HSPCs cultured as indicated (n=4). Student’s t-test. Fig. 3E shows estimation of relative HSC required for the observed reconstitution levels in Fig. 3C (n=4). Student’s t-test. Fig. 3F shows the number of colonies formed by BM-derived CD34+cells purified from mice in Fig. 3C at 15 weeks post-transplantation (n=4). Student’s t-test on total colony numbers. Fig. 3G shows percentage of human CD45+cells measured at 8 weeks in the PB of mice transplanted with HSPCs collected from the BM of primary recipients in Fig. 3C. Mann-Whitney test. Each dot represents an independent mouse and all the data are represented as Mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Fig. 3H shows percentage of human CD45+cells measured at 15 weeks in the BM of mice in Fig. 3G (n=5 per group). Mann- Whitney test, Each dot represents an independent mouse and all the data are represented as Mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Fig. 31 shows the percentage of human CD45+ cells measured at 15 w in the BM of mice in Fig. 3G (n=5 per group). Mann- Whitney test, Each dot represents an independent mouse and all the data are represented as Mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0034] Figs. 4A-4D show improved engraftment and progenitor output of adult-derived HSPCs upon Lip-1 treatment. Fig. 4A shows that mPB-derived HSPCs from a single donor were split into two groups and cultured for 4 days in a serum-free expansion medium either with or without Lip-1. Each condition was then transplanted into eight separate NBSGW recipient mice (n=8 per group). Fig. 4B shows the percentage of human CD45+cells measured at 12w in the BM of mice transplanted with HSPCs cultured as indicated (n=8 per group). Mann- Whitney test. Each dot represents an independent mouse and all the data are represented as Mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Fig. 4C shows the percentage of human CD34+45RA’90+cells measured at 12w in the BM of mice transplanted with HSPCs cultured as indicated (n=8 per group). Mann-Whitney test. Each dot represents an independent mouse and all the data are represented as Mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Fig. 4D shows estimation of relative HSC required for the observed reconstitution levels in Fig. 4B (n=8 per group). Mann- Whitney test. Each dot represents an independent mouse and all the data are represented as Mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0035] Figs. 5A-5K show Lip-1 reduces HSPCs proliferation and lipid peroxidation while increasing GPX4 expression as well as ribosomal and cholesterol synthesis pathways. Fig. 5A shows a growth curve of mPB-derived HSPCs cultured for up to 14 days in a serum-free expansion medium in the presence or absence of Lip- 1. Fold increase was calculated as the PCT / US25 / 47496 23 September 2025 (23.09.2025)

[0036] ATTORNEY DOCKET NO. 11624-021WQ1 number of cells counted at an indicated time point over the number of cells at day 0 (n=5 per condition). Student’s t-test. Fig. 5B shows number of cellular divisions performed by mPB- derived CD34+CD45RA CD90+on day 14. Up to 180 cells were analyzed. Mann- Whitney test. Fig. 5C shows relative expression of GPX4 gene at day 14. Fold change was calculated relatively to untreated condition (n=4). Student’s t-test. Each dot represents an independent mouse and all the data are represented as Mean + SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Fig. 5D shows representative flow cytometric histogram (top) of oxidized BODIPY dye of CD34+CD45RA CD90+treated with 25 pM Erastin for 6h on day 14. Quantification of lipid peroxidation level (bottom) measured as the ratio of oxidized and non-oxidized BODIPY dye (n=3 per condition). Each dot represents an independent mouse and all the data are represented as Mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Fig. 5E shows a volcano plot showing the differential expression between Untreated and Lip-1 cells, highlighting genes involved in ribosome biogenesis. Fig. 5F shows a representative flow cytometric histogram (top) showing O-propargyl-puromycin (OP-puro) incorporation to assess protein synthesis in mPB- derived CD34+CD45RA CD90+cells cultured for 4 days in serum-free expansion medium with or without Lip-1. Quantification (bottom) of fold change in translation rates is shown. Cycloheximide (CX) was used as a negative control to block protein synthesis (n=3 per condition). One sample t-test. Each dot represents an independent mouse and all the data are represented as Mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Fig. 5G shows a volcano plot showing the differential expression between Untreated and Lip-1 cells, highlighting genes involved in cholesterol biosynthetic process. Fig. 5H shows a schematic representation of Cholesterol production pathway. In italics are reported the enzymes responsible for the specific conversion and the arrows indicate the trend of their expression in the Lip-1 condition. Fig. 51 shows relative quantification of 7-DHC and cholesterol concentrations in CD34+CD45RA CD90’ and CD34+CD45RA CD90+cells sorted from untreated or Lip-1 samples (n=4,4,2,2). Student’s t-test. Each dot represents an independent mouse and all the data are represented as Mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Figure 5J shows the percentage of LT-HSCs (CD34+CD45RA CD133+CD90+EPCR+ITGA+) in mPB cells at day 7 and 14 cultured in serum-free medium supplemented with the indicated doses of 7-DHC (n=3 per condition). Friedman test followed by Dunn’s multiple comparison test. Each dot represents an independent mouse and all the data are represented as Mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Fig. 5K shows the fold-change of LT-HSCs (CD34+CD45RA CD133+CD90+EPCR+ITGA+) in mPB cells at day 7 and 14 cultured in serum- free medium supplemented with the indicated doses of 7-DHC (n=3 per condition). Friedman PCT / US25 / 47496 23 September 2025 (23.09.2025)

[0037] ATTORNEY DOCKET NO. 11624-021WO1 test followed by one sample t-test. Each dot represents an independent donor unless otherwise indicated and all the data are represented as Mean + SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0038] Figs. 6A-6N show Lip-1 enhances the expansion of genome engineered HSCs without impacting editing or phenotypic outcomes. Fig. 6A shows the percentage of edited HSPCs on day 14 (n=4 per condition). Each dot represents an independent donor unless otherwise indicated and all the data are represented as Mean + SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Fig. 6B shows the fold change of LT-HSC at day 14 calculated as the ratio of Lip-1 group %LT-HSC / untreated group %LT-HSC (n=4 per condition). One sample t-test. Each dot represents an independent donor unless otherwise indicated and all the data are represented as Mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Fig. 6C shows the percentage of cells expressing CD33 surface marker (n=3 per condition). Fig. 6D shows the Fold change of LT-HSC at day 14 calculated as the ratio of Lip-1 group %LT-HSC / untreated group %LT-HSC (n=4 per condition). One sample t-test. Each dot represents an independent donor unless otherwise indicated and all the data are represented as Mean + SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Fig. 6E shows the percentage of edited HSPCs on day 21(n=3 per condition.). Each dot represents an independent donor unless otherwise indicated and all the data are represented as Mean + SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Fig. 6F shows cell viability analysis of CB-HSPCs on day 14 conducted using flow cytometry. The Apotracker probe allowed the detection of dead cells (n=3 per condition). Fig. 6G shows the percentage of LT-HSCs (CD34+CD45RA CD133+CD90+EPCR+ITGA3+) at day 21 (n=3 per condition). Student’s t-test. Each dot represents an independent donor unless otherwise indicated and all the data are represented as Mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Fig. 6H shows the fold change of LT-HSC at day 21 calculated as the ratio of Lip-1 group %LT-HSC / untreated group %LT-HSC (n=3 per condition). One sample t- test. Each dot represents an independent donor unless otherwise indicated and all the data are represented as Mean + SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Fig. 61 shows the experimental workflow of in vitro erythroid differentiation experiments. mPB -derived HSPCs were cultured for 2 days in a serum- free expansion medium in the presence or absence of Lip-1. Cells were Cas9-edited targeting BCL11A enhancer (DHS +58) and immediately seeded either in red blood cells (RBC) differentiation medium or HSC medium. Fig. 6J shows the percentage of edited cells on day 7 of HSC culture or day 12 of RBC differentiation (n=3 per condition). Each dot represents an independent donor unless otherwise indicated and all the data PCT / US25 / 47496 23 September 2025 (23.09.2025)

[0039] ATTORNEY DOCKET NO. 11624-021WO1 are represented as Mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; **:|:*p < 0.0001. Fig. 6K shows the fold change of LT-HSC at day 21 calculated as the ratio of Lip-1 group %LT-HSC / untreated group %LT-HSC (n=3 per condition). One sample t-test. Each dot represents an independent donor unless otherwise indicated and all the data are represented as Mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Fig. 6L shows the analysis of culture composition during divergent phases of RBC differentiation (n=3 per condition). Fig. 6M shows the percentage of / -globin genes at day 17 (Phase III) of erythroid differentiation (n=3 per condition). Kruskal-Wallis’s test. Each dot represents an independent donor unless otherwise indicated and all the data are represented as Mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Fig. 6N shows HbF levels measured by flow cytometry on day 17 (Phase III) of erythroid differentiation (n=3 per condition). Kruskal- Wallis test. Each dot represents an independent donor unless otherwise indicated and all the data are represented as Mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0040] Figs. 7A-7B show phenotypic characterization of in vitro cultured HSPCs. Fig. 7A shows representative plots and gating strategy for subset composition of HSPCs cultured according to the standard protocol. Firstly, dead cells were eliminated (Apotracker+) from the subsequent analysis, then, gating on live cells (Apotracker ), CD34+CD45RA" cells were identified. Within this gate, CD133+CD90+(primitive HSPCs) were further gated and discriminated between EPCRHTGA3’ (ST-HSC) and EPCR+ITGA3+(LT-HSC) subsets. Fig. 7B shows representative plots and gating strategy for subset composition of HSPCs cultured according to the chemical defined cytokine-free protocol. Firstly, dead cells were eliminated (Apotracker+) from the subsequent analysis, then, gating on live cells (Apotracker ), CD34+CD45RA cells were identified. Within this gate, CD 133+CD90+(primitive HSPCs) were further gated to discriminate between EPCR+ITGA3’ (ST-HSC) and EPCR+ITGA3+(LT-HSC) subsets.

[0041] Figs. 8A-8R show LT-HSC expansion is uniquely driven by RTAs, not by other metabolic inputs. Fig. 8A shows percentage of CD34+CD45RA" in mPB cells at day 14 (n=3). Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean ± SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Fig. 8B shows percentage of ST-HSC (CD34+CD45RA CD133+CD90+EPCR+) in mPB cells at day 14 (n=3). Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean ± SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Fig. 8C shows cell viability analysis of mPB-HSPCs on day 14 conducted using flow cytometry. The Apotracker probe allowed the detection of dead cells (n=3). Fig. 8D shows percentage of ST-HSC (CD34+CD45RA CD133+CD90+EPCR+) in mPB cells at day 14 (n=3,3,3 ,3 ,3,3,2). Kruskal- PCT / US25 / 47496 23 September 2025 (23.09.2025)

[0042] ATTORNEY DOCKET NO. 11624-021WO1

[0043] Wallis test. Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean ± SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Fig. 8E shows percentage of LT-HSC (CD34+CD45RA CD133+CD90+EPCR+ITGA3+) in mPB cells at day 14 (n=3,3,3,3,3,3,2). Kruskal-Wallis test. Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean ± SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Fig. 8F fold change of LT-HSC at day 14 calculated as the ratio if Lip-1 group %LT-HSC / untreated group %LT-HSC (n=3,3,3,3,3,3,2). One sample t-test. Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean ± SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Fig. 8G shows cell viability analysis of mPB-HSPCs on day 14 conducted using flow cytometry. The Apotracker probe allowed the detection of dead cells (n=3 per condition). Fig. 8H shows percentage of CD34+CD45RA" in mPB cells at day 14 (n=3 per condition). Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean ± SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Fig. 81 shows percentage of ST-HSC (CD34+CD45RA_CD133+CD90+EPCR+) in mPB cells at day 14 (n=3 per condition). Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean ± SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Fig. 8J shows percentage of LT-HSC (CD34+CD45RA CD133+CD90+EPCR+ITGA3+) in mPB cells at day 14 (n=3 per condition). Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean ± SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Fig. 8K shows a western blot analysis of GPX4 in CD34+CD45RA’90+cells sorted on day 4 of culture in the presence of 250 nM SS (n=4 per condition). Actin was used as a loading control, and fold change relative to control is reported. Fig. 8L shows cell viability analysis of mPB-HSPCs on day 14 conducted using flow cytometry. The Apotracker probe allowed the detection of dead cells (n=3 per condition). Fig. 8M shows percentage of CD34+CD45RA" in mPB cells at day 14 (n=3 per condition). Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean + SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Fig. 8N shows percentage of ST-HSC (CD34+CD45RA CD133+CD90+EPCR+) in mPB cells at day 14 (n=3 per condition). Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean ± SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Fig. 80 shows percentage of LT-HSC (CD34+CD45RA CD133+CD90+EPCR+ITGA3+) in mPB cells at day 14 (n=3 per condition). Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean + SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Fig. 8P shows GSH levels measured in CD34+CD45RA’90+cells immediately after sorting. PCT / US25 / 47496 23 September 2025 (23.09.2025)

[0044] ATTORNEY DOCKET NO. 11624-021WO1

[0045] Cells were collected after 4 days of expansion in medium supplemented with 100 pM P-ME (n=3 per condition). Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean ± SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Fig. 8Q shows percentage of CD34+CD45RA" in CB cells at day 14 (n=6,4,4,6,4)- Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean ± SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Fig. 8R shows percentage of ST-HSC (CD34+CD45RA CD133+CD90+EPCR+) in CB cells at day 14 (n=6,4,4,6,4). Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean ± SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0046] Figs. 9A-9L shows LT-HSC expansion-mediated by Lip-1 and Fer-1 in cytokine-free culture conditions. Fig. 9A shows cell viability analysis of CB-HSPCs at indicated time points conducted using flow cytometry. The Apotracker probe allowed the detection of dead cells (n=4 per condition). Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Fig. 9B shows percentage of CD34+CD45RA" in CB cells at indicated time points (n=4 per condition). Student’s t-test. Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Fig. 9C shows percentage of primitive HSPCs (CD34+CD45RA CD90+) in CB cells at indicated time points (n=4 per condition). Student’s t-test. Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Fig. 9D shows percentage of ST-HSC (CD34+CD45RA‘ CD133+CD90+EPCR+) in CB cells at indicated time points (n=4 per condition). Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Fig. 9E shows percentage of LT-HSC (CD34+CD45RA CD133+CD90+EPCR+ITGA3+) in CB cells at indicated time points (n=4 per condition). Student’s t-test. Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean + SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Fig. 9F shows fold change of LT-HSCs at day 14 calculated as the ratio of Lip- 1 group %LT-HSC / untreated group %LT-HSC (n=4 per condition). One sample t-test. Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Fig. 9G shows cell viability analysis of CB-HSPCs cultured in the presence of 5pM Fer-1 conducted at indicated time points using flow cytometry. The Apotracker probe allowed the detection of dead cells (n=4,4,3,4,4,4). Fig. 9H shows percentage of CD34+CD45RA" in CB cells at indicated time points PCT / US25 / 47496 23 September 2025 (23.09.2025)

[0047] ATTORNEY DOCKET NO. 11624-021WO1

[0048] (n=4,4,3,4,4,4). Student’s t-test. Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Fig. 91 shows percentage of primitive HSPCs (CD34+CD45RA CD90+) in CB cells at indicated time points (n=4,4,3,4,4,4). Student’s t-test. Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Fig. 9J shows percentage of ST-HSC (CD34+CD45RA_CD133+CD90+EPCR+) in CB cells at indicated time points (n=4,4,3,4,4,4). Student’s t-test. Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Fig. 9K shows percentage of LT-HSC (CD34+CD45RA CD133+CD90+EPCR+ITGA3+) in CB cells at indicated time points (n=4,4,3,4,4,4). Student’s t-test. Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Fig. 9L shows fold change of LT-HSCs at day 14 calculated as the ratio of Fer- 1 group %LT-HSCs / untreated group %LT-HSCs (n=4,4,3,4,4,4). One sample t-test. Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0049] Figs. 10A-10F show that results align with previous analyses. Fig. 10A shows a heatmap showing log2 enrichment of Lip-1 treated cells compared to the untreated control across cell types. Significance was calculated using a one-sided Fisher’s test, and corrected using Benjamini & Hochberg (BH) method (*: adjusted P , 10'5) (n=2 per condition). Fig. 10B shows a UMAP showing the cell density of control samples (left) and Lip-1 treated samples (right) (n=2 per condition). Fig. 10C shows a box plot showing the HSC signature score of Lip-1 treated HSCs compared to the untreated control, based on individual biological replicates (n=2 per condition). The significance of differences in the two conditions was calculated based on a two-sided Wilcoxon rank-sum test. Fig. 10D shows UMAP of scRNA-seq cells from Sakurai and collegues (Sakurai 2023) showing 10-day-expanded CD34+ cells in three culture conditions of PCL- PVAc-PEG-based 3a medium, StemSpan with SR-1 medium, or StemSpan with UM171 medium. Fig. 10E shows UMAPs of the reference-based integration of scRNA-seq cells from Nakauchi’s group (Sakurai 2023) and the present application, illustrating the distribution of cells from each culture condition. Fig. 10F shows bar plots showing the proportion of scRNA-seq cells in individual culture conditions from Sakurai’s study (Sakurai 2023) and the current application.

[0050] Figs. 11A-11I show Lip-1 supplementation does not impact hematopoietic lineages in several organs. Fig. 11A shows representative plots and gating strategy for in vivo studies. PCT / US25 / 47496 23 September 2025 (23.09.2025)

[0051] ATTORNEY DOCKET NO. 11624-021WO1

[0052] Firstly, live cells (Apotracker ) were gated, then human CD45+cells were distinguished from murine cells (mCD45+). Within this gate, CD19+(B cells) and CD33+(Myeloid cells) were further gated, subsequently, in the double negative gate, CD3+(T cells) were identified. Cells negative for all the lineage markers used were defined as “others”. Fig. 1 IB shows percentage of human CD45+cells measured at 15w in the SP of mice transplanted with HSPCs cultured as indicated (n=4 per group). Each dot represents an independent mouse and all data are presented as Mean ± SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Fig. 11 C shows percentage of B cells (CD19+), Myeloid (CD33+), T cells (CD3+), and other cells within the PB of mice transplanted with HSPCs cultures as indicated (n=4 per group). Student’s t-test. Fig. 11D shows percentage of B cells (CD19+), Myeloid (CD33+), T cells (CD3+), and other cells within the BM of mice transplanted with HSPCs cultured as indicated (n=4 per group). Student’s t-test. Fig. HE shows percentage of B cells (CD19+), Myeloid (CD33+), T cells (CD3+), and other cells within the SP of mice transplanted with HSPCs cultured as indicated (n=4 per group). Student’s t-test. Fig. 1 IF shows percentage of human primitive HSPCs (hCD45+CD45RA CD19 CD34+CD90+) cells measured at 15w in the BM of mice transplanted with HSPCs cultured as indicated (n=4 per group). Each dot represents an independent mouse and all the data are represented as Mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Fig. 11G shows percentage of human CD45+ cells measures at 15 weeks in the SP of mice in Fig. 3G (n=5 per group). Student’s t-test. Each dot represents an independent mouse and all the data are represented as Mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Fig. 11H shows percentage of B cells (CD19+), Myeloid (CD33+), T cells (CD3+), and other cells within the BM of mice in Fig. 3H (n=5 per group). Fig. 1 II shows the number of colonies formed by BM-derived CD34+cells purified from mice in Fig. 11H at 12 weeks post-transplantation (n=8 per group).

[0053] Fig. 12 shows that targeted sequencing does not report any reliable CHIP-associated mutations. From VANTAGE analysis, three variants were reported as recurrent. From the top: a missense mutation in TP53 with a VAF close to 0.5 is a germline variant, being present in all four samples from donor CB-52. ASXL1 and IDH2 genes were found to have single-base insertions in homopolymeric tracts; as they are present across multiple independent samples from this study and healthy donor bone marrow samples from an unrelated study (gray bars), they were considered as PCR artifacts.

[0054] Figs. 13A-13B show analysis of copy number variation in scRNA-seq data. Fig. 13A shows copy number profiles of HSPCs cultured in presence or absence of Lip-1 inferred from scRNA-seq using Numbat (Gao 2023). Each row represents one cell and each line (colored by CNV type) represents a distinct inferred CNV in the cell. Events were filtered by posterior CNV PCT / US25 / 47496 23 September 2025 (23.09.2025)

[0055] ATTORNEY DOCKET NO. 11624-021WO1 probability > 0.95. Fig. 13B shows fraction of genome altered by expression-inferred CNV in each sample. Only autosomes are included in the analysis. Events were filtered by posterior CNV probability > 0.95. Replicates from each donor were combined for this analysis.

[0056] Figs. 14A-14M show HSPCs cultured in the presence of Lip-1 exhibit slower cell proliferation and higher expression of ferroptosis protective, ribosome-, and cholesterol-related genes. Fig. 14A shows the number of cellular divisions performed by mPB-derived CD34+CD45RA" on day 14. More than 4xl04cells were analyzed from 2 independent experiments. Mann- Whitney test. Fig. 14B shows number of cellular divisions performed by mPB-derived LT-HSCs (CD34+CD45RA CD133+CD90+EPCR+ITGA3+) on day 14. Up to 120 cells were analyzed. Fig. 14C shows the number of mPB-derived LT-HSCs calculated at day 14. Cell counts were estimated based on an initial population of 105cells and multiplying the fold increase in Fig. 3A by the percentage of LT-HSCs at the corresponding time point (n=5 per condition). Mann-Whitney test. Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean ± SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Fig. 14D shows relative expression of GPX4 gene at day 7. Fold change was calculated relatively to untreated condition (n=3 per condition). Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean ± SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Fig. 14E shows relative expression of GPX4 gene in HSPCs cultured with or without Lip-1 and treated with 25 pM Erastin or RSL-3 for 6h at day 14. Fold change was calculated relative to untreated condition (n=3 technical replicates). Fig. 14F shows quantification of lipid peroxidation level in CD34+CD45RA" treated with 25 pM Erastin for 6h on day 14 measured by the ratio of oxidized and non-oxidized BODIPY dye (n=3 per condition). Fig. 14G shows representative violin plots of iron transport, storage, and metabolism gene expression in untreated or Lip-1 transcriptionally defined HSCs (n=2 per condition). Fig. 14H shows representative violin plots of AIFM2, GCH1, and ALDH7A1 gene expression in untreated or Lip-1 transcriptionally defined HSCs (n=2 per condition). Fig. 141 shows a scatter plot showing the GSEA-enriched biological pathways based on RNA expression between Lip-1 and Untreated group using the 6,035 gene sets from GO Biological Process 2021 database. Fig. 14J shows GSEA bubble plot showing differential pathways (cholesterol biosynthetic process and ribosome biogenesis) between Lip- 1 -treated and untreated cells across cell types (n=2 per condition). Fig. 14K shows GSEA enrichment plot showing that the genes involved in ribosome biogenesis are significantly upregulated in the Lip-1 group (n=2 per condition). The significance was calculated based on an adaptive multi-level split Monte-Carlo scheme. Fig. 14L shows GSEA enrichment plot showing that the genes involved in cholesterol PCT / US25 / 47496 23 September 2025 (23.09.2025)

[0057] ATTORNEY DOCKET NO. 11624-021WO1 biosynthetic process are significantly upregulated in the Lip-1 group (n=2 per condition). The significance was calculated based on an adaptive multi-level split Monte-Carlo scheme. Fig. 14M shows relative expression of ribosomal protein genes (left) and cholesterol biosynthesis- related genes (right) in CD34+CD45RA CD90+sorted HSPCs cultured in cytokine-free expansion medium with or without Fer-1 for 10 days. Fold change was calculated relatively to untreated condition (n=3 per condition). One sample t-test. Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean ± SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0058] Figs. 15A-15D show trends of cholesterol, other intermediates, and phospholipids in HSCs cultured in the presence of Lip-1. Fig. 15A shows concentration of (from the top) 7-DHC, cholesterol, lanosterol, and 7-dehydro-desmosterol in sorted CD34+CD45RA CD90‘ and CD34+CD45RA CD90+cells cultured with or without Lip-1 (n=4,4,2,2). Student’s t-test. Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean ± SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Fig. 15B shows percentage of primitive HSPCs (CD34+CD45RA CD90+) in mPB cells at day 7 and 14 cultured in serum-free medium supplemented with the indicated doses of 7-DHC (n=3 per condition). Friedman test. Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean ± SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Fig. 15C shows percentage of ST-HSCs (CD34+CD45RA CD144+CD90+EPCR+) in mPB cells at day 7 and 14 cultured in serum-free medium supplemented with the indicated doses of 7-DHC ( n=3 per condition). Friedman test. Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean + SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Fig. 15D shows a heatmap describing the relative levels of phospholipids in CD34+CD45RA" CD90+cells cultured with or without Lip-1, showing a reduction of phospholipids with polyunsaturated fatty acids and polyunsaturated plasmalogens.

[0059] Figs. 16A-16M show culture composition of gene-edited HSPCs cultured with or without Lip-1 supplementation. Fig. 16A shows percentage of CD34+CD45RA" in mPB-derived HSPCs edited by Cas9 targeting the AAVS1 locus at day 14 (n=4). Student’s t-test. Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean +SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Fig. 16B shows percentage of ST-HSC (CD34+CD45RA CD133+CD90+EPCR+) in mPB-derived HSPCs edited by Cas9 targeting the AAVS1 locus at day 14 (n=4). Student’s t-test. Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean +SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Fig. 16C shows percentage of LT-HSC (CD34+CD45RA_ PCT / US25 / 47496 23 September 2025 (23.09.2025)

[0060] ATTORNEY DOCKET NO. 11624-021WO1

[0061] CD133+CD90+EPCR+ITGA3+) in mPB-derived HSPCs edited by Cas9 targeting the AAVS1 locus at day 14 (n=4). Student’s t-test. Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean +SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Fig. 16D shows percentage of CD34+CD45RA" in mPB-derived HSPCs edited by ABE8 targeting the CD33 locus at day 14 (n=3 per condition). Student’s t-test. Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean +SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Fig. 16E shows percentage of ST-HSC (CD34+CD45RA CD133+CD90+EPCR+) in mPB-derived HSPCs edited by ABE8 targeting the CD33 locus at day 14 (n=3 per condition). Student’s t-test. Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean +SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Fig. 16F shows percentage of LT-HSC (CD34+CD45RA CD133+CD90+EPCR+ITGA3+) in mPB-derived HSPCs edited by ABE8 targeting the CD33 locus at day 14 (n=3 per condition). Student’s t-test. Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean +SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Fig. 16G shows percentage of CD34+CD45RA" in CB-derived HSPCs cultured in cytokine-free medium and edited by Cas9 targeting the AAVS1 locus at day 14 (n=3 per condition). Student’s t-test. Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean +SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.Fig. 16H shows percentage of primitive HSPCs (CD34+CD45RA'CD90+) in CB-derived HSPCs cultured in cytokine-free medium and edited by Cas9 targeting the AAVS1 locus at day 14 (n=3 per condition). Student’s t-test. Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean +SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Fig. 161 shows percentage of ST- HSC (CD34+CD45RA CD133+CD90+EPCR+) in CB-derived HSPCs cultured in cytokine-free medium and edited by Cas9 targeting the AAVS1 locus at day 14 (n=3 per condition). Student’s t-test. Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean +SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Fig. 16J shows percentage of CD34+CD45RA" in mPB-derived HSPCs edited by Cas9 targeting the BCL11A enhancer (DHS +58) at day 14 ( n=3 per condition). Student’s t-test. Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean +SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Fig. 16K shows percentage of ST-HSC (CD34+CD45RA CD133+CD90+EPCR+) in mPB-derived HSPCs edited by targeting the

[0062] BCL11A enhancer (DHS +58) at day 14 (n=3 per condition). Student’s t-test. Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean +SEM. PCT / US25 / 47496 23 September 2025 (23.09.2025)

[0063] ATTORNEY DOCKET NO. 11624-021WO1

[0064] *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Fig. 16L shows percentage of LT-HSC (CD34+CD45RA CD133+CD90+EPCR+ITGA3+) in mPB-derived HSPCs edited by Cas9 targeting the BCL11A enhancer (DHS +58) at day 14 (n=3 per condition). Student’s t-test. Each dot represents an independent donor unless otherwise indicated and all the data are presented as Mean +SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Fig. 16M shows relative expression of HBG gene at day 17 (Phase III) of erythroid differentiation. Fold change was calculated relative to untreated condition. One sample t-test. Each dot represents an independent donor unless otherwise indicated and all the data are represented as Mean + SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0065] DETAILED DESCRIPTION

[0066] Definitions

[0067] In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:

[0068] Throughout the description and claims of this specification the word “comprise” and other forms of the word, such as “comprising” and “comprises,” means including but not limited to, and is not intended to exclude, for example, other additives, components, integers, or steps.

[0069] The term “about” as used herein when referring to a measurable value such as an amount, a percentage, and the like, is meant to encompass variations of +20%, +10%, +5%, or +1% from the measurable value.

[0070] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like.

[0071] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a PCT / US25 / 47496 23 September 2025 (23.09.2025)

[0072] ATTORNEY DOCKET NO. 11624-021WO1 value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed the “less than or equal to 10” as well as “greater than or equal to 10” is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0073] "Comprising" is intended to mean that the compositions, methods, etc. include the recited elements, but do not exclude others. "Consisting essentially of' when used to define compositions and methods, shall mean including the recited elements, but excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. "Consisting of' shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions provided and / or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0074] A “control” is an alternative subject or sample used in an experiment for comparison purposes. A control can be "positive" or "negative."

[0075] The term “detect” or “detecting” refers to an output signal released for the purpose of sensing of physical phenomenon. An event or change in environment is sensed and signal output released in the form of light.

[0076] As used herein, “diagnose”, “diagnosed”, “diagnosing”, and any grammatical variations thereof as used herein, refers to the act of process of identifying the nature of an illness, disease, disorder, or condition in a subject by examination or monitoring of symptoms.

[0077] “Effective amount” of an agent refers to a sufficient amount of an agent to provide a desired effect. The amount of agent that is “effective” will vary from subject to subject, depending on many factors such as the age and general condition of the subject, the particular agent or agents, and the like. Thus, it is not always possible to specify a quantified “effective amount.” However, an appropriate “effective amount” in any subject case may be determined by PCT / US25 / 47496 23 September 2025 (23.09.2025)

[0078] ATTORNEY DOCKET NO. 11624-021WO1 one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an “effective amount” of an agent can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts. An “effective amount” of an agent necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0079] “Pharmacologically active”, “functionally active” (or simply “active”), as in a “functionally active” derivative or analog, can refer to a derivative or analog (e.g., a salt, ester, amide, conjugate, metabolite, isomer, fragment, etc.) having the same type of pharmacological activity as the parent compound (e.g., an antibody) and approximately equivalent in degree.

[0080] “Therapeutic agent” refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition (e.g., a chronic muscle disease developed after a physical injury). The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the terms “therapeutic agent” is used, then, or when a particular agent is specifically identified, it is to be understood that the term includes the agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, derivatives, etc.

[0081] As used herein, “enhance”, “enhanced”, “enhancement”, “enhancing”, and any grammatical variations thereof as used herein, refers to an act of intensifying, increasing, or further improving the quality, value, or extent of a biological function, composition, compound, cell, or tissue.

[0082] As used herein, “hematopoietic stem cells” refers to stem cells that can differentiate into the hematopoietic lineage and give rise to all blood cell types such as white blood cells and red blood cells, including myeloid (e.g., monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells), and lymphoid lineages (e.g., T-cells, B-cells, NK-cells). “Stem cells” are defined by their ability to form multiple cell types (multipotency) and their ability to self-renew. Hematopoietic stem cells can be identified, for example by cell surface markers such as CD34-, CD133+, CD48-, CD150+, CD244-, cKit+, PCT / US25 / 47496 23 September 2025 (23.09.2025)

[0083] ATTORNEY DOCKET NO. 11624-021WO1

[0084] Scal+, and lack of lineage markers (negative for B220, CD3, CD4, CD8, Macl, Grl, and Teri 19, among others). Methods of identifying and analyzing hematopoietic stem cells has been reviewed by Wogunum et al. (see e.g., “Identification and Isolation of Hematopoietic Stem Cells,” Archives of Medical Reseach. 2003; 34(6):461-475, incorporated herein by reference in its entirety).

[0085] Hematopoietic stem cells are optionally obtained from blood products. A blood product includes a product obtained from the body or an organ of the body containing cells of hematopoietic origin. Such sources include unfractionated bone marrow, umbilical cord, placenta, peripheral blood, or mobilized-peripheral blood. All of the aforementioned crude or unfractionated blood products can be enriched for cells having hematopoietic stem cell characteristics in a number of ways. For example, the more mature, differentiated cells are selected against, via cell surface molecules they express. Optionally, the blood product is fractionated by positively selecting for CD34+cells. CD34+cells include a subpopulation of hematopoietic stem cells capable of self-renewal, multi-potency, and that can be reintroduced into a transplant recipient whereupon they home to the hematopoietic stem cell niche and reestablish productive and sustained hematopoiesis. Hematopoietic stem cells can also be optionally obtained from differentiated embryonic stem cells, differentiated induced pluripotent stem cells, or from other reprogrammed mature cells types.

[0086] “Expanding” or “stem cell expansion” as used herein refers to the process of increasing the number of stem cells in a population through in vivo, in vitro, or ex vivo culture techniques, while maintaining their undifferentiated state and functional characteristics. The process typically involves the use of specialized culture media, growth factors, extracellular matrices, and / or bioreactor systems designed to promote proliferation without inducing differentiation. Stem cell expansion is critical for generating sufficient quantities of stem cells for therapeutic, research, or industrial applications. Hematopoietic stem cell expansion aims to mimic the bone marrow niche in order to preserve the multipotency and self-renewal capacity of HSCs.

[0087] The term “increased” or “increase” as used herein generally means an increase by a statically significant amount; for the avoidance of any doubt, “increased” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10- fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference PCT / US25 / 47496 23 September 2025 (23.09.2025)

[0088] ATTORNEY DOCKET NO. 11624-021WO1 level so long as the increase is statistically significant.

[0089] "Inhibit," "inhibiting," and "inhibition" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

[0090] As used herein, the terms “may,” “optionally,” and “may optionally” are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation “may include an excipient” is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient.

[0091] As used herein, the term “preventing” a disease, a disorder, or unwanted physiological event in a subject refers to the prevention of a disease, a disorder, or unwanted physiological event or prevention of a symptom of a disease, a disorder, or unwanted physiological event. As used herein, “preventing loss of’ means reducing the amount of a composition lost compared to an untreated control. This reduction can be 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, and all values in between. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed.

[0092] As used herein, the term “radical trapping antioxidants” (RTAs) refers to chemical compounds that inhibit oxidative degradation by neutralizing reactive free radicals. These antioxidants function by donating electrons or hydrogen atoms to free radicals, thereby converting them into more stable, non-reactive species. This mechanism interrupts the chain reactions typically initiated by reactive oxygen species (ROS) or other radical species, which can otherwise lead to the deterioration of materials, biological tissues, or chemical formulations. RTAs reduce ferroptosis in cells.

[0093] The term “reduced”, “reduce”, “reduction”, or “decrease” as used herein generally means a decrease by a statistically significant amount. However, for avoidance of doubt, “reduced” means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (i.e. absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level so long as the decrease is statistically significant. PCT / US25 / 47496 23 September 2025 (23.09.2025)

[0094] ATTORNEY DOCKET NO. 11624-021WO1

[0095] As used throughout, by a "subject" (or a “host”) is meant an individual. Thus, the "subject" can include, for example, domesticated animals, such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.) mammals, non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animal. The subject can be a mammal such as a primate or a human. Administration of the therapeutic agents can be carried out at dosages and for periods of time effective for treatment of a subject.

[0096] “Therapeutically effective amount” or “therapeutically effective dose” of a composition refers to an amount that is effective to achieve a desired therapeutic result. Therapeutically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject. The term can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the agent and / or agent formulation to be administered (e.g., the potency of the therapeutic agent, the concentration of agent in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art. In some instances, a desired biological or medical response is achieved following administration of multiple dosages of the composition to the subject over a period of days, weeks, or years.

[0097] As used herein, the terms “treating” or “treatment” of a subject includes the administration of a drug to a subject with the purpose of curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving, stabilizing or affecting a disease or disorder, or a symptom of a disease or disorder. The terms “treating” and “treatment” can also refer to reduction in severity and / or frequency of symptoms, elimination of symptoms and / or underlying cause, and improvement or remediation of damage.

[0098] Disclosed herein are the components to be used to prepare the disclosed compositions as to be used in the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. If a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. PCT / US25 / 47496 23 September 2025 (23.09.2025)

[0099] ATTORNEY DOCKET NO. 11624-021WO1

[0100] Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods.

[0101] General Description

[0102] The present disclosure provides methods for enhancing stem cell expansion by culturing hematopoietic stem cells ex vivo in the presence of radical trapping antioxidants. Improved ex vivo human hematopoietic stem cell (HSC) expansion would advance HSC transplantation and therapeutic genome engineering. Most efforts to achieve this have focused on preventing differentiation or optimizing cytokine stimulation. There have been many advances in culture conditions to maintain and expand human HSCs ex vivo. Initially, it was found that serum can promote HSC differentiation, and serum-free conditions using albumin or polymers were identified that improved maintenance. In addition, small molecules that prevent differentiation have been discovered, including the pyrimidoindole derivative, UM171, that alters the epigenetic state of hematopoietic cells. Modulation of cytokine composition and concentration to maximally maintain HSCs has also shown value. Recently, the development of chemically- defined cytokine-free culture conditions has enabled the expansion of human HSCs over the course of weeks. However, all of the advances in HSC expansion to date have focused on either preventing differentiation or maximizing quiescence. Given the complexity of the endogenous HSC niche, it is likely that culture conditions fail to appropriately recapitulate all of the support present and thereby healthy HSCs might be lost as a result. Furthermore, it has remained unclear whether the loss of human HSCs ex vivo is due to insufficient support in culture is preventable.

[0103] Recently it has been shown that human HSCs display a unique vulnerability to loss by ferroptosis, a metabolically programmed form of cell death, in the setting of bone marrow failure. The present invention discloses that targeting this distinct vulnerability of human HSCs to ferroptosis can be exploited to improve expansion of human HSCs. It is herein demonstrated that blockade of ferroptosis with radical trapping antioxidant small molecules significantly augments the expansion of human HSCs derived from either cord blood or adult sources using diverse methods, including widely applied serum-free cultures and recently reported chemically- defined conditions. The expansion of phenotypic and molecularly-defined HSCs, as well as improved human hematopoietic reconstitution after xenotransplantation was observed. PCT / US25 / 47496 23 September 2025 (23.09.2025)

[0104] ATTORNEY DOCKET NO. 11624-021WO1

[0105] Importantly, there were no signs of aberrant hematopoiesis or genotoxicity. Mechanistically, human HSCs treated with radical trapping antioxidants, liproxstatin-1 or ferrostatin-1 display increased ribosome biogenesis and cholesterol biosynthesis, accompanied by elevated 7- dehydrocholesterol levels - a potent endogenous inhibitor of ferroptosis (Wilkinson 2020, Frangoul 2024). Finally, the effectiveness of mitigating ferroptosis to enhance human HSC expansion in the setting of therapeutic genome modifications is demonstrated, paving the path to clinical applications.

[0106] Radical Trapping Antioxidants

[0107] Radical trapping antioxidants (RTAs) are chemical compounds that inhibit oxidative degradation by neutralizing reactive free radicals. These antioxidants function by donating electrons or hydrogen atoms to free radicals, thereby converting them into more stable, non- reactive species. This mechanism interrupts the chain reactions typically initiated by reactive oxygen species (ROS) or other radical species, which can otherwise lead to the deterioration of materials, biological tissues, or chemical formulations.

[0108] Unlike preventive antioxidants, which act by inhibiting the formation of radicals (e.g., through metal chelation or decomposition of peroxides), radical trapping antioxidants act directly on existing radicals. Their effectiveness is determined by factors such as reaction rate constants with radicals, stability of the resulting antioxidant-derived radicals, and solubility or compatibility with the medium in which they are applied.

[0109] Radical trapping antioxidants can reduce ferroptosis in cells. Naturally produced RTAs that are known to inhibit ferroptosis include, but are not limited to, vitamin E, vitamin K, and glutathione. Synthetic RTAs include but are not limited to Ferrostatin-1 (Fer-1), Liproxstatin-1 (Lip-1), UAMC-3203, phenothiazine (PTZ), phenoxazine (PNX), ferfluor-1, Srsll-92, spiroquinoxaline, C15-THN, MM-12-Urea, and their derivatives, see e.g., Chen et al. ( “Srsl l- 92, a ferrostatin-1 analog, improves oxidative stress and neuroinflammation via Nrf2 signal following cerebral ischemia / reperfusion injury”, CNS Neurosci Ther. 2033; 29(6): 1667-1677), Chen et al. (“Ferroptosis: A Novel Therapeutic Target for Ischemia-Reperfusion Injury”, Front Cell Dev Biol. 2021 9:688605), Devisscher et al. (“Discovery of Novel, Drug-Like Ferroptosis Inhibitors with in Vivo Efficacy”, J Med Chem. 2018 Nov 21 ;61(22): 10126- 10140, and US15 / 707,729 incorporated herein by reference in their entirety. Also disclosed for use with the methods herein are any other functional derivatives of the radical trapping antioxidants disclosed herein. PCT / US25 / 47496 23 September 2025 (23.09.2025)

[0110] ATTORNEY DOCKET NO. 11624-021 WO 1

[0111] Liproxstatin-1 (Lip-1), C19H21CIN4, is a small molecule inhibitor able to inhibit ferroptosis. Lip-1 halts oxidative damage in the process of ferroptosis by neutralizing reactive lipid radicals. Lip-1 is stable and active under physiological conditions and highly soluble. In some embodiments, ex vivo expansion of hematopoietic stem cells is increased with Lip-1 treatment.

[0112] Liproxstatin-1 Chemical Structure

[0113] Ferrostatin-1 (Fer-1), C15H22N2O2, is a synthetic compound able to inhibit ferroptosis. Fer-1 halts oxidative damage in the ferroptosis by neutralizing lipid peroxyl radicals. Fer-1 is less metabolically stable than Lip-1. In some embodiments, ex vivo expansion of hematopoietic stem cells is increased with Fer-1 treatment.

[0114] Ferrostatin-1 Chemical Structure

[0115] Methods

[0116] The present disclosure provides methods for increasing ex vivo hematopoietic stem cell expansion. In some embodiments, a method for treating a blood disorder is disclosed, wherein, isolated hematopoietic stem cells are expanded in the presence of radical trapping antioxidants, for example, Lip-1 or Fer-1, the cells are then genetically modified, and administered to a subject.

[0117] By “expanded” it is meant that the amount of HSCs in a given culture is increased by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% relative to a control, or any amount PCT / US25 / 47496 23 September 2025 (23.09.2025)

[0118] ATTORNEY DOCKET NO. 11624-021WO1 below, above, or between these values. In certain embodiments, the expansion can also be by 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100 fold or more, or any amount below, above, or between these amounts. The control can be identical in every manner (culture solution, temperature, length of culture, etc.), except that the control is not exposed to the radical trapping antioxidant(s).

[0119] The disclosed methods may be employed in the treatment of a wide variety of hematologic, metabolic, immunologic, and genetic disorders where stem cell transplantation is indicated. Such conditions include, without limitation, severe acquired immune deficiency, metabolic diseases, muscular dystrophy, myotonic dystrophy, genetic blood disorders, congenital neutropenia, Diamond-Blackfan anemia, Shwachman-Diamond syndrome, telomerase disorders, acquired aplastic anemia, bone marrow failure syndromes, immunodeficiency syndromes, lysosomal storage diseases, hemoglobinopathies including sickle cell disease and P-thalassemia, Fanconi anemia, leukodystrophies, congenital dyserythropoietic anemia, severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome, chronic granulomatous disease, osteopetrosis, mucopolysaccharidoses, Gaucher disease, Niemann-Pick disease, Hurler syndrome, Krabbe disease, and X-linked adrenoleukodystrophy. In these contexts, the modified stem cells can be autologous or allogeneic hematopoietic stem cells, mesenchymal stromal cells, or induced pluripotent stem cell-derived hematopoietic progenitors. In some embodiments, expanded HSCs are genetically edited to treat a subject with a blood disorder. In an additional embodiment, treatment of HSCs with a radical trapping antioxidant preserves genetic editing.

[0120] Examples of radical trapping antioxidants are given above, and any of the above examples, either alone or in combination of 2, 3, 4, 5, 6, or more radical trapping antioxidants, can be used with the methods disclosed herein.

[0121] In one embodiment, the method can additionally include exposing the cells to 7- dehydrocholesterol (7-DHC). 7-DHC is a naturally produced sterol that plays a role in vitamin D synthesis and cholesterol synthesis. Treatment of hematopoietic stem cells with Lip-1 or Fer-1 increases amounts of 7-DHC. In some embodiments, ex vivo expansion of hematopoietic stem cells is increased with treatment with 7-DHC. The increase in HSC expansion can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater or any amount below, above, or between these values. In certain embodiments, the increase in expansion can also be by 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100 fold or more, or any amount below, above, or between PCT / US25 / 47496 23 September 2025 (23.09.2025)

[0122] ATTORNEY DOCKET NO. 11624-021WO1 these amounts. In some embodiments, amounts of 7-DHC in HSCs are measured in response to Lip-1 or Fer-1 treatment and compared to 7-DHC amounts from untreated cells.

[0123] Glutathione Peroxidase 4 (GPX4) is an antioxidant enzyme that protects cells from oxidative stress and prevents ferroptosis. In some embodiments, treatment of hematopoietic stem cells with Lip-1 or Fer-1 increases GXP4. In an additional embodiment, the concentration of GPX4 in HSCs is measured in response to Lip-1 or Fer-1 treatment and compared to GPX4 concentrations in untreated cells. The increase in GPX4 can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or any amount below, above, or between these values greater than a control HSC, not treated with a radical trapping antioxidant. The increase in GPX4 can also be measured via fold change compared to a control. The increase in GPX4 can be 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100 fold or more, or any amount below, above, or between these amounts compared to a control.

[0124] In some embodiments, cell proliferation and / or lipid peroxidation are measured in HSCs treated with Lip-1 or Fer-1 and compared to levels of cell proliferation and / or lipid peroxidation in untreated cells. In an additional embodiment, levels of ribosome biogenesis and / or cholesterol biosynthesis are measured in HSCs treated with Lip-1 or Fer-1 and compared to corresponding levels in untreated cells.

[0125] Kits

[0126] An composition, or combination of compositions, as described herein can be provided in a kit. In a specific embodiment, the kit includes (a) the composition(s) can include a radical trapping agent, and (b) informational material. The informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein and / or the use of the agent for the methods described herein. For example, the informational material describes methods for enhancing expansion of hematopoietic stem cells including information on cell culture conditions and concentrations.

[0127] In one embodiment, the informational material can include instructions to administer the agent in a suitable manner, e.g., in a suitable dose, dosage form, or mode of administration (e.g., a dose, dosage form, or mode of administration described herein). In another embodiment, the informational material can include instructions for selecting a suitable subject, e.g., a human, e.g., an adult human. The informational material of the kits is not limited in its form. In many cases, the informational material, e.g., instructions, is provided in printed matter, e.g., a printed text, drawing, and / or photograph, e.g., a label or printed sheet. However, the informational PCT / US25 / 47496 23 September 2025 (23.09.2025)

[0128] ATTORNEY DOCKET NO. 11624-021WO1 material can also be provided in other formats, such as Braille, computer readable material, video recording, or audio recording. In another embodiment, the informational material of the kit is a link or contact information, e.g., a physical address, email address, hyperlink, website, or telephone number, where a user of the kit can obtain substantive information about the modulator and / or its use in the methods described herein. Of course, the informational material can also be provided in any combination of formats.

[0129] In addition to the agent or the composition of the kit can include other components, such as a solvent or buffer, a stabilizer or a preservative, and / or a second agent for treating a condition or disorder described herein, e.g. a disease requiring transplantation of mobilized peripheral blood stem cells. Alternatively, the other ingredients can be included in the kit, but in different compositions or containers than the agent. In such embodiments, the kit can include instructions for admixing the agent and the other ingredients, or for using the modulator together with the other ingredients.

[0130] The agent can be provided in any form, e.g., liquid, dried or lyophilized form. It is preferred that the agent be substantially pure and / or sterile. When the agent is provided in a liquid solution, the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being preferred. When the agent is provided as a dried form, reconstitution generally is by the addition of a suitable solvent. The solvent, e.g., sterile water or buffer, can optionally be provided in the kit.

[0131] In some embodiments, the agent comprises a standard serum-free medium for culturing stem cells. The standard serum-free medium may consist of serum-free StemSpan SFEM II medium supplemented with 1% L-glutamine, 1% penicillin / streptomycin, IX CC100 (containing the cytokines FLT3L, SCF, IL3, and IL6), 100 ng / ml recombinant thrombopoietin (TPO), and 35 nM UM171. In other embodiments, the agent comprises a chemically -defined cytokine- free medium for culturing stem cells. The chemically-defined cytokine-free medium may consist of Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 1% insulin- transferrin- selenium-ethanolamine (ITSX), 1% L-glutamine, 1% penicillin / streptomycin, 1 mg / ml polyvinyl alcohol (PVA), 1 M 740Y-P, 0.1 ,u M butyzamide, and 70 nM UM171.

[0132] EXAMPLES

[0133] To further illustrate the principles of the present disclosure, the following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compositions, articles, and methods claimed herein are made and evaluated. They are intended to be purely exemplary of the invention and are not intended to PCT / US25 / 47496 23 September 2025 (23.09.2025)

[0134] ATTORNEY DOCKET NO. 11624-021WO1 limit the scope of what the inventors regard as their disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.); however, some errors and deviations should be accounted for. Unless indicated otherwise, temperature is °C or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of process conditions that can be used to optimize product quality and performance. Only reasonable and routine experimentation will be required to optimize such process conditions.

[0135] EXAMPLE 1: Human hematopoietic stem cell expansion by preventing ferroptosis

[0136] Improved ex vivo expansion of human hematopoietic stem cells (HSCs) provides the opportunity to considerably advance transplantation and genome-engineered therapies, yet existing culture methods still allow substantial HSC loss. The present application demonstrates this attrition is driven largely by ferroptosis, a metabolically-regulated, iron-dependent cell-death pathway, and that it can be blocked to augment HSC expansion. Inhibiting ferroptosis with liproxstatin-1 or ferrostatin-1 markedly increases expansion of cord blood and adult HSCs consistently across donors in both widely used serum-free cultures and recently reported chemically-defined conditions. Expanded cells retain phenotypic and molecular stem cell identity and mediate improved durable, multilineage engraftment in xenotransplanted mice without genotoxicity or aberrant hematopoiesis. Mechanistically, ferroptosis blockade is accompanied by up-regulated ribosome biogenesis and cholesterol synthesis, elevating 7- dehydrocholesterol - a potent endogenous ferroptosis inhibitor that itself promotes HSC expansion. Crucially, this approach enhances yields of therapeutically genome-modified HSCs, paving a path for clinical applications.

[0137] Tremendous advances in allogeneic hematopoietic transplantation from diverse cell sources and genome engineering of autologous hematopoietic stem cells (HSCs) have resulted in curative treatments for hundreds of thousands of patients (Appelbaum 2007, Ferrari 2021). However, the application of these approaches has been limited by inadequate cell doses in many instances (Shpall 2021). The ability to maintain, expand, and genetically modify HSCs ex vivo without compromising their functional properties for effective transplantation enables therapies for a broader range of patients (Liggett 2020, Wilkinson 2020). Even in successful trials, such as the recently reported and now approved use of CRISPR / Cas9 genome editing of the BCL11A +58 enhancer to cure sickle cell disease, all patients required between 3-18 days of apheresis after HSC mobilization to collect sufficient numbers of cells (Frangoul 2024). Such observations emphasize the limitations of current approaches to collect and manipulate human HSCs. PCT / US25 / 47496 23 September 2025 (23.09.2025)

[0138] ATTORNEY DOCKET NO. 11624-021WO1

[0139] There have been many advances in culture conditions to maintain and expand human HSCs ex vivo. Initially, it was found that serum can promote HSC differentiation, and serum-free conditions using albumin or polymers were identified that improved maintenance (Meaker 2024). In addition, small molecules that prevent differentiation have been discovered, including the pyrimidoindole derivative, UM171, that alters the epigenetic state of hematopoietic cells (Wilkinson 2020, Chagraoui 2021). Modulation of cytokine composition and concentration to maximally maintain HSCs has also shown value (Zonari 2017, Tsutsumi 2023, Kobayashi 2019). Recently, the development of chemically-defined cytokine-free culture conditions has enabled the expansion of human HSCs over the course of weeks (Sakurai 2023). However, all of the advances in HSC expansion to date have focused on either preventing differentiation or maximizing quiescence. Given the complexity of the endogenous HSC niche (Crane 2017, Wu 2024, Baccin 2020), it is likely that culture conditions fail to appropriately recapitulate all of the support present and thereby healthy HSCs can be lost as a result. Therefore, preventing the loss of HSCs enables improved ex vivo HSC expansion. In the setting of bone marrow failure, human HSCs display a unique vulnerability to loss by ferroptosis, a metabolically programmed form of cell death (Zhao 2017). The present application investigates whether targeting this distinct vulnerability of human HSCs to ferroptosis can be exploited to improve expansion of human HSCs.

[0140] Evaluating blockade of ferroptosis to enhance diverse culture systems

[0141] Though a number of inhibitors of ferroptosis have been identified, non-specific effects and undesirable properties limit the utility of many of these molecules. Radical trapping antioxidants such as ferrostatin-1 (Fer-1) or liproxstatin-1 (Lip-1) have been identified through high-throughput screens to potently prevent lipid peroxidation and ferroptosis (Zilka 2017, Dixon 2012, Friedmann 2014). Studies have demonstrated the utility of both Lip-1 and Fer-1 in preventing ferroptosis in human HSCs (Zhao 2023), and therefore it was sought to apply such molecules in the context of existing human HSC ex vivo expansion approaches. Initially, supplemented standard serum- free cultures were used for human adult HSC maintenance with varying doses of Lip-1 and HSC content was examined in the cultures using the phenotypic marker combination of CD34+CD45RA CD90+CD133+EPCR+that are known to enrich for bona fide human HSCs, even after ex vivo culture (Bao 2020, Tomellini 2019, Voit 2023, Zhao 2024) (Fig. 1A). This marker combination can be further stratified to separate long-term (LT-) from short-term (ST-) reconstituting HSCs based on ITGA3 surface expression on the former (Fig. 7A). It was found that Lip-1 did not cause cell toxicity, except at the highest dose of 25 pM (Fig. IB). No major differences in the percentages of progenitors (CD34+CD45RA_cells) or ST-HSCs PCT / US25 / 47496 23 September 2025 (23.09.2025)

[0142] ATTORNEY DOCKET NO. 11624-021WO1 compared to untreated cells were noted (Figs. 8A-8B). Importantly, there was dose-dependent preservation of LT-HSCs, with a ~4-fold increase in LT-HSCs at the 10 pM Lip-1 dose compared to controls after two weeks of culture (Figs. 1C-1D). Notably, similar expansion was achieved when the same serum-free culture medium was supplemented with Fer-1 (Figs. 8C-8F).

[0143] While radical-trapping antioxidants (RTAs) like Lip-1 and Fer-1 are potent inhibitors of ferroptosis, other metabolic inputs into this process may be manipulated to optimize the ex vivo culture of HSCs. The glutathione peroxidase GPX4 is a key seleno-enzyme involved in ferroptosis. It was attempted to maximize GPX4 activity (Ingold 2018, Saito 2003) by optimizing selenium concentrations in cells with sodium selenite (SS, Na2SeO3) supplementation, as has been shown previously in other cell contexts (Ingold 2018, Yao 2024, Alim 2019). However, no improvement in HSC maintenance, nor any change in GPX4 protein levels was observed, suggesting that selenium concentrations and GPX4 levels were already optimal in HSCs (Figs. 8G-8K). GPX4 utilizes reduced glutathione (GSH) to detoxify lipid peroxides and cystine is known to be rate-limiting for the production of GSH. Different concentrations of P-mercaptoethanol (P-ME), which serves as a cystine donor to promote GSH biosynthesis (Yang 2014, Conrad 2012) were added, but once again observed no improvement in human HSCs maintenance ex vivo (Figs. 8L-8O). Importantly, -ME supplementation did not increase GSH levels in human HSCs, demonstrating that GSH availability is also not limiting in the culture conditions to maximally support GPX4 activity (Fig. 8P). Therefore, these findings show that radical-trapping antioxidants that inhibit ferroptosis are uniquely able to prevent HSC loss in culture, and other specific metabolic inputs - selenium and GSH levels - do not appear to be limiting in cultured human HSCs.

[0144] Having shown improved ex vivo HSC maintenance and expansion in standard serum-free cultures with cells derived from adult sources, it was examined whether this was also the case for HSCs obtained from cord blood (CB). CB HSCs are frequently used for clinical hematopoietic transplantation and cell numbers obtained from CB units are often limited (Shpall 2021). Akin to the results with adult human HSCs, a ~4-fold expansion of CB-derived LT-HSCs was observed after two weeks of culture with 10 pM Lip-1, without signs of toxicity or impacts on other subpopulations, indicating that blocking ferroptosis can broadly enable HSC expansion across a variety of cell sources (Figs. 1E-1G and Figs. 8Q-8R).

[0145] While serum-free culture approaches are commonly applied in clinical gene therapy and genome editing applications (Ferrari 2021, Frangoul 2024), recent advances have been reported in the development of chemically-defined cytokine-free human HSC expansion conditions that enable culture of the cells over a few weeks (Sakurai 2023). Remarkably, while these cultures PCT / US25 / 47496 23 September 2025 (23.09.2025)

[0146] ATTORNEY DOCKET NO. 11624-021WO1 enable more human HSCs to be preserved, it was found that by adding 10 pM Lip-1, LT-HSCs were expanded by ~50-fold, while also expanding ST-HSCs and primitive progenitors (CD90+) after 3 weeks in these culture conditions (Figs. 2A-2E and Fig. 7B and Figs. 9A-9F). Similar results were obtained when using the structurally distinct radical- trapping antioxidant Fer-1, further supporting the robustness and generalizability of the findings (Figs. 9G-9L). These results reveal how even in conditions optimized for maximal HSC expansion (Sakurai 2023), the preservation of HSCs is likely suboptimal and can be further enhanced by inhibiting ferroptosis.

[0147] While these results across a range of human HSC sources and culture approaches are promising, the fidelity of surface markers has been relied upon to quantify these cells. Such surface markers can display variability with some perturbations. Therefore, to further analyze the impact of inhibiting ferroptosis on human HSCs, single-cell RNA sequencing (scRNA-seq) was performed on CD34+CD45RA CD90+cells cultured with or without Lip-1 supplementation for 10 days in chemically-defined conditions (see Materials and Methods). A total of 29,096 cells collected from 2 different donors were profiled (with an average of 18,170 individual cells per condition) and 10 cell clusters corresponding to known hematopoietic populations were annotated (Zeng 2023), which were visualized using Uniform Manifold Approximation and Projection (UMAP) (Fig. 2F). The comparison of cluster composition and cellular density plots showed significant enrichment of molecularly-defined HSCs in the Lip-1 treated condition (Figs. 2G-2I and Figs. 10A-10C). Importantly, the findings of cell states were consistent with those observed in the original paper describing the use of chemically-defined culture conditions, demonstrating that the augmentation seen arose in a setting consistent with the previous study (Sakurai 2023) (Figs. 10D-10F).

[0148] Lip-1 augments in vivo repopulation capacity without signs of compromised hematopoiesis and does not cause detectable genotoxicity

[0149] While earlier results suggest promise for inhibiting ferroptosis as a strategy to preserve and expand more human HSCs ex vivo across different culture approaches, it is necessary to ensure that this preserves appropriate stem cell functionality and does not result in aberrant hematopoiesis. Following 7 days of culture with or without Lip-1 in chemically-defined conditions, CB-derived HSPCs were transplanted into the NOD.Cg- Kitw-4iJTyr+PrkdCscidll2rgtmlWjl / ThomJ (NBSGW) strain of immunodeficient and Kzf-mutant mouse recipients (Zhao 2023, Voit 2023, McIntosh 2015) (Fig. 3A). Once long-term engraftment was achieved at 15 weeks post-transplantation from equivalent starting cell numbers, greater repopulation capacity was observed over time in the peripheral blood as well as in the bone marrow and spleen with the Lip-1 treated cells (Figs. 3B-3C and Figs. 11A-11B). Analysis of PCT / US25 / 47496 23 September 2025 (23.09.2025)

[0150] ATTORNEY DOCKET NO. 11624-021WO1 hematopoiesis in these engrafted mice showed a similar composition of myeloid and lymphoid cells without any notable alterations among diverse hematopoietic lineages upon Lip-1 treatment (Figs. 11C-1 IE). Importantly, the mice did not display any signs of impaired hematopoiesis or malignancy.

[0151] The NBSGW recipients transplanted with the Lip-l-treated HSPCs showed more CD34+HSPCs in the bone marrow after long-term reconstitution and a trend was also observed for more primitive CD90+HSPCs (Fig. 3D and Fig. 1 IF). Estimates of HSC numbers using maximum likelihood estimation from the degree of bone marrow engraftment observed, demonstrated that HSPCs expanded with Lip-1 required ~ 100-fold fewer HSCs to achieve similar levels of engraftment in comparison with controls (Fig. 3E). Moreover, when BM-derived CD34+cells were enriched for at 15 weeks post-transplant and their clonogenic potential was tested, a higher number of colonies generated by the Lip-1 groups were observed, with colonies representative of all major lineages that were similar to controls (Fig. 3F).

[0152] Additionally the serial repopulating capacity of long-term HSCs was tested by performing secondary transplants with BM-derived CD34+cells from the primary CB- transplanted mice (Fig. 3 A). Despite transplanting similar numbers of CD34+HSPCs, higher human hematopoietic engraftment among the Lip-1 treated group was observed in all organs analyzed, rigorously demonstrating the more robust HSC expansion by mitigating ferroptosis (Figs. 3G-3H and Fig. 11G). Moreover, while the output in the different lineages was similar between the two groups, a higher fraction of stem and progenitor cells in the Lip-1 treated group were reported (Fig. 31 and Fig. 11H).

[0153] To extend these results to other cell sources and culture methods, mPB-derived CD34+HSPCs were cultured for only 4 days in serum-free culture conditions, akin to commonly applied approaches in gene and cell therapy protocols. Consistent with the findings from transplanting CB-derived HSPCs in chemically-defined conditions, similar results were obtained by transplanting shorter-term expanded mPB-derived cells into NBSGW mice (Fig. 4A). At 12 weeks post-transplantation, higher human chimerism was observed in the Lip-l-treated group, with a notable increase in the frequency of more primitive progenitor cells (Figs. 4B-4C). In line with these outcomes, maximum likelihood estimation confirmed that Lip- 1 -expanded HSPCs required ~100-fold fewer input cells to reach engraftment levels comparable to controls (Fig. 4D). Furthermore, BM-derived CD34+cells obtained from the Lip-1 group exhibited greater colony output in methylcellulose assays (Fig. 111). Collectively, these results demonstrate that Lip-1 treatment consistently improves the long-term repopulating capacity of both cord blood and adult-derived HSPCs, even in short-term serum-free cultures, validating its potential for PCT / US25 / 47496 23 September 2025 (23.09.2025)

[0154] ATTORNEY DOCKET NO. 11624-021WO1 clinically-relevant HSC expansion.

[0155] While these results suggest that hematopoiesis is preserved and appears ostensibly normal, even after transplantation, it was sought to ensure that there were no signs of aberrant hematopoiesis. To assess genotoxicity in-depth, 137 amplicons were profiled, covering >95% of clonal hematopoiesis of indeterminate potential mutations (Mack 2024) across three different donors in samples cultured ex vivo with or without Lip-1 over several weeks (the same samples were analyzed after 1 and 3 weeks of culture). While a single germline benign polymorphism was detected in TP53 in one donor (Methods), no somatic variants were identified and no signs of clonal expansions were noted in this analysis (Fig. 12). Additionally, the scRNA-seq data was examined to assess for major structural variants or aneuploidies using a sensitive tool for haplotype- aware copy number analyses, Numbat (Gao 2023). No signs of detectable copy number variation in Lip- 1 -treated hematopoietic cells were observed in comparison to controls (Figs. 13A-13B). Collectively, these results strongly support the contention that while blockade of ferroptosis with Lip-1 improves human HSC expansion ex vivo, this does not appear to promote genotoxicity in the cells.

[0156] Dissecting mechanisms of Lip-1 treatment impact upon human HSCs

[0157] It was desired to more fully understand mechanisms underlying the improved expansion of HSCs observed by preventing ferroptosis. Interestingly, when cells were cultured with Lip-1 (at 10 M ), overall decreased cell expansion in the cultures was observed (Fig. 5A and Fig. 14A) and concomitantly, it was found that HSC-enriched fractions (CD34+CD45RA'CD90+) underwent fewer divisions (Fig. 5B), with a similar trend seen among phenotypic LT-HSCs (Fig. 14B). While this could be attributable to either a direct slowing of cell divisions or improved maintenance of LT-HSCs, the numbers of LT-HSCs observed in the cultures were higher upon Lip-1 treatment (Fig. 14C). Therefore, ferroptosis prevention helps to better preserve and expand bona fide quiescent LT-HSCs that would generally fail to be appropriately supported in such ex vivo cultures.

[0158] Lip-1 treatment was associated with increased expression of the key ferroptosis protective gene GPX4 for up to 14 days in serum-free cultures (Fig. 5C and Fig. 14D). Notably, even after the induction of ferroptosis by the molecules erastin and RSL-3 (Dixon 2012, Yang 2014), Lip-1 supplementation protected human HSPCs with higher GPX4 expression and subsequently decreasing lipid peroxidation in progenitor and HSC-enriched subpopulations (Fig. 5D and Figs. 14E-14F). Assessment of the scRNA-seq data from molecularly-defined HSC populations showed an upregulation of GPX4 and iron binding genes, all of which are expected to protect against ferroptosis, as well as reductions in the expression of genes involved in oxidant PCT / US25 / 47496 23 September 2025 (23.09.2025)

[0159] ATTORNEY DOCKET NO. 11624-021WO1 detoxification, as expected under reduced oxidative stress (Fig. 14G). Moreover, other major ferroptosis-regulating antioxidant genes (Kraft 2020, Doll 2019, Yang 2025)- AIFM2 (also known as FSP1), GCH1, and ALDH7A1 - were expressed at low or undetectable levels in human HSCs. These findings support the notion that ferroptosis inhibition in human HSCs is primarily mediated through the GPX4 axis, while other ferroptosis regulators may have a less dynamic role in these cells under standard ex vivo culture conditions (Fig. 14H).

[0160] While Lip-1 and Fer-1 function as radical trapping antioxidants within the cell membrane that thereby prevent lipid peroxidation (Zilka 2017), it was heretofore unappreciated that there are secondary adaptations in human HSCs that promote further protection from ferroptosis and enable improved HSC expansion. To examine this possibility, the upregulated gene sets from the molecularly-defined HSCs in the scRNA-seq data were queried and unexpectedly an enrichment of only two major pathways: ribosomal biogenesis / translation regulation and cholesterol metabolism was found (Fig. 141). Notably, the upregulation of cholesterol biosynthetic pathways was primarily confined to the HSC compartment, suggesting a cell type-specific transcriptional response in the setting of Lip-1 treatment (Fig. 14J).

[0161] In particular, a substantial number of ribosomal protein genes - including both large and small subunit components - were significantly upregulated, suggesting a coordinated increase in ribosome production (Fig. 5E and Fig. 14K). To directly assess whether protein synthesis rates were altered, CD34+CD45RA“CD90+cells were labeled with O-propargyl-puromycin (OP-puro), a puromycin analog that gets incorporated into and terminates nascent polypeptide chains, to assess protein synthesis rates (Zhao 2023, Singer 2014, Hidalgo 2020). Lip-l-treated HSCs had a ~1.5-fold increase in protein synthesis in comparison with untreated cells (Fig. 5F). While HSCs are known to typically maintain low and highly regulated protein synthesis rates (Singer 2014, Hidalgo 2020), prior work has shown that slightly increased translation within human HSCs can protect from ferroptosis (Zhao 2023), aligning with current observations.

[0162] The upregulation of cholesterol biosynthesis was unanticipated and noteworthy given recent studies demonstrating a role for intermediates in this pathway, particularly B -ring- unsaturated sterols such as 7-dehydrocholesterol (7-DHC), in protecting cells from ferroptosis (Li 2024, Freitas 2024). Remarkably, upregulated gene expression for almost all components of the cholesterol biosynthesis pathway was noted (Mitsche 2015) (Figs. 5G-5H and Fig. 14L). Importantly, upregulation of ribosomal protein and cholesterol biosynthesis genes were also observed upon Fer-1 treatment, suggesting that these gene expression changes reflect a general adaptation of HSCs that are protected from ferroptosis (Fig. 14M).

[0163] To examine the impact of these alterations, lipidomic analysis was performed on sorted PCT / US25 / 47496 23 September 2025 (23.09.2025)

[0164] ATTORNEY DOCKET NO. 11624-021WO1

[0165] HSC-enriched (CD34+CD45RA CD90+) and other hematopoietic progenitors (CD34+CD45RA‘ CD90 ) in chemically-defined culture conditions. While the levels of cholesterol and other measured intermediates were only slightly elevated or unchanged in the HSC-enriched population and were reduced in progenitors, the amount of 7-DHC was significantly elevated, consistent with a state that can robustly protect cells from lipid peroxidation and resultant ferroptosis (Fig. 51 and Fig. 15A). Notably, some of the observed upregulation in 7-DHC levels could be attributed to increased radical scavenging by Lip-1 and Fer-1, which thereby preserve greater 7-DHC levels in the membrane, although the observed gene expression changes suggest at least some impact from altered cholesterol biosynthesis. Direct supplementation with 7-DHC expanded HSC-enriched subpopulations, with an increase in primitive progenitors, as well as ST- and LT-HSCs, resembling the effect of Lip-1 or Fer-1 addition (Figs. 5J-5K and Figs. 15B- 15C). Furthermore, the diversity of phospholipids within human HSCs cultured with Lip-1 was also examined and a reduction of phospholipids with polyunsaturated fatty acids and plasmalogens was observed, which would reduce the propensity for a cell to undergo ferroptosis (Zou 2020) (Fig. 15D). These results point to a remarkable metabolic adaptation within cultured human HSCs that increases expression of the protein synthesis machinery and alters cholesterol biosynthesis, while also reducing polyunsaturated phospholipids, to protect from ferroptosis.

[0166] Prevention of ferroptosis to improve HSC yields with genome engineering

[0167] Though earlier results support the notion that human HSC expansion with different cell sources and culture approaches can be improved by preventing ferroptosis, one of the most immediate applications of this advance is the clinical use of genome engineering approaches in HSCs cultured ex vivo. This is particularly important, as genome editing of human HSCs involves additional manipulations, such as the introduction of recombinant Cas9 protein or derivates, as well as the electroporation of cells, which might promote further cell loss.

[0168] First, mPB -derived HSPCs were cultured in serum-free cultures supplemented with Lip- 1. Two days after thawing, HSPCs were nucleofected with Cas9 ribonucleoprotein complexes (RNP) pre-assembled with synthetic base-modified single-guide RNAs (sgRNAs) targeting the benign safe harbor AAVS1 (adeno-associated virus integration site 1) locus. After confirming that Lip-1 supplementation had no impact on editing efficiency a ~2-fold increase in the fraction of LT-HSC was observed, without notable changes in other subpopulations analyzed (Figs. 6A-6B and Figs. 16A-16C).

[0169] The CD33 surface protein is an attractive immunotherapy target for acute leukemia, but is also expressed on normal hematopoietic cells and therefore epitope editing of CD33 in HSCs allows for the application of CD33 -targeting immunotherapies concomitant with the continuation PCT / US25 / 47496 23 September 2025 (23.09.2025)

[0170] ATTORNEY DOCKET NO. 11624-021WO1 of normal hematopoiesis (Kim 2018, Martin-Rufina 2023, Borot 2019, Borot 2023). Therefore it was next examined what occurs when targeting the CD33 locus with base editing. When using recombinant base editor protein-based RNPs, comparable loss of CD33 protein expression and level of editing was observed, while there was an ~8-fold improved expansion of LT-HSCs with Lip-1 treatment (Figs. 6C-6D and Figs. 16D-16F). These results demonstrate that the blockade of ferroptosis improves HSC maintenance in the presence of various types of genome engineering manipulations.

[0171] Next, genome editing of CB -derived HSPCs cultured in chemically-defined cytokine-free medium was examined (Sakurai 2023). After a week in this culture, Cas9 RNPs and sgRNAs targeting the A4VS7 locus were introduced again and high and comparable levels of edited cells were obtained (Fig. 6E). Crucially, Lip-1 supplementation protected cells from loss post-editing and led to a marked expansion of HSC-enriched subpopulations (CD90+ cells), along with substantial increases in ST- and LT-HSCs, which exceeded 50-fold after three weeks of culture (Figs. 6F-6H and Figs. 16G-16I).

[0172] Genome engineering of HSCs has now been clinically tested and approved for the treatment of sickle cell disease and P-thalassemia by using Cas9-based genome editing to disrupt the BCL11A +58 enhancer (Frangoul 2024, Locatelli 2024). However, HSC numbers for these procedures are often limiting. The approach of inhibiting ferroptosis was tested to augment HSC retention in cultures akin to those applied clinically, while also showing similar editing efficiency. Cas9 RNPs targeting the BCL11A +58 enhancer were introduced into mPB-derived HSPCs, in a similar manner to what is being done clinically, and then HSCs were either retained in HSC expansion conditions or underwent erythroid differentiation (Giani 2016) (Fig. 61). Efficient and comparable editing in the HSPC population was observed, but surprisingly the edits were better preserved during erythroid differentiation of Lip-1 treated cells, suggesting that blocking ferroptosis also better preserves edited cells (Fig. 6J). Importantly, LT-HSC expansion was improved by ~3-fold with minimal impact on other subpopulations (Fig. 6K and Figs. 16J- 16L). Additionally, early Lip-1 supplementation did not compromise erythroid differentiation capabilities of edited or unedited cells, while a similar extent of fetal hemoglobin induction was seen across all conditions (Figs. 6L-6N and Fig. 16M). These data from conditions that mimic what is being clinically-applied in approved genome editing therapies highlight a potential path for integration of ferroptosis blockade to improve HSC expansion in clinical settings.

[0173] Discussion

[0174] While significant progress has been made to improve the maintenance and expansion of human HSCs ex vivo, this remains a significant challenge (Meaker 2024). This is due to the fact PCT / US25 / 47496 23 September 2025 (23.09.2025)

[0175] ATTORNEY DOCKET NO. 11624-021WO1 that minimal components present in culture fail to appropriately mimic the highly -regulated niche within the bone marrow in which these cells ordinarily reside throughout life (Morrison 2014, Tikhonova 2019, Baryawno 2019). Moreover, most ex vivo HSC culture protocols not only seek to maintain these cells that ordinarily undergo self-renewal once or twice a year in the bone marrow, but also try to expand these cells over a short period in culture (Liggett 2020). Therefore, the existing culture methods fail to fully support human HSCs under the stressful conditions required for expansion. Based on prior studies demonstrating a unique vulnerability of human HSCs to loss via ferroptosis (Zhao 2023), the present invention centers on the finding that ferroptosis can be mitigated in diverse and commonly applied culture conditions in order to improve the expansion of human HSCs. Remarkably, consistent impacts across a range of approaches were observed without any signs of cell toxicity or indication that hematopoiesis is adversely impacted.

[0176] These observations can have an impact on clinical and translational efforts to maintain and expand HSCs ex vivo for gene therapy and genome editing approaches in autologous settings, where harvested cell numbers can be limiting, as well as in the setting of using allogeneic hematopoietic cell sources, where product availability can be limited for many individuals (Sanchez-Petitto 2023). Given the ease with which radical-trapping antioxidants can be added to existing culture methods without compromising cell viability, this approach can be readily integrated into existing protocols. This enables hematopoietic cell therapy approaches to be applied in more patients and require fewer cells to be harvested, which significantly advances treatment availability and options.

[0177] Furthermore, how key intermediates of this pathway, such as 7-DHC, can enhance human HSC expansion in tandem with other metabolic alterations, such as reductions in polyunsaturated phospholipid levels were previously unappreciated. Additionally, changes in cholesterol biogenesis enzymes may also impact other metabolic pathways, contributing to broader cellular adaptations. These findings reveal critical metabolic regulators involved in stem cell biology, paving the path toward additional advances in this field.

[0178] Methods

[0179] Primary cell culture: Human CD34+HSPCs from mobilized peripheral blood of healthy adults were obtained from the Cooperative Center of Excellence in Hematology at the Fred Hutchinson Cancer Research Center. After thawing, HSPCs were seeded at the concentration of 5xl05cells / ml in serum-free StemSpan SFEM II medium (StemCell Technologies) supplemented with 1% L-glutamine (Thermo Fisher Scientific), 1% penicillin / streptomycin (Life Technologies), IX CC100 (containing the cytokines FLT3L, SCF, IL3, and IL6; StemCell PCT / US25 / 47496 23 September 2025 (23.09.2025)

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[0181] Technologies), 100 ng / ml recombinant thrombopoietin (TPO; PeproTech), and 35 nM UM171 (StemCell Technologies), as we have described previously (Zhao 2023, Voit 2023, Martin- Rufino 2023).

[0182] Human CD34+HSPCs were also sourced from cord blood, obtained from the Dana- Farber Cancer Institute or Brigham and Women’s Hospital as discarded deidentified samples. CD34+HSPCs were enriched from the cord blood by the EasySep Human Cord Blood CD34+positive selection kit (StemCell Technologies) according to the manufacturer’s instructions. These cells were cultured either in serum-free StemSpan SFEM II medium (StemCell Technologies) with the aforementioned supplements or in Iscove's Modified Dulbecco's Medium (IMDM; Life Technologies). The IMDM was supplemented with 1% insulin-transferrin- selenium-ethanolamine (ITSX; Life Technologies), 1% L-glutamine (Thermo Fisher Scientific), 1% penicillin / streptomycin (Life Technologies), 1 mg / ml polyvinyl alcohol (PVA; Sigma- Aldrich), 1 |iM 740Y-P (MedChemExpress), 0.1 pM butyzamide (MedChemExpress), and 70 nM UM171. This latter culture method follows the protocol outlined by Sakurai and colleagues (Sakurai 2023). In this condition, human CB CD34+ cells were seeded at a density of 7xl04to IxlO5cells / ml in either 1 ml of medium per well in a 24-well plate or 5 ml of medium per well in a 6-well CellBind plate.

[0183] Where indicated, HSPCs were treated with liproxstatin-1 (Lip-1; Caymen Chemicals), ferrostatin-1 (Fer-1; MedChemExpress), sodium selenite (Sigma- Aldrich), P-mercaptoethanol (Thermo Fisher Scientific), RSL-3 (SelleckChem), Erastin (SelleckChem), and 7- dehydrocholesterol (7-DHC; Sigma- Aldrich).

[0184] In vitro erythroid differentiation: 48h post-thawing, mPB-derived HSPCs were electroporated with Cas9 RNPs targeting the BCL11A +58 enhancer (Frangoul 2024, Locatelli 2024, Canver 2015) and were immediately transferred into erythroid differentiation medium (Giani 2016). The differentiation medium consisted of IMDM (Life Technologies) supplemented with 2% human AB plasma (SeraCare), 3% human AB serum (Atlanta Biologicals), 1% penicillin / streptomycin (Life Technologies), 3 U / mL heparin (Hospira), and 10 pg / mL recombinant human insulin (Lilly). During Phase I of differentiation (days 0-6), the medium was further supplemented with 200 pg / mL holo-human transferrin (Sigma), 1 ng / mL of recombinant human IL- 3 (Peprotech), 10 ng / mL human SCF (Peprotech), and 3 U / mL erythropoietin (Amgen). In Phase II (days 7-11), IL-3 was removed from the medium. During Phase III (days 12-21), both IL-3 and SCF were omitted, and the holo-transferrin concentration was increased to 1 mg / mL. PCT / US25 / 47496 23 September 2025 (23.09.2025)

[0185] ATTORNEY DOCKET NO. 11624-021WO1

[0186] Xenotransplantation and animal models: All animal procedures were performed under a protocol approved by the Boston Children’s Hospital Institutional Animal Care and Use Committee (IACUC). CD34+cells derived from cord blood (CB) were cultured in cytokine-free conditions, and after 7 days of expansion in culture, 20,000 input cells (yielding approximately 50,000 total cells) were injected per mouse via tail vein into NBSGW immunodeficient and Kit mutant mice (JAX#026622). For secondary transplants, CD34+cells purified from the BM of primary recipients were pooled according to the experimental group and similar numbers of cells were transplanted into five separate NBSGW recipient mice. Mobilized peripheral blood (mPB)- derived CD34+cells were cultured in serum-free conditions, and after 4 days of expansion in culture, 150,000 input cells (yielding approximately 1,400,000 total cells) were injected per mouse via tail vein into NBSGW mice.

[0187] To prevent infections, the mice were provided with autoclaved sulfatrim antibiotic water, which was changed weekly. To monitor engraftment, peripheral blood was collected at 4, 8, 12, and 15-weeks post-transplantation through retro-orbital sampling. At 12 or 15 weeks posttransplantation, the animals were euthanized, and their bone marrows and spleens were collected for analysis. Bone marrow cells were obtained by flushing the bilateral femurs and tibias, while spleens were carefully minced. Human chimerism in the collected samples was assessed by flow cytometry using anti-human CD45 and anti-mouse CD45 antibodies. The composition of specific cell lineages in the organs was evaluated using lineage- specific markers: hCD3 (T cells), hCD33 (myeloid cells), and CD19 (B cells).

[0188] The relative number of HSCs required for a specific extent of engraftment is calculated by the maximum likelihood estimation method (MLE) using the following formula: HSCrequired = -log(l - fraction stem cells) * total_cells_transplanted / mean(log(l-donor_chimerism)), where the fraction of stem cell frequency here is assumed to be 0.005 across all groups. While not providing a precise estimate of the number of HSCs in a mixed and heterogeneous population, as can be achieved with limiting dilution analyses, this approach provides an estimate of how many HSCs are required to achieve a specified level of engraftment (Zhao 2024, Berger 1996).

[0189] Colony-forming unit cell assay: The CFU-C assay was performed using CD34+ cells derived from the bone marrow (BM) of mice transplanted with cord blood (CB) cells, cultured either in the presence or absence of Lip-1. A total of 2,500 cells were plated in a methylcellulose-based medium (MethoCult H4434, StemCell Technologies) that contains the cytokines IL-3, SCF, GM-CSF, and EPO and which is supplemented with 100 lU / ml penicillin and 100 pg / ml streptomycin. Two weeks post-plating, colonies were counted in a blinded PCT / US25 / 47496 23 September 2025 (23.09.2025)

[0190] ATTORNEY DOCKET NO. 11624-021WO1 fashion, and erythroid, myeloid, and mixed colonies were identified according to morphological criteria.

[0191] CRISPR / Cas9 RNP nucleofection: The Cas9 ribonucleoprotein (RNP) complexes were assembled by combining 2.1 pL of DPBS, 1.2 pL of lOOmM sgRNA in IDTE pH 7.5 (IDT) and 1.7 pL of 62mM Alt-R S.p. HiFi Cas9 Nuclease V3 (IDT, 1081061), and incubating at room temperature for 10-30 min. The assembled RNP complex was then mixed with Lonza P3 primary cell nucleofection reagent (Lonza, V4XP-3032) in the presence of 1 pl of lOOmM stock of IDT nucleofection enhancer. The mixture was delivered into CD34+HSPCs by nucleofection using the Lonza 4D nucleofector system with the EO100 program 2 or 7 days after thawing in serum- or cytokine-free culture medium, respectively. The cells were harvested for genomic DNA extraction at least 72 hours post- nucleofection and PCR fragments flanking the editing site (at least 250 bp upstream and downstream) were amplified and sent for Sanger sequencing to assess editing efficiencies. Sanger traces were imported to TIDE CRISPR version 3.2.0 for indel measurement with 100 bp left boundary and automatically set at break site -10 bp as alignment window, 115-515 bp decomposition window, 40 bp indel size range, and 0.05 P-value.

[0192] Base editor protein electroporation: RNP complexes were assembled by combining 2.1 pL of DPBS, 1.57 pL of chemically-modified sgRNAs (IDT) resuspended at 100 mM in IDTE pH 7.5 (IDT, 11-01-02-02), and the ABE8e protein, and incubating at room temperature for 10- 30 min. ABE8e protein was purified as previously described (Martin- Rufino 2023) while the final amount of base editor protein per electroporation ranged between 20-40 pg and was optimized using the base editing activity of the batch as assessed by titration experiments in primary HSPCs. The mixture was delivered into CD34+HSPCs by nucleofection using the Lonza 4D nucleofector system with the EO100 program. The cells were harvested, and genomic DNA was extracted at least 72 hours post-nucleofection for NGS-based calculation of editing efficiency.

[0193] Real-time PCR analysis: The total RNA was obtained using the Quick-DNA / RNA Microprep Plus (Zymo research) purification kit according to the manufacturer’s instructions. 100 to 500 ng of total RNA was used for reverse transcription using iScript cDNA synthesis kit (Biorad). The cDNA product was used for real-time PCR analysis using iQ SYBR green supermix (Biorad). Three technical replicates were performed for each sample, and the mean value was selected for further analysis. The relative expression of each target gene was first normalized to ACTB housekeeping gene expression and then represented as fold changes (2ddCt) relative to the indicated control conditions. PCT / US25 / 47496 23 September 2025 (23.09.2025)

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[0195] Immunophenotypic and apoptosis analysis: For immunophenotypic analyses (performed on LSRII or LSRFortessa; BD Pharmingen) of ex vivo cultured HSPCs, cellular suspension (up to IxlO6cells) was incubated for 30 min with different fluorescent-labeled antibodies: 1:100 dilution of anti-human CD34 APC-Cy7 (BioLegend), 1:50 dilution of antihuman CD133 Super Bright 436 (Invitrogen), 1:100 dilution of anti-human CD90 PE-Cy7 (BD Biosciences), 1:50 dilution of anti-human CD45RA AlexaFluor-700 (BioLegend), 1:100 dilution of anti-human CD201 (EPCR) PE (BioLegend), and 1:40 dilution of anti-human CD49c (ITGA3) APC (BioLegend). Immunophenotypic staining was combined with 1:200 dilution of Apotracker Green (BioLegend) viability staining according to the manufacturer’s.

[0196] For erythroid differentiation analysis, cells at the indicated stage of differentiation were harvested and incubated with the following fluorescent-labeled antibodies: 1:150 dilution of antihuman CD235a APC-Cy7 (BioLegend) and 1:150 dilution of anti-human CD71 BV421 (BioLegend). Dead cells were excluded according to their positivity to Apotracker Green (BioLegend) staining.

[0197] For immunophenotypic analyses (performed on LSRII or LSRFortessa; BD Pharmingen) of cells retrieved from in vivo organs, cells were stained with different fluorescent-labeled antibodies:

[0198] Peripheral blood and Spleen: 1 :200 dilution of Apotracker Green (BioLegend), 1:100 dilution of anti-human CD45 APC (BioLegend), 1:50 dilution of anti-mouse CD45 PE (BioLegend), 1:100 dilution of anti-human CD19 APC-Cy7 (BD Biosciences), 1:100 dilution of anti-human CD33 BV421 (BioLegend), and 1:100 dilution of anti-human CD3 BV605 (BD Biosciences);

[0199] Bone marrow: 1:200 dilution of Apotracker Green (BioLegend), 1:100 dilution of anti -human CD45 APC (BioLegend), 1:50 dilution of anti-mouse CD45 PE (BioLegend), 1:100 dilution of anti-human CD19 APC-Cy7 (BD Biosciences), 1: 100 dilution of anti-human CD34 BV421 (BioLegend), 1:100 dilution of anti-human CD90 PE-Cy7 (BD Biosciences), and 1:50 dilution of anti-human CD45RA AlexaFluor-700 (BioLegend).

[0200] Single-stained cells were used as controls and SPHERO™ Rainbow Calibration Particles (Spherotech) were used to calibrate instrument. Data were analyzed using the FlowJo software.

[0201] Western blot: For immunoblot analysis, HSPCs were cultured in serum-free expansion medium for 4 days in the presence or absence of SS. Viable cells (negative to Apotracker) were sorted with a 100 mm nozzle - on a BD FACS Aria Fusion (BD Biosciences) - according to the surface expression of CD34, CD45RA, andCD90 markers. Sorted CD34+CD45RA CD90+cells were washed twice with cold PBS and lysed on ice for 30 minutes in RIPA buffer (Thermo PCT / US25 / 47496 23 September 2025 (23.09.2025)

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[0203] Fisher Scientific, 89900), supplemented with protease and phosphatase inhibitor mini tablets (Thermo Fisher Scientific, A32965). Lysates were centrifuged at 16,000 x g for 10 minutes at 4°C, and protein concentration in the supernatants was quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, 23225), according to the manufacturer’s instructions. Equal amounts of protein were separated on 10% Mini-PROTEAN® TGX™ Gels (Bio-Rad, 4561036) and transferred onto PVDF membranes (Millipore, IPVH00010) using the Trans-Blot® Turbo™ Transfer System (Bio-Rad). Membranes were blocked in Intercept blocking buffer (LICORbio, 927-70001) and incubated overnight at 4°C with primary antibodies against GPX4 (CellSignaling Technology, 52455; 1:1000) and Actin (Santa Cruz, sc-8432; 1: 1000). After washing, membranes were incubated for 1 hour at room temperature with HRP-conjugated antirabbit or anti-mouse secondary antibodies (Bio-Rad, 1706515 and 1706516, respectively; 1:5000). Signal detection was performed using Clarity-Western ECL Substrate (Bio-Rad, 170- 5061), and images were acquired using a ChemiDoc Imaging System (Bio-Rad).

[0204] Glutathione Measurement: Total glutathione levels were measured using the GSH- Glo™ Glutathione Assay (Promega, V6911) according to the manufacturer’s instructions. Briefly, HSPCs were cultured in serum- free expansion medium for 4 days in the presence or absence of -ME. Viable cells (negative to Apotracker) were sorted with a 100 mm nozzle - on a BD FACSAria Fusion (BD Biosciences) - according to the surface expression of CD34, CD45RA, and CD90 markers. Sorted CD34+CD45RA CD90+cells were resuspended in 50[iL of PBS and plated in white 96-well plates (Thermo Fisher Scientific, 15042) at a density of 10,000 cells / well in technical triplicates. An equal volume of GSH-Glo™ Reagent, containing a luciferin derivative and glutathione S-transferase, was added to each well and plates were incubated for 30 minutes at room temperature to allow conversion of the substrate in the presence of intracellular glutathione. After this incubation, Luciferin Detection Reagent was added, and following a 15-minute incubation at room temperature, luminescence was measured using a microplate luminometer (CLARIOstar). Glutathione concentrations were determined by comparing luminescence values to a standard curve generated using known concentrations of GSH.

[0205] Protein Synthesis Measurement: Protein synthesis rates were assessed using O- Propargyl-puromycin (OP-puro, Cayman Chemical, 601100) incorporation. HSPCs were cultured for 4 days in serum-free expansion medium in the presence or absence of Liproxstatin-1 (Lip-1). On the day of analysis, OP-puro (1:400 dilution from stock) was added to the culture medium and cells were incubated at 37°C for 2 hours. After incubation, cells were washed with PBS and fixed using Cell-Based Assay Fixative for 5 minutes at room temperature. To detect PCT / US25 / 47496 23 September 2025 (23.09.2025)

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[0207] OP-puro incorporation, cells were resuspended in FAM- Azide staining solution and incubated for 30 minutes at room temperature in the dark. Following staining, cells were washed with Cell- Based Assay Wash Buffer and analyzed using an LSRFortessa flow cytometer (BD Biosciences). As a negative control, cells were pretreated with cycloheximide (50 pg / mL) for 1 hour before OP-puro addition to inhibit protein synthesis. Data were analyzed with FlowJo software, and mean fluorescence intensity (MFI) was used to quantify relative translation activity.

[0208] Fetal hemoglobin detection in cells: Erythroid differentiation Phase III cellular suspension of approximately 3-5xl04cells was washed with 3 mL of 2% FBS-DPBS and fixed with 4% paraformaldehyde (Santa Cruz Biotechnology) at room temperature for 15 minutes. Following fixation, the cells were washed again with 3 mL of 2% FBS-DPBS and then permeabilized with 0.2% tween 20 in PBS for 5 minutes. Subsequently, the cells were stained with a 1:200 dilution of anti-human HbF PE fluorescent antibody for 30 minutes at room temperature. After a final wash with 2% FBS in PBS, sample acquisition was performed using an LSRFortessa flow cytometer. The collected data were then analyzed using FlowJo software.

[0209] Cell proliferation: After thawing, HSPCs were stained with Cell Trace CFSE (Thermo Scientific) according to the manufacturer’s instructions. The cells were resuspended in lx DPBS at a concentration of 106cells / mL. 1 pL of Cell Trace solution was added per mL of cell suspension, resulting in a final concentration of 5 pM. Cells were incubated for 20 minutes at 37°C, protected from light. Following this, cells were incubated with five times the original staining volume of lx DPBS + 2% FBS for 5 minutes. After centrifugation, the cells were resuspended in the appropriate culture medium volume and incubated for at least 30 minutes before analysis. Sample acquisition was performed on an LSRFortessa flow cytometer (BD Pharmingen), and the collected data were analyzed using FlowJo software.

[0210] Lipid peroxidation analysis: HSPCs were initially stained with anti-human CD34 BV421 (BioLegend), anti-human CD90 PE-Cy7 (BD Biosciences), and anti-human CD45RA AlexaFluor-700 (BioLegend) as previously described. Following this, the cells were incubated with the BODIPY Cll lipid probe (Invitrogen) according to the manufacturer’s instructions. In brief, cells were stained with 5 pM BODIPY 581 / 591 Cl l reagent in PBS at 37°C for 30 minutes. After staining, the labeled cells were washed and analyzed using an LSRFortessa flow cytometer (BD Pharmingen).

[0211] The lipid peroxidation state of each group was quantified by calculating the ratio of the mean fluorescence intensity (MFI) of oxidized lipids (detected in the green channel) to that of reduced lipids (detected in the red channel). PCT / US25 / 47496 23 September 2025 (23.09.2025)

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[0213] C8-pos: Reversed-phase C8 chromatography / positive ion mode MS detection to measure lipids: Analyses of polar and non-polar lipids were conducted using an LC-MS system comprised of a Shimadzu Nexera X2 U-HPLC (Shimadzu Corp.) coupled to an Exactive Plus orbitrap mass spectrometer (Thermo Fisher Scientific). HSPCs were cultured in chemically defined cytokine-free conditions (Sakurai 2023) for 10 days in the presence or absence of Lip-1. Viable cells (negative to Apotracker) were sorted with a 100 mm nozzle - on a BD FACSAria Fusion (BD Biosciences) - according to the surface expression of CD34, CD45RA, and CD90 markers. 5xl05Sorted CD34+CD45RA CD90+and CD34+CD45RA CD90" cells were collected in 1.5 ml Eppendorf tubes containing 100 pl of Isopropanol. Samples were centrifuged at 10,000 ref for 10 minutes and 10 pL of supernatant was injected directly onto a 100 x 2.1 mm, 1.7 pm ACQUITY BEH C8 column (Waters). The column was eluted isocratically with 80% mobile phase A (95:5:0.1 vol / vol / vol lOmM ammonium acetate / methanol / formic acid) for 1 minute followed by a linear gradient to 80% mobile-phase B (99.9:0.1 vol / vol methanol / formic acid) over 2 minutes, a linear gradient to 100% mobile phase B over 7 minutes, then 3 minutes at 100% mobile-phase B. MS analyses were carried out using electrospray ionization in the positive ion mode using full scan analysis over 220-1100 m / z at 70,000 resolution and 3 Hz data acquisition rate. Other MS settings were: sheath gas 50, in source CID 5 eV, sweep gas 5, spray voltage 3 kV, capillary temperature 300°C, S-lens RF 60, heater temperature 300°C, microscans 1, automatic gain control target le6, and maximum ion time 100 ms. Raw data were processed using TraceFinder software (Thermo Fisher Scientific) for targeted peak integration and manual review of a subset of identified lipids and using Progenesis QI (Nonlinear Dynamics) for peak detection and integration of both lipids of known identity and unknowns. Lipid identities were determined based on comparison to reference plasma extracts and are denoted by total number of carbons in the lipid acyl chain(s) and total number of double bonds in the lipid acyl chain(s).

[0214] Clonal hematopoiesis mutation amplicon sequencing and data analysis: HSPCs derived from 4 different donors were cultured in chemically defined cytokine-free conditions (Sakurai 2023) for up to 3 weeks in the presence or absence of Lip-1. To assess the potential acquisition of a CHIP mutation, an early (7-10 days) and a late (17-25 days) sample from the same cultures were analyzed by deep amplicon sequencing. The presence of mutations in donor- derived cultures was assessed using a targeted gene panel that has been previously described (Mack 2024). The panel consists of 137 amplicons across 24 genes, covering >95% of observed clonal hematopoiesis and indeterminate potential (CHIP) mutations. One sample (#14) failed sequencing for technical reasons, while for all the others paired-end sequencing yielded 116-256reads per sample. Each sample was processed by an in-house variant calling pipeline. PCT / US25 / 47496 23 September 2025 (23.09.2025)

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[0216] Specifically, sequencing adapters were trimmed using cutadapt (Martin 2011) and assessed in FastQC. Trimmed reads were mapped to hg38 using BWA mem. Then, duplicates were marked using Picard, base qualities were recalibrated using GATK (McKenna 2010), and Mutect2 (Benjamin 2019) was run with default parameters to call somatic variants. The produced VCF file (unfiltered Mutect2 results) was filtered by FilterMutectCalls (with -max-events-in-region 3) and BCFtools (Benjamin 2019, Danecek 2021), to select variants with sequencing depth >= 50, >= 5 reads supporting the variant, and allele frequency >= 0.01. Additionally, the commonly artifactual ASXL1 c,1934dupG frameshift was assessed with allele frequency > 0.05, which often fails the ‘slippage’ filter of Mutect2 but was reported as a true variation in some samples (Alberti 2018). Called variants were then annotated using the Ensembl Variant Effect Predictor (McLaren 2016) and manually inspected in the Interactive Genome Viewer (Robinson 2011) to remove germline variants and false positive calls with low support or polymerase slippage. Additionally, a cross-abundance analysis was conducted to assess the number of occurrences of each selected variant in unfiltered Mutect2 results across all samples in this sequencing run, including unrelated samples. Three variants were recurrent (Figs. 11A-11I). One of them, a missense mutation in TP53 with a VAF close to 0.5 is a germline variant, being present in all four samples from donor CB-52. This specific variant (NM_000546.6(TP53):c.869G>A (p.Arg290His)) has been annotated by the ClinGen TP53 Variant Curation Expert Panel and other expert panels to be a benign polymorphism found in the population (Fortuno 2021). The two other variants are single-base insertions in homopolymeric tracts within ASXL1 and IDH2 genes; they are considered PCR artifacts as they are present across multiple independent samples from the present application and healthy donor bone marrow samples from an independent study (gray bars) (Fig. 12). Of three additional variants that were reported, two were found to be artifacts upon manual inspection because sequencing reads supporting these variants were also present in several unrelated samples, and one was a synonymous variant in ZBTB33 with VAF<2%, supported by less than 10 reads. Overall, the targeted sequencing of 137 amplicons did not show any reliable CHIP-associated mutations at a VAF threshold >1%.

[0217] Single-cell RNA-sequencing and analysis: Droplet-based digital 3 ’-end scRNA-Seq was performed on a Chromium Single-Cell Controller (10X Genomics) using the Chromium Next GEM Single Cell 3’ Reagent Kit v3.1 according to the manufacturer’s instructions. CB HSPCs derived from 2 different donors were cultured in chemically defined cytokine-free conditions (Sakurai 2023) for 10 days in the presence or absence of Lip-1. Viable cells (negative to Apotracker) were sorted with a 100 mm nozzle - on a BD FACSAria Fusion (BD Biosciences) - according to the surface expression of CD34+CD45RA CD90+markers. Sorted PCT / US25 / 47496 23 September 2025 (23.09.2025)

[0218] ATTORNEY DOCKET NO. 11624-021WO1 cells were collected in 1.5 ml Eppendorf tubes containing 100 pl of IxDPBS. The sorted cells were counted with Trypan Blue solution 0.4% (GIBCO) and roughly 2xl04viable cells from each sample were utilized for the subsequent procedure (estimated recovery: 104cells / sample). Briefly, single cells were partitioned in Gel Beads in Emulsion (GEMs) and lysed, followed by RNA barcoding, reverse transcription, and PCR amplification (11 cycles). scRNA-Seq libraries were prepared according to the manufacturer’s instructions, checked, and quantified on LabChip GX Touch HT (Perkin Elmer) and Qubit 3.0 (Invitrogen) instruments. Sequencing was performed on a Nova Seq S2 (Illumina). The raw scRNA-seq FASTQ files were processed with the CellRanger (v8.0.1) pipeline to map in the reference genome (GRCh38). cells with unique molecular identifier (UMI) counts less than 1,000, or mitochondrial UMI fraction higher than 20% were excluded, and potential doublets were removed by a threshold of doublet score > 0.2 using ScrubletR, which resulted in a total of 29,096 cells for the Lip-1 (replicate 1 = 5,991 and replicate 2 = 4,935) and Untreated (replicate 1 = 8,991 and replicate 2 = 9,179) groups.

[0219] Symphony R package (Kang 2021) was used to project the cells on the human bone marrow reference (Zeng 2023), and the scRNA-seq reference built from 10-day-expanded CD34+cells in cytokine-free medium, and the predicted cell type was further curated to match the 12 hematopoietic cell types presented from Sakurai and collegues (Sakurai 2023). A standard Seurat framework (v4.4.0) was used to conduct normalization, principal component analysis (PCA), and dimensionality reduction. The feature-barcode matrix was normalized by the total read count and log-transformed, and the top 3,000 variable features were selected by the vst method in the FindVariableFeatures function. The normalized expression was scaled by Seurat’s ScaleData function, and PCA was performed using the RunPCA function (npc = 30). The sampledependent technical variation was corrected by using Harmony (Korsunsky 2019). Uniform Manifold Approximation and Projection (UMAP) was conducted to reduce dimensions to embed the cells into two-dimensional space. The HSC signature score was measured by applying AUCell (Aibar 2017) using CD34 and HLF RNA expression. Seurat’s FindMarkers function using “wilcox” method was applied to the HSC compartment to identify differentially expressed genes between Lip-1 and Untreated cells with a significance threshold of Benjamini & Hochberg (BH)-adjusted P < 0.05, log2 fold change > 0.1, and minimum percent of expressed cells > 10%. Gene set enrichment analysis was performed using the fGSEA package using GO Biological Process 2021 database. Profiling of chromosome copy numbers was assessed by Numbat (Gao 2023) (vl.4.0), run using default parameters. The Sakurai et al. dataset (Sakurai 2023) was used as the expression reference for all samples. Figures were generated using R (v4.4). PCT / US25 / 47496 23 September 2025 (23.09.2025)

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[0221] Quantification and statistical analysis: In all experiments, data were presented as mean ± standard error of mean. When comparing two samples, a two-tailed Student’s t-test was used to test statistical significance. When one of the two samples was a default value (as in fold change comparison), the one sample t- and Wilcoxon test was applied. When comparing three or more samples, Levene’s test was first used to test equality of variance. If the variance across all samples were tested insignificantly differed, one-way or two-way ANOVA with Dunnett’s test (for multiple comparisons where no reference group is defined) or Tukey’s test (for multiple comparisons where reference group is defined) as post-hoc analysis was used. If the variance across samples was tested to be significantly different, the Kruskal- Wallis test was used instead of ANOVA, with the Dunn test as the post-hoc multiple comparison test. All statistical tests were performed in Graphpad software or R when statistical tests were not available through Graphpad.

[0222] Data and materials availability: All raw and processed scRNA-seq data for Lip-1 and control untreated CD34+HSPCs have been deposited in the GEO repository (GSE276160, reviewer token: olkrgsiylpspfuf), and will be made publicly available as of the publication date. The raw scRNA-seq of 10 day-expanded CD34+cells from PCL-PVAc-PEG-based 3a medium, StemSpan with SR-1 medium, or StemSpan with UM171 medium culture conditions was obtained from GEO repository GSE192519. There is no original code generated in this study. Any information required to reanalyze the data reported in this study or other information is available from V.G.S. upon request.

[0223] Lastly, it should be understood that while the present disclosure has been provided in detail with respect to certain illustrative and specific aspects thereof, it should not be considered limited to such, as numerous modifications are possible without departing from the broad spirit and scope of the present disclosure as defined in the appended claims.

[0224] It will be apparent to those skilled in the art that various modifications and variations can be made in the present disclosure without departing from the scope or spirit of the invention. Other embodiments of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the methods disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

[0225] The following patents, applications and publications as listed below and throughout this document are hereby incorporated by reference in their entirety herein. PCT / US25 / 47496 23 September 2025 (23.09.2025)

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Claims

ATTORNEY DOCKET NO. 11624-021WO1CLAIMSWhat is claimed is:

1. A method of reducing ferroptosis in a hematopoietic stem cell (HSC) population, the method comprising administering at least one radical trapping antioxidant (RTA) to a population of HSCs, wherein the RTA reduces ferroptosis within the HSC population, thereby allowing expansion of the cells.

2. The method of claim 1, wherein the RTA comprises liproxstatin-1 (Lip-1), ferrostatin- 1 (Fer-1), or a functional derivative thereof.

3. The method of claim 1 or 2, wherein the ferroptosis is reduced by at least 10% within the cell population compared to a control in which an RTA is not administered.

4. The method of any one of claims 1-3, wherein cells are expanded by at least 10% compared to a control in which an RTA is not administered.

5. The method of any one of claims 1-4, wherein the HSCs are ex vivo, in vitro, or in vivo.

6. The method of any one of claims 1-5, wherein the HSCs are isolated from cord blood, peripheral blood, or bone marrow of a subject.

7. The method of any one of claims 1-6, wherein administrating an RTA to the HSC population increases expression of ferroptosis protective gene GPX4 in an HSC compared to a control.

8. The method of any one of claims 1-7, wherein administrating an RTA to the HSC population increases 7-dehydrocholesterol levels in the HSC compared to a control, wherein 7-dehydrocholesterol is an inhibitor of ferroptosis.

9. The method of any one of claims 1-8, wherein administrating an RTA to the HSC decreases cell proliferation and / or lipid peroxidation in the HSC compared to a control.ATTORNEY DOCKET NO. 11624-021WO110. The method of any one of claims 1-9, wherein administrating an RTA to the HSC population increases ribosome biogenesis and / or cholesterol biosynthesis in the HSC compared to a control.

11. The method of any one of claims 1-10, wherein the method is carried out ex vivo, and further wherein the subject is a human.

12. The method of any one of claims 1-11, wherein the HSC population is supplemented with at least one additional composition.

13. The method of claim 12, wherein the additional composition comprises 7- dehydrocholes terol.

14. A method of treating a blood disorder in a subject, the method comprising: a. isolating a hematopoietic stem cell (HSC) from a sample from a subject; b. administering at least one radical trapping antioxidant (RTA) to the HSC in culture, wherein the RTA can reduce ferroptosis in the HSC; c. genetically engineering the HSC, and d. administering to a subject in need thereof, the engineered hematopoietic stem cell.

15. The method of claim 14, wherein the RTA comprises liproxstatin-1 (Lip-1), ferrostatin-1 (Fer-1), or a functional derivative thereof.

16. The method of claims 14 or 15, wherein ferroptosis is reduced by at least 10% within the cell population compared to a control in which an RTA is not administered.

17. The method of any one of claims 14-16, wherein cells are expanded by at least 10% compared to a control in which an RTA is not administered.

18. The method of any one of claims 14-17, wherein administering an RTA protects HSCs from loss-post editing and preserves edited cells.

19. The method of any one of claims 14-18, wherein the genetic engineering of the HSC improves the function of blood cells.ATTORNEY DOCKET NO. 11624-021WO120. The method of any one of claims 14-19, wherein genetically engineering the HSC comprises introducing a mutation in an adeno- associated virus integration site 1 (AAVS1) locus, a CD33 locus, or a BCL11A +58 enhancer.

21. The method of any one of claims 14-20, wherein the genetic engineering increases expansion of HSCs compared to a control.

22. The method of any one of claims 14-21, wherein the sample is cord blood, peripheral blood, or bone marrow.

23. The method of any one of claims 14-22, wherein the blood disorder comprises severe acquired immune deficiency, metabolic diseases, muscular dystrophy, myotonic dystrophy, genetic blood disorders, congenita; neutropenia, Diamond-Blackfan anemia, Shwachman-Diamond syndrome, telomerase disorders, acquired aplastic anemia, bone marrow failure syndromes, immunodeficiency syndromes, lysosomal storage diseases, hemoglobinopathies including sickle cell disease and P-thalassemia, Fanconi anemia, leukodystrophies, congenital dyserythropoietic anemia, servere combined immunodeficiency (SCID), Wiskott-Aldrich syndrome, chronic granulomatous disease, osteopetrosis, mucopolysaccharidoses, Gaucher disease, Niemann-Pick disease, Hurler syndrome, Krabbe disease, metachromatic leukodystrophy, pyruvate kinase deficiency, and X-linked adrenoleukodystrophy.

24. The method of any one of claims 14-23, wherein the ex vivo genetic engineering comprises administering an endonuclease and single-guide RNA (sgRNAs) to the HSC.

25. The method of claim 24, wherein the endonuclease is Cas9.

26. The method of any one of claims 14-25, wherein the HSCs are supplemented with at least one additional composition.

27. The method of claim 26, wherein the additional composition comprises 7- dehydrocholesterol.ATTORNEY DOCKET NO. 11624-021WO128. The method of any one of claims 14-27, wherein the treatment is autologous.

29. The method of any one of claims 14-27, wherein the treatment is allogenic.

30. A kit for use in the expansion of hematopoietic stem cells, the kit comprising a medium appropriate for culturing stem cells and a radical trapping antioxidant (RTA).

31. The kit of claim 30, wherein the RTA comprises liproxstatin- 1 (Lip-1) or ferrostatin- 1 (Fer-1), or a functional derivative thereof.

32. The kit of claim 30 or 31, wherein the medium appropriate for culturing stem cells is a standard serum-free medium.

33. The kit of claim 32, wherein the medium appropriate for culturing stem cells is a chemically-defined cytokine-free medium.

34. The kit of any one of claims 30-33, wherein the kit consists of at least one additional composition.

35. The kit of claim 34, wherein the additional composition comprises 7- dehydrocholesterol.

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