Methods for regaining self-renewing capacity in exhausted hematopoietic stem cells (HSC)

Abstract

In alternative embodiments, methods as provided herein are used to maintain or extend the self-renewing capacity of HSCs in long term culture, where the HSCs can be isolated from bone-aged marrow donors. In alternative embodiments, methods as provided herein use a three-dimensional (3D) nanobioreactor. In alternative embodiments, provided are methods for regaining of the self-renewing capacity by hematopoietic stem cell (HSC) that is lost after long-term culture, where the methods comprise placing the HSC that have lost self-renewing capacity (so-called "exhausted HSCs) into a supporting, nourishing microenvironment for the additional co-culture period; and in alternative embodiments the methods comprise long term culture of HSCs isolated from bone aged marrow donors.

Description

[0001] PATENT 0321.158068PCT / SD2024-024PCT

[0002] METHODS FOR REGAINING SELF-RENEWING CAPACITY IN EXHAUSTED HEMATOPOIETIC STEM

[0003] CELLS (HSC)

[0004] RELATED APPLICATIONS

[0005] This Patent Convention Treaty (PCT) International Application claims the benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 693,935, Sept 12, 2024. The aforementioned application is expressly incorporated herein by reference in its entirety and for all purposes. All publications, patents, patent applications cited herein are hereby expressly incorporated by reference for all purposes.

[0006] STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0007] This invention was made with government support under NNJ13ZBG00 IN awarded by NASA; R01CA205944 awarded by NCI; R01DK114468-01 awarded by National Institutes of Health; AG068635 awarded by National Institutes of Health. The government has certain rights in the invention.

[0008] TECHNICAL FIELD

[0009] This invention generally relates to stem cell technology and space biology. In alternative embodiments, provided are methods for regaining of the self-renewing capacity by hematopoietic stem cell (HSC) that is lost after long-term culture, where the methods comprise placing the HSC that have lost self-renewing capacity (so- called “exhausted HSCs) into a supporting, nourishing microenvironment for the additional co-culture period; and in alternative embodiments the methods comprise long term culture of HSCs isolated from bone aged marrow donors. In alternative embodiments, methods as provided herein are used to maintain or extend the selfrenewing capacity of hematopoietic stem cell (HSCs) in long term culture, where the HSCs can be isolated from bone-aged marrow donors. In alternative embodiments, methods as provided herein use a three-dimensional (3D) nanobioreactor.

[0010] BACKGROUND

[0011] Life-long blood cell production and effective immune responses are predicated on the maintenance of healthy hematopoietic stem cells (HSCs) that can reside dormant in protective niches; mobilize following a “fight or flight” response; selfrenew (regenerate); and differentiate into all blood cell types. Hallmarks of stem cell aging include reduced self-renewal capacity, loss of dormancy, increased mobilization PATENT 0321.158068PCT / SD2024-024PCT from bone marrow niches, telomere attrition and reduced telomere maintenance, genomic instability, mitochondrial stress, accumulation of clonal mutations, and retrotransposon derepression, which triggers interferon-related inflammation- associated aging (inflammaging). These hallmarks of stem cell aging can be acquired in response to chronic or acute macroenvironmental exposures or stressors.

[0012] In addition to promoting hematopoietic stem and progenitor cell (HSPC) inflammaging, derepression of retrotransposons, which comprise approximately 35% of the human genome, can contribute to genomic and epitranscriptomic instability by activating DNA and RNA editing enzymes. Under homeostatic conditions, base deaminases, including adenosine deaminase associated with RNA (AD ARI) and apolipoprotein B mRNA editing catalytic polypeptide 3 (APOBEC3) family enzy mes, protect HSPC genomic integrity by restricting activation of retrotransposons, including human endogenous retroviruses (hERVs) with long terminal repeat (LTR) elements, Long INnterspersed Elements (LINEs) encoding reverse transcriptase, and the Short INnterspersed Elements (SINEs) that utilize LINE-encoded reverse transcriptase for retrotransposition, such as Alu sequences. However, protracted activation of base deaminases has been linked to clonal APOBEC3C-induced cytosine-to-thymine (C-to-T) single base substitution (SBS) DNA mutations that fuel pre-malignant myeloproliferative neoplasm (MPN) HSPC proliferation and ADAR1- induced adenosine to inosine (A-to-I) RNA editing alterations that drive malignant regeneration in at least 20 malignancies. Thus, early detection and reversal of microenvironmental and macroenvironmental stressors that accelerate HSPC aging are vital for preserving human health and longevity.

[0013] Pioneering astronaut research, including the National Aeronautics and Space Administration (NASA) Twins Study from a 1-year long mission and The Space Omics and Medical Atlas (SOMA) studies from short-term missions, has shown that spaceflight serves as a profound stressor, capable of reshaping the immune system, altering physiologic function, and causing prolonged molecular and cellular changes in many tissues21. Moreover. NASA research utilizing animal models, simulated microgravity, and human-derived cell cultures has revealed changes in inflammatory cytokines and shifts in immune cell populations, including natural killer (NK) cells, monocytes, granulocytes, and T-cells typical of aging. Previously, we investigated whether immune deficits arise from HSPC dysfunction in space in four month-long NASA-supported integrated space stem cell orbital research (ISSCOR) center (SpX- PATENT 0321.158068PCT / SD2024-024PCT

[0014] 24, SpX-25, SpX-26 and SpX-27) missions to the International Space Station (ISS) that utilized novel Al-driven 3D nanobioreactors seeded with aged normal bone marrow-derived HSPCs and stromal cells. In these ISSCOR studies, we observed niche-dependent HSPC dysfunction following post-spaceflight return compared with ground-based controls, which included reduced HSPC self-renewal capacity, base deaminase deregulation, reduced telomere maintenance gene expression, and dynamic retrotransposon derepression. Although we observed significant effects of spaceflight from in vitro nanobioreactor models of human HSPC aging, the temporal sequence and long-term impact of space associated stem cell hallmarks of aging in astronauts had not been addressed to date.

[0015] SUMMARY

[0016] In alternative embodiments, provided are methods for maintaining or extending the self-renewing capacity of hematopoietic stem cell (HSCs) in long term culture, or for regaining self-renewing capacity by hematopoietic stem cell (HSC) that that have lost their self-renewing capacity (so-called “exhausted HSCs) after longterm culture, the method comprising:

[0017] (a) providing a three-dimensional (3D) nanobioreactor;

[0018] (b) plating the HSCs that have been in long-term culture or the exhausted HSCs on an autologous primary stroma established from a CD34-negative fraction of aged bone marrow (ABM) cells or an Hs27a cell line monolayer for an additional coculture period comprising about at least 2 weeks, or between about 2 weeks and 4 weeks.

[0019] In alternative embodiments, the HSCs are derived from or isolated from human aged bone marrow donors.

[0020] In alternative embodiments, provided are methods for maintaining or extending the self-renewing capacity’ of hematopoietic stem cell (HSCs) in long term culture, or for regaining self-renewing capacity by hematopoietic stem cell (HSC) that that have lost their self-renewing capacity (so-called “exhausted HSCs) after longterm culture, the method comprising:

[0021] (a) providing a plurality of HSCs that have been in long-term culture and / or are exhausted HSCs;

[0022] (b) providing a three-dimensional (3D) nanobioreactor; PATENT 0321.158068PCT / SD2024-024PCT

[0023] (c) establishing or culturing in the 3D nanobioreactor: (i) an autologous primary stroma derived from a CD34-negative fraction of aged bone marrow (ABM) cells; (ii) an Hs27a cell line monolayer; or, (iii) both (i) and (ii);

[0024] (d) plating or culturing in the 3D nanobioreactor on the autologous primary stroma and / or on the Hs27a cell line monolayer the plurality of HSCs that have been in long-term culture and / or are exhausted HSCs; and

[0025] (e) culturing the plurality of HSCs that have been in long-term culture and / or are exhausted HSCs in the 3D nanobioreactor for at least 2 weeks, or between about 2 weeks and 4 weeks, or between one week and 5 weeks, or between 6 days and 6 weeks, wherein culturing the plurality of HSCs that have been in long-term culture and / or are exhausted HSCs in the 3D nanobioreactor for at least 2 weeks, or between about 2 weeks and 4 weeks, or between one and 5 weeks, maintains or extends the self-renewing capacity of the plurality of HSCs, or, results in the plurality of HSCs that that have lost their self-renewing capacity (so-called "‘exhausted HSCs) to regain their self-renewing capacity or to substantially regain their self-renewing capacity.

[0026] In alternative embodiments of methods as provided herein the plurality of HSCs are derived from or isolated from human aged bone marrow donors.

[0027] In alternative embodiment, provided are hematopoietic stem cell (HSC) selfrenewing capacity generating three-dimensional (3D) nanobioreactors made by a method comprising:

[0028] (a) providing HSCs that have been in long-term culture and / or are exhausted HSCs;

[0029] (b) providing a three-dimensional (3D) nanobioreactor;

[0030] (c) establishing or culturing in the three-dimensional (3D) nanobioreactor: (i) an autologous primary stroma derived from a CD34-negative fraction of aged bone marrow' (ABM) cells; (ii) an Hs27a cell line monolayer; or, (iii) both (i) and (ii), and

[0031] (d) plating or culturing in the 3D nanobioreactor on the autologous primary stroma and / or on the Hs27a cell line monolayer HSCs that have been in long-term culture and / or are exhausted HSCs.

[0032] In alternative embodiments of the 3D nanobioreactors as provided herein: the HSCs are derived from or isolated from human aged bone marrow' donors.

[0033] In alternative embodiments, provided are uses of a hematopoietic stem cell (HSC) self-renewing capacity generating three-dimensional (3D) nanobioreactor as PATENT 0321.158068PCT / SD2024-024PCT provided herein to: maintain or extend the self-renewing capacity of a plurality of HSCs, or, have or convert HSCs that that have lost their self-renewing capacity (so- called “exhausted HSCs) to regain their self-renewing capacity or to substantially regain their self-renewing capacity.

[0034] In alternative embodiments, provided are hematopoietic stem cell (HSC) selfrenewing capacity generating three-dimensional (3D) nanobioreactors for use in: maintaining or extending the self-renewing capacity of a plurality of HSCs, or. for use in converting HSCs that that have lost their self-renewing capacity (so-called “exhausted HSCs) HSCs that have regained their self-renewing capacity or to substantially regain their self-renewing capacity.

[0035] In alternative embodiment, provided are products of manufacture made by a method comprising:

[0036] (a) providing HSCs that have been in long-term culture and / or are exhausted HSCs;

[0037] (b) providing a three-dimensional (3D) bioreactor: and

[0038] (c) establishing or culturing in the three-dimensional (3D) bioreactor: (i) an autologous primary stroma derived from a CD34-negative fraction of aged bone marrow' (ABM) cells; (ii) an Hs27a cell line monolayer; or, (iii) both (i) and (ii).

[0039] (d) plating or culturing in the 3D bioreactor on the autologous primary stroma and / or on the Hs27a cell line monolayer HSCs that have been in long-term culture and / or are exhausted HSCs.

[0040] In alternative embodiment, provided are products of manufacture made by a method comprising:

[0041] (a) providing HSCs that have been in long-term culture and / or are exhausted HSCs;

[0042] (b) providing a three-dimensional (3D) bioreactor; and

[0043] (c) establishing or culturing in the 3D bioreactor: (i) an autologous primary stroma derived from a CD34-negative fraction of aged bone marrow (ABM) cells; (ii) an Hs27a cell line monolayer; or, (iii) both (i) and (ii).

[0044] In alternative embodiment, provided are methods for maintaining or extending the self-renewdng capacity of hematopoietic stem cell (HSCs) in long term culture, or for regaining self-renewing capacity by hematopoietic stem cell (HSC) that that have lost their self-renewing capacity (so-called “exhausted HSCs) after long-term culture, the method comprising: PATENT 0321.158068PCT / SD2024-024PCT

[0045] (a) providing a product of manufacture as provided herein;

[0046] (b) plating or culturing in the 3D nanobioreactor on the autologous primary stroma and / or on the Hs27a cell line monolayer a plurality of HSCs that have been in long-term culture and / or are exhausted HSCs, and

[0047] (e) culturing the plurality of HSCs that have been in long-term culture and / or are exhausted HSCs in the 3D nanobioreactor for at least 2 weeks, or between about 2 weeks and 4 weeks, or between one week and 5 weeks, or between 6 days and 6 weeks, wherein culturing the plurality of HSCs that have been in long-term culture and / or are exhausted HSCs in the 3D nanobioreactor for at least 2 weeks, or between about 2 weeks and 4 weeks, or between one and 5 weeks, maintains or extends the self-renewing capacity of the plurality of HSCs, or, results in the plurality of HSCs that that have lost their self-renewing capacity (so-called “exhausted HSCs) to regain their self-renewing capacity7or to substantially regain their self-renewing capacity7.

[0048] The details of one or more exemplary embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0049] All publications, patents, patent applications, American Type Culture Collection (ATCC) deposits and NCBI reference sequences cited herein are hereby expressly incorporated by reference in their entireties for all purposes.

[0050] DESCRIPTION OF DRAWINGS

[0051] The drawings set forth herein are illustrative of exemplary embodiments provided herein and are not meant to limit the scope of the invention as encompassed by the claims.

[0052] FIG. 1 A-B graphically illustrate summarized data of Survival and SelfRenewal of 4 paired Space / Ground aged bone marrow (ABM) samples: 4 paired Space (HSC) (FIG. IB) and Ground (GND) (FIG. IB) ABM samples were cultured in a nanobioreactor (BR) for 6 weeks (w) and then subjected to Survival and Selfrenewal assays to compare to the intact (before flight) original levels considered to be 100%; each bar represents Mean+ / - SE for triplicate conditions as described in detail in Example 1 , below. PATENT 0321.158068PCT / SD2024-024PCT

[0053] FIG. 2 graphically illustrates summarized data for 5 aged bone marrow (ABM) samples Self-Renewal after 4w in Ground BR plus 4 w on various stroma co-culture. 5 ABM samples were cultured in Ground RR for 4 weeks and then transferred to various stroma for an additional 4 w co-culture; then the cells were subjected to Survival and Self-renewal assays to compare to the intact (before flight) original levels considered to be 100% as described in detail in Example 1. below.

[0054] FIG. 3A-D illustrate images of the self -renewal of SAK-290 cells after 2 w co-culture with autologous or HS27a stroma (1 of 2 SpX-27 ABM samples): the FUCCI (Fluorescent Ubiquitinati on-based Cell Cycle Indicator)-labeled ABM SAK- 290 cells were cultured for 6 weeks in a Space nanobioreactor (BR) (SAK-290F) or Ground RR (SAK-290G) and additionally co-cultured for 2 weeks with autologous (290 stroma) or Hs27a ( Hs27a stroma) monolayers:

[0055] FIG. 3A SAK-290F and 290 stroma;

[0056] FIG. 3B SAK-290F and Hs27a stroma;

[0057] FIG. 3C SAK-290G and 290 stroma: and

[0058] FIG. 3D SAK-290F and Hs27a stroma; as described in detail in Example 1, below.

[0059] FIG. 4A-D graphically illustrate survival and self-renewal: Space / Ground RR cells before and after 2 weeks (w) stroma co-culture recovery: 2 paired Space (F) and Ground (G) aged bone marrow (ABM) samples were cultured in a nanobioreactor (BR) for 6 w and then subjected to Survival and Self-renewal assays to compare to the intact (before flight) original levels considered to be 100%; each bar represents Mean+ / - SE for triplicate conditions, where the bars represent: no stroma; Hs27a stroma; and, autologous stroma:

[0060] FIG. 4A graphically illustrates survival of SAK-290F cells and SAK-290G cells as percent of intact control cells;

[0061] FIG. 4B graphically illustrates survival of SAK-291F cells and SAK-291G cells as percent of intact control cells;

[0062] FIG. 4C graphically illustrates self-renewal of SAK-290F cells and SAK- 290G cells as percent of intact control cells; and

[0063] FIG. 4D graphically illustrates self-renewal of SAK-291F cells and SAK- 291G cells as percent of intact control cells; as described in detail in Example 1, below. PATENT 0321.158068PCT / SD2024-024PCT

[0064] FIG. 5A-F illustrates nanostring analysis of Differentially Expressed genes (DEgenes) in hematopoietic cells recovered after 12d co-culture with autologous primary or HS27a stroma, in particular, illustrates gene expression (NANOSTRING™, Seattle, WA) differences between Flight and Ground nABM stem cells recovered with the stromal cell line HS27a:

[0065] FIG. 5 A illustrates a Volcano plot of gene expression of flight sample (FHS) versus (v) ground sample (GHS), where Log fold change (LogFC) is shown on the x- axis with the negative log of the nominal p-value of the Benjamini -Hochberg method on the y-axis, and red dots represent genes with nominal p-value less than (<) 0.05;

[0066] FIG. 5B graphically illustrates significant genes upregulated in ground autologous stroma (Gauto) (Auto is autologous stroma used as a control for comparing Gauto / Fauto) (Gauto is ground autologous stroma, Fauto is flight autologous stroma);

[0067] FIG. 5C graphically illustrates gene expression (NanoString) differences between autologous nABM stroma and HS27a;

[0068] FIG. 5D illustrates a Volcano plot of gene expression of flight sample (FHS) versus (v) ground sample (GHS), as explained in FIG. 5A (Gauto is ground autologous stroma, Fauto is flight autologous stroma);

[0069] FIG. 5E-F graphically illustrate significant genes expressed in flight-HS27a versus ground-HS27a, Boxplots of the top 12 significant genes expressed in flight- autologous stroma versus ground-autologous stroma;

[0070] FIG. 5G graphically illustrates a Violin Plot of the distribution of expression of the 750 genes in the NANOSTRING™ panel across all samples; Dark Orange distributions represent nABM (aged bone marrow (ABM)) Stem Cells (Flight or Ground) recovered on individual matched Autologous Stroma while green distributions are the same nABM samples recovered on the stomal cell culture HS27a; both HS27a and Autologous Stroma had similar effects on the flight samples while the recovery of the Ground samples displayed a trend of lower overall gene expression;

[0071] FIG. 5H-M illustrate Gene expression (NANOSTRING™, Seattle, WA) differences between autologous nABM stroma and HS27a;

[0072] FIG. 5H circle graphically illustrates genes downregulated in nABM auto stroma and illustrates genes upregulated in nABM auto stroma;

[0073] FIG. 51 graphically illustrates HS versus ABM, upregulated in ABM; PATENT 0321.158068PCT / SD2024-024PCT

[0074] FIG. 5J graphically illustrates enrichment of complement activation genes;

[0075] FIG. 5K graphically illustrates genes downregulated in nABM auto stroma, and genes upregulated in nABM auto stroma;

[0076] FIG. 5L graphically illustrates HS versus ABM and genes downregulated in ABM; and

[0077] FIG. 5M graphically illustrates enrichment of TLR signaling genes; as described in detail in Example 1, below.

[0078] FIG. 6A-I illustrates Hematopoietic Stem and Progenitor Cell Functionally Organized Multi-omics Aging (HSPC-FOMA) Analyses of Astronauts:

[0079] FIG. 6A schematically illustrates an exemplary experimental design of hematopoietic stem and progenitor cell (HSPC) Functionally Organized Multi-omics Aging Analyses (HSPC-FOMA) of Astronauts, as provided herein; and the right-hand images illustrate a cytokine array analysis, as discussed in detail in Example 2, below;

[0080] FIG. 6B graphically illustrates a single cell RNA-sequencing (scRNA-seq) UMAP analysis of CD34+HSPCs from Axiom Mission 2 (Ax-2) over time (x-axis);

[0081] FIG. 6C graphically illustrates scRNA-seq UMAP of CD34+HSPCs from Ax- 3 over time (x-axis);

[0082] FIG. 6D graphically illustrates a bar plot of cell cycle phases (Gl, S, G2M) analyzed by scRNA-seq over time of CD34+ cells normalized to total CD34+ cells captured from Ax-2 (N=4);

[0083] FIG. 6E graphically illustrates a bar plot of cell cycle phases (Gl , S, G2M) analyzed by scRNA-seq over time of CD34+ cells normalized to total CD34+ cells captured from Ax-3 (N=3);

[0084] FIG. 6F-G graphically illustrate summarized colony survival data of n=4 Ax-2 samples (FIG. 6F) and n=3 Ax-3 (FIG. 6F) CD34+ cells;

[0085] FIG. 6H-I graphically illustrate summarized self-renewal data of n=4 Ax-2 samples (H) and n=3 Ax-3 (I) CD34+ cells, as discussed in detail in Example 2, below.

[0086] FIG. 7A-F illustrate space-associated Immune Repertoire Alterations:

[0087] FIG. 7A-B graphically illustrate immunophenotyping by FACS analysis of CD34+ cells (CD3-CD19-CD56-CD14-), CD3+ T cells, CD19+ B Cells, CD56+ NK cells, and CD14+ monocytes from bulk PBMCs isolated from Ax-2 (FIG. 7A) and Ax-3 (FIG. 7B) astronauts; PATENT 0321.158068PCT / SD2024-024PCT

[0088] FIG. 7C graphically illustrates immunophenotyping by FACS analysis of T- cells from bulk peripheral blood mononuclear cells from cryopreserved samples from Ax-2, and analysis includes CD4+ (top) and CD8+ (bottom) T cells, naive T cells, activated T cells, central memory T cells, and effector memory T cells;

[0089] FIG. 7D graphically illustrates a dot plot of T cell gene expression based on CD3+ T cells from Ax-2 scRNA-seq analysis;

[0090] FIG. 7E graphically illustrates immunophenotyping by FACS analysis of T- cells from bulk PBMCs from freshly processed samples from Ax-3 mission, and analysis includes CD4+ (top) and CD8+ (bottom) T cells, naive T cells, activated T cells, central memory T cells, and effector memory T cells; and

[0091] FIG. 7F graphically illustrates a dot plot of T cell gene expression based on CD3+ T cells from Ax-3 scRNA-seq analysis, as described in detail in Example 2, below.

[0092] FIG. 8A-J illustrates space- Associated Hematopoietic Stem and Progenitor Cell Telomere, Mitochondrial and Inflammatory’ Cytokine Deregulation:

[0093] FIG. 8A illustrates a bar graph illustrating whole genome sequencing analysis of the average TELOMERECAT™-estimated overall telomere length in base pairs (bp) of CD34+ cells derived from Ax-2 samples;

[0094] FIG. 8B illustrates a bar graph illustrating whole genome sequencing analysis of the average TELOMERECAT™-estimated overall telomere length in bp of CD34+ cells derived from Ax-3 samples;

[0095] FIG. 8C illustrates tables of significant differences between timepoints of telomerase maintenance gene expression in all CD34+ HSPCs in Ax-2 (left) and Ax-3 (right) by scRNA-seq; ’

[0096] FIG. 8D illustrates Violin plots of mitochondrial stress-related gene expression in CD34+CD38- cells from normal control samples derived from normal aged- matched peripheral blood, young and aged bone marrow, and Ax-2 and Ax-3 astronauts. SSCI 8-21 are normal young and middle-aged donor PBMCs (PB) or bone marrow (BM) SAK527 and SAK528 are samples derived from normal aged BM; telomerase pathway genes include TPP1, POLD2 , POLA1, NHP2, TERFI, and PRIM1;

[0097] FIG. 8E illustrates a bar graph of average mitochondrial copy number estimates (y-axis) from Ax-2 samples over time (x-axis) measured by WGS, with PATENT 0321.158068PCT / SD2024-024PCT timepoints include L-45 days, n=4; L-2 days, n=4; inflight, n=4; R+l day, n=3; R+42- 55 days, n=4; R+l year, n=4;

[0098] FIG. 8F illustrates a bar graph of average mitochondrial copy number estimates (y-axis) from Ax-3 samples over time (x-axis) measured by WGS, and timepoints include inflight, n=3; R+0 days, n=3; R+21 days, n=3; R+l year, n=2;

[0099] FIG. 8G illustrates tables of significant differences between timepoints of mitochondrial stress-related gene expression in all CD34+ HSPCs in Ax-2 (top) and Ax-3 (bottom) by scRNA-seq. Statistics determined by Mann-Whitney U test with FDR < 0.05.

[0100] FIG. 8H illustrates Violin plots of mitochondrial stress-related gene expression in CD34+CD38- cells from normal control samples derived from normal aged- matched peripheral blood, young and aged bone marrow, and Ax-2 and Ax-3 astronauts. SSCI 8-21 are normal young and middle-aged donor PBMCs (PB) or bone marrow (BM) SAK527 and SAK528 are samples derived from normal aged BM, and telomerase pathway genes include PRKN, OPTN, and PINK1; and

[0101] FIG. 8I-J illustrate for the sample groups R+l day and R+42-55 days from Ax-2 (FIG. 81) and R+0 days and R+21 days from Ax-3 (FIG. 8J), pseudobulk counts were used to generate a heatmap for the expressed genes in the Mitochondrial translation category; as described in detail in Example 2, below.

[0102] FIG. 9A-K illustrates space-associated Somatic Mutations and Clonal Hematopoietic Stem and Progenitor Cell Mutations:

[0103] FIG. 9A illustrates in table form total radiation averaged from seven locations on the International Space Station for the duration of Ax-2 and Ax-3 missions;

[0104] FIG. 9B illustrates a bar plot showing the total amount of somatic mutations (SBS and indels) from CD34+ cells acquired across the study timepoints in Ax -2, including L-2 days, inflight, R+l day, R+42-55 days, and R+l year;

[0105] FIG. 9C illustrates a bar plot showing the total number of somatic single base substitutions (SBS) from CD34+ cells acquired across the study timepoints in Ax-2, including L-2 days, inflight, R+l day, R+42-55 days, and R+l year;

[0106] FIG. 9D illustrates a bar plot showing the total number of somatic insertions and deletions (indels) from CD34+ cells acquired across the study timepoints in Ax-2, including L-2 days, inflight, R+l day, R+42-55 days, and R+l year; PATENT

[0107] 0321.158068PCT / SD2024-024PCT

[0108] FIG. 9E illustrates patterns of single base substitution (SBS) for the Ax-2 samples are shown using the SBS96 classification scheme on the x-axis, and the y- axis is scaled differently in each plot to optimally show each mutational pattern with the y-axis reflecting the number of mutations for the respective mutational scheme;

[0109] FIG. 9F illustrates a bar plot showing the total amount of somatic mutations (SBS and indels) from CD34+ cells acquired across the study timepoints in Ax-3, including R+0 days, R+21 days, and R+l year;

[0110] FIG. 9B illustrates a bar plot showing the total number of somatic single base substitutions (SBS) from CD34+ cells acquired across the study timepoints in Ax-3, including R+0 days, R+21 days, and R+l year;

[0111] FIG. 9H illustrates a bar plot showing the total number of somatic insertions and deletions (indels) from CD34+ cells acquired across the study timepoints in Ax-3, including R+0 days, R+21 days, and R+l year;

[0112] FIG. 91 illustrates patterns of single base substitution (SBS) for the Ax-3 samples are shown using the SBS96 classification scheme on the x-axis, and the y- axis is scaled differently in each plot to optimally show each mutational pattern with the y-axis reflecting the number of mutations for the respective mutational scheme;

[0113] FIG. 9J illustrates an oncoplot displaying clonal hematopoiesis (CH) mutated genes acquired in CD34+ cells from each individual per timepoint. Sample SSCI-004 at R+l day could not be collected; and

[0114] FIG. 9K illustrates an oncoplot displaying clonal hematopoiesis (CH) mutated genes acquired in CD34+ cells from each individual per timepoint. Sample SSCI-007 at R+l year has not been collected; as described in detail in Example 2, below.

[0115] FIG. 10A-J illustrate space-associated AD ARI and APOBEC3C Base Deaminase Deregulation:

[0116] FIG. 10A illustrates a dot plot (left image) of hematopoietic stem cell gene expression based on CD34+ cells from Ax-2 scRNA-seq analysis, and a table (right image) of significant differences between timepoints of hematopoietic stem cell gene expression in all CD34+ HSPCs in Ax-2 by scRNA-seq;

[0117] FIG. 10B illustrates a dot plot (left image) of hematopoietic stem cell gene expression based on CD34+ cells from Ax-3 scRNA-seq analysis, and a table (right image) of significant differences between timepoints of hematopoietic stem cell gene expression in all CD34+ HSPCs in Ax-3 by scRNA-seq; PATENT 0321.158068PCT / SD2024-024PCT

[0118] FIG. 10C-D illustrates Violin plots showing key gene expression based on CD34+ cells from Ax-2 (FIG. IOC) and Ax-3 (FIG. 10D) by scRNA-seq analysis. Genes include CD34, CD38, PROCR, PIEZO1, ADAR, and APOBEC3C;

[0119] FIG. 10E-F illustrate Log Expression of ADAR in pseudobulk generated counts from single cell RNA-Seq of CD34 positive cells derived from the peripheral blood of individuals at timepoints before, during, or after time spent in low earth orbit (LEO), and data from Ax-2 mission (FIG. 10E) and Ax-3 mission (FIG. 1 OF);

[0120] FIG. 10G-H illustrate an Oncoplot visualization shows the top 20 edited genes across all time points in Ax-2 (FIG. 10G) and Ax-3 (FIG. 10H); and

[0121] FIG. 101- J graphically illustrate Log Expression of APOBEC3C in pseudobulk generated counts from single cell RNA-Seq of CD34 positive cells derived from the peripheral blood of individuals at timepoints before, during, or after time spent in LEO. Data from Ax-2 mission (FIG. 101) and Ax-3 mission (FIG. 10J), as described in detail in Example 2, below.

[0122] FIG. 11A-E illustrates Space-associated Retrotransposon Deregulation:

[0123] FIG. 11 A illustrates Waterfall plots of the expression of the top 40 significant repeat elements (and L1ME3A) in each comparison of time points (L-2days vs L- 45days, left; inflight vs L-2days, center; inflight vs L-45days, right), and the y-axis represents the log fold change for the comparison while the x-axis indicates the repeat element with the bar colored by the family of the repeat;

[0124] FIG. 1 I B illustrates dot plots generated from scTE analysis of LINEs expression of CD34+CD38- cells from Ax-2 and Ax-3, and the top 50 significantly differentially expressed (adj. p value < 0.05) retrotransposons with the highest absolute log-fold change at any timepoint and control CD34+CD38- HSCs from healthy donor PBMCs;

[0125] FIG. 11C illustrates a Violin plot of the LINEs total expression scores generated with AddModuleScore in all CD34+ HSPCs for each individual astronaut across timepoints and normal control samples derived from peripheral blood and bone marrow CD34+ HSPCs. SSCI 8-21 are normal young and middle-aged donor PBMCs (PB) or bone marrow (BM). SAK527 and SAK528 are samples derived from normal aged BM;

[0126] FIG. 1 ID illustrates dot plots generated from scTE analysis of hERVs expression of CD34+CD38- cells from Ax-2 and Ax-3. The top 50 significantly differentially expressed (adj. p value greater than (<) 0.05) retrotransposons with the PATENT 0321.158068PCT / SD2024-024PCT highest absolute log-fold change at any timepoint and control CD34+CD38- HSCs from healthy donor PBMCs; and

[0127] FIG. 1 IE illustrates a Violin plot of HERV total expression scores generated with ADDMODULESCORE™ in all CD34+ HSPCs for each individual astronaut across timepoints and normal control samples derived from peripheral blood and bone marrow CD34+ HSPCs. SSCI 8-21 are normal young and middle-aged donor PBMCs (PB) or bone marrow (BM). SAK527 and SAK528 are samples derived from normal aged BM, as described in detail in Example 2, below.

[0128] FIG. 12A-H (also called Figure SI) illustrate Hematopoietic Stem and Progenitor Cell Functionally Organized Multi-omics Aging (HSPC-FOMA) Analyses of Astronauts, related to Figure 6:

[0129] FIG. 12A schematically illustrates summarizing the types of cells and downstream analyses performed per individual per timepoint, and analyses included 90X whole genome sequencing, whole transcriptome sequencing, single cell RNA sequencing, functional clonogenic assay, and immunophenotyping. where circles indicate CD34+ HSPC fraction, squares indicate CD34- cell fraction, and triangles indicate bulk PBMCs, and half circle or squares indicate that cells were recombined after enrichment to ensure high capture efficiency on the 10X scRNA-seq platform;

[0130] FIG. 12B illustrates a table of hematopoietic stem and progenitor cell functionally organized multi-omics aging (HSPC-FOMA) study characteristics;

[0131] FIG. 12C-D graphically illustrate bar plots of n=4 Ax-2 and n=3 Ax-3 individual survival (FIG. 12C) and self-renewal (FIG. 12D) capacity7collected over time (x-axis). and survival (FIG. 12C) capacity measured as percent of multilineage colonies on the y-axis, and self-renewal (FIG. 12D) capacity measured as percent change from baseline set to 100% of first collection timepoint (Ax-2 L-45 days and Ax-3 L-30 days) on the y-axis;

[0132] FIG. 12E-F graphically illustrate bar plots comparing n=4 Ax-2 and n=3 Ax-3 survival (FIG. 12E) and self-renewal (FIG. 12F) capacity collected at similar timepoints from each mission (L-45 days vs L-30 days; L-2 days vs L-l day; inflight day 10 vs inflight day 21; R+l day or R+0 days; R+42-55 days vs R+21 days); and

[0133] FIG. 12G-H graphically illustrate stacked bar plots of CD34+ labeled cells by scRNA-seq for Ax-2 (FIG. 12G) and Ax-3 (FIG. 12H), where the y-axis indicates percent of cells from each labeled timepoint. Ax-2 timepoints include L-2 days. PATENT 0321.158068PCT / SD2024-024PCT

[0134] Inflight, R+l day, R+42-55 days, and R+l year. Ax-3 timepoints include L-30 days, L-l day, Inflight. R+0 days, and R+21 days. as described in detail in Example 2, below.

[0135] FIG. 13A-C (also called Figure S2) illustrate Space-associated Immune Repertoire Alterations, related to Figure 7:

[0136] FIG. 13A illustrates a table of top 15 significantly differentially expressed genes from all CD34+ cells in Ax-2 determined by scRNA-seq using MAST and FindAllMarkers. Timepoints include L-2 days, inflight, R+l day, R+42-55 days, and R+l year;

[0137] FIG. 13B illustrates a table of top 15 significantly differentially expressed genes from all CD34+ cells in Ax-3 determined by scRNA-seq using MAST and FindAllMarkers. Time points include L-30 days, L-l day, inflight, R+0 days, R+21 days, and R+l year; and

[0138] FIG. 13C illustrates a table of top 25 significantly differentially expressed genes in all CD34+ cells between Ax-2 and Ax-3 missions ranked by log-fold change determined by scRNA-seq using MAST and FINDALLMARKERS™. as described in detail in Example 2, below.

[0139] FIG. 14A-J (also called Figure S3) illustrate Space- Associated Hematopoietic Stem and Progenitor Cell Telomere, Mitochondrial and Cytokine Deregulation, related to Figure 8:

[0140] FIG. 14A illustrates a line graph illustrating whole genome sequencing analysis of the average TELOMERECAT™-estimated overall telomere length in base pairs (bp) of CD34+ cells derived from individual Ax-2 (top) and Ax-3 (bottom) samples;

[0141] FIG. 14B illustrates a line graph illustrating whole genome sequencing analysis of the relative telomere content on the y-axis of CD34+ cells derived from individual Ax-2 (top) and Ax-3 (bottom) samples;

[0142] FIG. 14C illustrates a line graph showing average mitochondrial copy number estimates from Ax-2 (N=4, top) and Ax-3 (N=3. bottom) samples;

[0143] FIG. 14D illustrates Violin plots of telomere maintenance gene expression in CD34+CD38- cells from normal control samples derived from normal aged-matched peripheral blood, young and aged bone marrow, and Ax-2 and Ax-3 astronauts. SSCI 8-21 are normal young and middle-aged donor PBMCs (PB) or bone marrow (BM) PATENT 0321.158068PCT / SD2024-024PCT

[0144] SAK527 and SAK528 are samples derived from normal aged BM, and the telomerase pathway genes include HIST2H2BE, RUVBL1. POLD3, CTC1, STM1, and POTI;

[0145] FIG. 14E-F illustrate data from a gene set enrichment analysis (GSEA) that was performed on pseudobulk data for the comparison of Launch-2 days vs Inflight and Retum+1 day vs Retum+42-55 days during Ax-2 Mission (FIG. 14E), and Launch- 1 day vs Inflight and Retum+0 days vs Retum+21 days during Ax-3 (FIG. 14F);

[0146] FIG. 14G-H illustrate bar plots of cytokine expression (IL- / ?, IL-10, IL-15, and IL-23) measured from diluted plasma in Ax-2 (FIG. 14G) and Ax-3 (FIG. 14H); and

[0147] FIG. 14 1-J illustrate boxplots of inflammatory cytokine (FIG. 141) and cytokine receptor (FIG. 14J) expression levels in CD34+ cells by whole transcriptome sequencing, as described in detail in Example 2, below.

[0148] FIG. 15A-N (also called Figure S4) illustrate Space-associated Clonal Hematopoietic Stem and Progenitor Cell Mutations, related to Figure 9:

[0149] FIG. 15A illustrates bar plots presenting single base substitution mutations in their 6 possible substitution context and the cumulative C>T mutations at CpG sites and other sites in L-2 days, inflight, R+l day, and R+42-55 days versus L-45 days for Ax-2 samples;

[0150] FIG. 15B graphically illustrates patterns of small insertions and deletions (indels) for the Ax-2 samples are show n using the ID-83 classification scheme on the x-axis, and the y-axis is scaled differently in each plot to optimally show each mutational pattern with the y-axis reflecting the number of mutations for the respective mutational scheme;

[0151] FIG. 15C graphically illustrates patterns of double base substitution (DBS) for the Ax-2 samples are shown using the DBS78 classification scheme on the x-axis, and the y-axis is scaled differently in each plot to optimally show each mutational pattern with the y-axis reflecting the number of mutations for the respective mutational scheme;

[0152] FIG. 15D graphically illustrates a line graph showing the total amount of somatic mutations (SBS and indels) from CD34+ cells acquired across the study timepoints in Ax-2, including R+l day, R+42-55 days, and R+l year: PATENT 0321.158068PCT / SD2024-024PCT

[0153] FIG. 15E graphically illustrates a line graph showing the total number of somatic single base substitutions (SBS) from CD34+ cells acquired across the study timepoints in Ax-2, including R+l day, R+42-55 days, and R+l year;

[0154] FIG. 15F graphically illustrates a line graph showing the total number of somatic insertions and deletions (indels) from CD34+ cells acquired across the study timepoints in Ax-2, including R+l day, R+42-55 days, and R+l year;

[0155] FIG. 15G graphically illustrates a line graph showing the total amount of somatic mutations (SBS and indels) from CD34+ cells acquired across the study timepoints in Ax-3, including R+0 days, R+21 days, and R+l year;

[0156] FIG. 15H graphically illustrates a line graph showing the total number of somatic single base substitutions (SBS) from CD34+ cells acquired across the study timepoints in Ax-3, including R+0 days, R+21 days, and R+l year;

[0157] FIG. 151 graphically illustrates a line graph showing the total number of somatic insertions and deletions (indels) from CD34+ cells acquired across the study timepoints in Ax-3, including R+0 days. R+21 days, and R+l year;

[0158] FIG. 15J illustrates bar plots presenting single base substitution mutations in their 6 possible substitution context and the cumulative C>T mutations at CpG sites and other sites in R+0 days, R+21 days, and R+l year versus inflight for Ax-3 samples;

[0159] FIG. 15K illustrates patterns of small insertions and deletions (indels) for the Ax-3 samples are shown using the ID-83 classification scheme on the x-axis, and the y-axis is scaled differently in each plot to optimally show each mutational pattern with the y-axis reflecting the number of mutations for the respective mutational scheme;

[0160] FIG. 15L illustrates patterns of double base substitution (DBS) for the Ax-3 samples are shown using the DBS78 classification scheme on the x-axis, where the y- axis is scaled differently in each plot to optimally show each mutational pattern with the y-axis reflecting the number of mutations for the respective mutational scheme;

[0161] FIG. 15M illustrates an Oncoplot displaying top 25 of the somatically mutated genes within Ax-2 samples; and

[0162] FIG. 15N illustrates an Oncoplot displaying top 25 of the somatically mutated genes within Ax-3 samples, as described in detail in Example 2, below. PATENT 0321.158068PCT / SD2024-024PCT

[0163] FIG. 16A-E (also called Figure S5). Space-associated AD ARI and APOBEC3C Base Deaminase Deregulation, related to Figure 10:

[0164] FIG. 16A illustrates a boxplot of AD ARI isoform expression LogCPM values in L-45 days, L-2 days, and inflight from Ax-2 mission;

[0165] FIG. 16B illustrates a boxplot of AD ARI editing in L-45 days, L-2 days, and inflight from Ax-2 mission;

[0166] FIG. 16C illustrates a boxplot of APOBEC3 expression LogCPM values in L- 45 days, L-2 days, and inflight from Ax-2 mission, and

[0167] FIG. 16D-E illustrate Log Expression of APOBEC3C in pseudobulk generated counts from scRNA-Seq of CD34+ cells derived from the peripheral blood of individuals at timepoints before, during, or after time spent in LEO. Data from Ax-2 mission (FIG. 16D) and Ax-3 mission (FIG. 16E), as described in detail in Example 2, below.

[0168] FIG. 17A-F (also called Figure S6) illustrate Space-associated Retrotransposon Deregulation, related to Figure 11 :

[0169] FIG. 17A-B illustrate Volcano plots of the expression of LINE elements (FIG. 17A) and HERV elements (FIG. 17B) in each comparison of time points (L-2days vs L-45days, left; inflight vs L-45days, center; inflight vs L-2days, right), and the x-axis shows the log fold change and y-axis represents the negative log of the comparison p- value, and red dots indicate those elements with p-value < 0.05;

[0170] FIG. 17C illustrates a heatmap of retrotransposons that are differentially expressed in any of the time point comparisons, and the hierarchical cluster along the left edge shows how the expression patterns of the repeat element relate while the cluster across the top shows that samples within time points cluster together based on the retrotransposon;

[0171] FIG. 17D illustrates a table of all retrotransposons represented in Fig. 6A waterfall plots with adj. p value < 0.1. retrotransposons are ranked based on top positive and negative log-fold change;

[0172] FIG. 17E illustrates a table of top 5 significantly differentially expressed LINEs in CD34+CD38- cells from Ax-2 and Ax-3 ranked by logFC; and

[0173] FIG. 17F illustrates a table of top 5 significantly differentially expressed HERVs in CD34+CD38- cells from Ax-2 and Ax-3 ranked by logFC, as described in detail in Example 2. below. PATENT 0321.158068PCT / SD2024-024PCT

[0174] FIG. 18 illustrates a dot plot of hematopoietic stem cell gene expression based on CD34+ cells from Ax-2 scRNA-seq analysis. Table of significant differences between timepoints of hematopoietic stem cell gene expression in all CD34+ HSPCs in Ax-2 by scRNA-seq. Statistics determined by Mann- Whitney U test with FDR < 0.05.

[0175] FIG. 19 illustrates a dot plot of hematopoietic stem cell gene expression based on CD34+ cells from Ax-3 scRNA-seq analysis. Table of significant differences between timepoints of hematopoietic stem cell gene expression in all CD34+ HSPCs in Ax-3 by scRNA-seq. Statistics determined by Mann- Whitney U test with FDR < 0.05.

[0176] FIG. 20 illustrates Violin plots showing key gene expression based on CD34+ cells from Ax-2 by scRNA-seq analysis. Genes include CD34, CD38, PROCR, PIEZO 1, ADAR, and APOBEC3C. Statistics w as determined by Mann-Whitney U test with an FDR < 0.05.

[0177] FIG. 21 illustrates Violin plots showing key gene expression based on CD34+ cells from Ax-3 by scRNA-seq analysis. Genes include CD34, CD38, PROCR. PIEZO1, ADAR, and APOBEC3C. Statistics was determined by Mann-Whitney U test with an FDR < 0.05.

[0178] FIG. 22 illustrates Violin plot of the LINEs total expression scores generated with AddModuleScore in all CD34+ HSPCs for each individual astronaut across timepoints and normal control samples derived from peripheral blood and bone marrow' CD34+ HSPCs. SSCI 8-21 are normal young and middle-aged donor PBMCs (PB) or bone marrow' (BM). SAK527 and SAK528 are samples derived from normal aged BM.

[0179] FIG. 23 illustrates a Violin plot ofHERV total expression scores generated with ADDMODULESCORE™ in all CD34+ HSPCs for each individual astronaut across timepoints and normal control samples derived from peripheral blood and bone marrow CD34+ HSPCs. SSCI 8-21 are normal young and middle-aged donor PBMCs (PB) or bone marrow (BM). SAK527 and SAK528 are samples derived from normal aged BM.

[0180] Like reference symbols in the various drawings indicate like elements. PATENT 0321.158068PCT / SD2024-024PCT

[0181] DETAILED DESCRIPTION

[0182] In alternative embodiments, provided are methods for regaining of the selfrenewing capacity by hematopoietic stem cell (HSC) that is lost after long-term culture, where the methods comprise placing the HSC that have lost self-renewing capacity (so-called “exhausted HSCs) into a supporting, nourishing microenvironment for the additional co-culture period; and in alternative embodiments the methods comprise long term culture of HSCs isolated from bone aged marrow donors.

[0183] Inventors have found that targeting of the microenvironment results in better survival and maintenance of normal HSCs and represents a novel avenue for treatment of certain malignancies. Microgravity and higher inflammogenic conditions in space potentially might change the normal steady-state of HSC / microenvironment interaction status. Therefore, inventors compared the self-renewal abilities of HSC in Space and on the Ground states and investigated the possible recovery of HSC ’s lost replating abili ty by placing them into more ‘supportive niche' and recreating the beneficial microenvironment. Example 1. below, presents data demonstrating that methods as provided herein can result in the regaining of self-renewing capacity by (exhausted) hematopoietic stem cell (HSC), the self-renewing capacity lost after long term culture, wherein the methods as provided herein comprise placing or culturing the exhausted HSC into a supporting, nourishing microenvironment for at least one additional co-culture period.

[0184] Nanobioreactors (BRs)

[0185] In alternative embodiments, provided are methods for maintaining or extending the self-renewing capacity of hematopoietic stem cell (HSCs) in long term culture, or for regaining self-renewing capacity by hematopoietic stem cell (HSC) that that have lost their self-renewing capacity (so-called “exhausted HSCs) after longterm culture, the method comprising use of a three-dimensional (3D) nanobioreactor (or BR).

[0186] In alternative embodiments, a BR used in methods as provided herein are as described in WO 2023 137107 Al, or are made as described in WO 2023 137107 Al.

[0187] In alternative embodiments, a BR used in methods as provided herein comprises a defined three dimensional (3D) stromal microenvironment for cell culturing, which can comprise: a container, enclosure or bag having gas-permeable (and optionally, liquid impermeable or liquid impervious) walls or sides, and a three PATENT 0321.158068PCT / SD2024-024PCT dimensional (3D) sponge matrix or a sponge-like material contained within the container, enclosure or bag, wherein: the sponge matrix or sponge-like material is infused with a cell culture media and a mixture of cells comprising stem cells and / or bone marrow matrix cells, the container, enclosure or bag interior is sterile, and the container, enclosure or bag comprises at least one liquid or cell input port, or at least two liquid or cell input ports. In alternative embodiments, a BR used in methods as provided herein comprises at least two liquid or cell input ports, one out port and one in port, optionally further comprises a micro-peristaltic pump for circulating liquid inside the container, enclosure or bag is operably connected to the in port and the out port. In alternative embodiments, the sponge or sponge-like material comprises: an absorbable gelatin sponge (optionally an absorbable human or porcine gelatin sponge), a solubilized or reconstituted basement membrane matrix, a solubilized or reconstituted laminin / collagen IV-rich basement membrane extracellular matrix, optionally, MATRIGEL™ (Coming Life Sciences) or GELTREX™ (ThermoFisher Scientific), a compressed sponge with adsorbable gelatin, optionally GELFOAM™ (Pfizer), a demineralized cancellous sponge (Berkeley Advanced Biomaterials (BAB), Berkeley CA), or VIASORB™ (Globus Medical, Audubon, PA); and / or a hydrogel -based macroporous sponge or porous hydroxipropylcellulose (HPC) scaffold, optionally CELLUSPONGE™, CELLUSPONGE-GAL™. or CELLUSPONGE-COL™ (Bio- Bybios), optionally a cancellous (or trabecular) bone sponge, for example, CANCELLOUS SPONGE™ (VMI Medical). In alternative embodiments, the sponge or sponge-like material further comprises: a demineralized cancellous bone matrix sponge, or demineralized bone matrix (DBM) components, optionally comprising OSTEOSPONGE™ (Xtant Medical).

[0188] Products of manufacture and Kits

[0189] Provided are products of manufacture and kits for practicing methods as provided herein; and optionally, products of manufacture and kits can further comprise instructions for practicing methods as provided herein.

[0190] Any of the above aspects and embodiments can be combined with any other aspect or embodiment as disclosed here in the Summary, Figures and / or Detailed Description sections. PATENT 0321.158068PCT / SD2024-024PCT

[0191] As used in this specification and the claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0192] Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive and covers both “or” and “and”.

[0193] Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About (use of the term “about”) can be understood as within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12% 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.”

[0194] Unless specifically stated or obvious from context, as used herein, the terms “substantially all”, “substantially most of’, “substantially all of’ or “majority of’ encompass at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%, or more of a referenced amount of a composition.

[0195] The entirety of each patent, patent application, publication and document referenced herein hereby is incorporated by reference. Citation of the above patents, patent applications, publications and documents is not an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. Incorporation by reference of these documents, standing alone, should not be construed as an assertion or admission that any portion of the contents of any document is considered to be essential material for satisfying any national or regional statutory disclosure requirement for patent applications. Notwithstanding, the right is reserved for relying upon any of such documents, where appropriate, for providing material deemed essential to the claimed subj ect matter by an examining authority or court.

[0196] Modifications may be made to the foregoing without departing from the basic aspects of the invention. Although the invention has been described in substantial detail with reference to one or more specific embodiments, those of ordinary skill in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, and yet these modifications and improvements are within the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", PATENT 0321.158068PCT / SD2024-024PCT "consisting essentially of, and "consisting of may be replaced with either of the other two terms. Thus, the terms and expressions which have been employed are used as terms of description and not of limitation, equivalents of the features show n and described, or portions thereof, are not excluded, and it is recognized that various modifications are possible within the scope of the invention. Embodiments of the invention are set forth in the following claims.

[0197] The invention will be further described with reference to the examples described herein; however, it is to be understood that the invention is not limited to such examples.

[0198] EXAMPLES

[0199] Unless stated otherwise in the Examples, all recombinant DNA techniques are earned out according to standard protocols, for example, as described in Sambrook et al. (2012) Molecular Cloning: A Laboratory Manual, 4th Edition. Cold Spring Harbor Laboratory Press, NY and in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology7, Current Protocols, USA. Other references for standard molecular biology techniques include Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY, Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK). Standard materials and methods for polymerase chain reactions can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory7Manual. Cold Spring Harbor Laboratory Press, and in McPherson at al. (2000) PCR - Basics: From Background to Bench, First Edition, Springer Verlag, Germany.

[0200] Example 1: Exhausted in Space Hematopoietic Stem Cells Recover Their SelfRenewal Capacity^ by Additional Stromal Co-Culture.

[0201] This example present data demonstrating that methods as provided herein can result in the regaining of self-renewing capacity7by (exhausted) hematopoietic stem cell (HSC), the self-renewing capacity lost after long term culture, wherein the methods as provided herein comprise placing or culturing the exhausted HSC into a supporting, nourishing microenvironment for at least one additional co-culture period. In alternative embodiments, methods as provided herein are used to maintain or PATENT 0321.158068PCT / SD2024-024PCT extend the self-renewing capacity of HSCs in long term culture, where the HSCs can be isolated from bone-aged marrow donors.

[0202] CD34 positive cells from aged normal bone marrow (BM) (ABM) were transduced with FUCCI BICISTRONIC VECTOR™ in order to observ e the cell cycle changes of those cells (2). Cells were cultured in three-dimensional (3D) nanobioreactor (3) for 4 weeks (w) in Space post 42 days (d) SpX-27 mission, and as a control in ground conditions. This nanobioreactor (BR) was designed with the purpose of supporting primary human hematopoietic stem cell culture on orbit aboard the International Space Station.

[0203] After that those cells were plated on the autologous primary stroma established from CD34 negative fraction of ABM or Hs27a cell line monolayer for additional 2 w coculture. Additionally, in similar experiments, 5 other ABM samples were cultured 4w in Ground BR and plates on primary allogenic ABM stroma or Hs27a monolayer for 4 more weeks of co-culture.

[0204] At the end of total 6 -8 w co-culture, cells were harvested and subsequently subjected to survival and self-renewal assays (4) and some of cultures were used for RNA transcriptome analysis.

[0205] Microgravity7and higher inflammogenic conditions in space potentially change the normal steady-state of HSC / microenvironment interaction status. Therefore, here we compared the self-renewal abilities of HSC in Space and on the Ground states and investigated the possible recovery of HSC’s lost replating ability by placing them into more ‘supportive niche’ and recreating the beneficial microenvironment.

[0206] We hypothesized (correctly) that plating the non-functional Space nanobioreactor (BR) samples on the stroma for additional co-culture might restore their ability to Self-renew. The data generated indicated that only 2 weeks of coculture with autologous primary stroma or Hs27a monolayer resulted in strong expansion of cell number and colony formation in both Survival and Self-renewal assays for 2 ABM samples. In Space nanobioreactor (BR) samples the Hs27a and even the autologous primary stroma were able to ‘recover’ self-renewal of ABM samples to the levels of intact cells; however, the ability7of hematopoietic cells to replate was significantly lower compared to Ground BR control or intact cell’s results.

[0207] Damage to the cells in Space or Ground nano-bioreactor (BR) long culture was not permanent, and could be reversed by plating them onto supporting, nourishing niche. Cells Self-renewal ability and cell cycle dormant status after 8w of PATENT 0321.158068PCT / SD2024-024PCT culture could be reversed to the levels of the original intact HSC, demonstrating full recovery.

[0208] Methods

[0209] CD34 positive cells from aged normal BM (ABM) were transduced with Fucci Bicistronic Vector in order to observe the cell cycle changes of those cells (2). Cells were cultured in a 3D nanobioreactor (3) for 4 weeks (w) in space post 42 days (d) SpX-27 mission, and as a control in Ground conditions. This nanobioreactor (BR) was designed with the purpose of supporting primary human hematopoietic stem cell culture on orbit aboard the International Space Station. After that those cells were plated on the autologous primary stroma established from CD34 negative fraction of ABM or Hs27a cell line monolayer for additional 2 w co-culture.

[0210] Additionally, in similar experiments, 5 other ABM samples were cultured 4w in Ground BR and plates on primary allogenic ABM stroma or Hs27a monolayer for 4 more weeks of co-culture.

[0211] At the end of total 6 -8 w co-culture, cells were harvested and subsequently subjected to survival and self-renewal assays (4) and some of cultures were used for RNA transcriptome analysis. Results

[0212] Analysis of 4 paired ABM samples after 6 w in Space or Ground BR revealed that in survival assays the total number of colonies and. importantly, the number of multilineage colonies were significantly lower compared to ground samples. In Selfrenewal assays none of the Space samples demonstrated the replating capacity. In Ground samples the number of replated colonies was lower than in original intact cells, demonstrating that small number remaining stem cells were active and could repopulate.

[0213] To model the conditions we plated Fucci labeled CD34+ cells from 5 ABM samples in Ground BR for 4 weeks and then, keeping the small part still continuously in BR, collected the majority of them and replated on various allogenic primary’ stroma or Hs27a monolayer for additional 4 weeks co-culture.

[0214] At the end of co-culture Ground BR cells were not functional, while additional coculture with Hs27a or primary' allogenic stroma monolayers resulted in strong expansion of cell number and colony formation in both Survival and Self-renewal assays for all ABM samples. Cell cycle analysis revealed that cells were in both proliferating and dormant stage after 75 d of total culture. Compared to allogenic PATENT 0321.158068PCT / SD2024-024PCT primary stroma, Hs27a demonstrated superior environment for the hematopoietic cells: Self-renewal was equivalent to the original, intact replating indicators.

[0215] We hypothesize that plating the non-functional Space BR samples on the stroma for additional co-culture might restore their ability to Self-renew. Our data indicated that only 2 weeks of co-culture with autologous primary stroma or Hs27a monolayer resulted in strong expansion of cell number and colony formation in both Survival and Self-renewal assays for 2 ABM samples. In Space BR samples the Hs27a and even the autologous primary stroma were able to ‘recover’ Self-renewal of ABM samples to the levels of intact cells, however the ability of hematopoietic cells to replate was significantly lower compared to ground BR control or intact cell’s results.

[0216] Conclusions:

[0217] Damage to the cells in Space or Ground BR long culture was not permanent, and could be reversed by plating them onto supporting, nourishing niche. Cells Selfrenewal ability and cell cycle dormant status after 8w of culture could be reversed to the levels of the original intact HSC. demonstrating full recovery.

[0218] Figure legends Example 1 :

[0219] FIG. 1 : Summarized data of Survival and Self-Renewal of 4 paired Space / Ground ABM samples: 4 paired Space (HSC) and Ground (GND) ABM samples were cultured in BR for 6 w and then subjected to Survival and Self-renewal assays to compare to the intact (before flight) original levels considered to be 100%. Each bar represents Mean+ / - SE for triplicate conditions. Statistical analysis included Student’s t-test and one-way Anova, including All Pairwise Multiple Comparison Procedures (Holm-Sidak method).

[0220] FIG. 2: Summarized data for 5 ABM samples Self-Renewal after 4w in Ground BR + 4 w on various stroma co-culture. 5 ABM samples were cultured in Ground RR for 4 weeks and then transferred to various stroma for an additional 4 w co-culture. Then cells were subjected to Survival and Self-renewal assays to compare to the intact (before flight) original levels considered to be 100%. Each bar represents Mean+ / - SD for n=5 conditions. Statistical analysis included Student’s t-test and one-way Anova, including All Pairwise Multiple Comparison Procedures (Holm-Sidak method).

[0221] FIG. 3: Self -renewal of SAK-290 after 2 w co-culture with autologous or HS27a stroma (1 of 2 SpX-27 ABM samples): Fucci-labeled ABM SAK-290 was cultured for 6 weeks in Space BR (SAK-290F) or Ground RR (SAK-290G) and additionally PATENT

[0222] 0321.158068PCT / SD2024-024PCT co-cultured for 2 weeks with autologous (290 stroma) or Hs27a ( Hs27a stroma) monolayers. Images (lOx) were taken in bright field and in GRN and Red fluorescent light fields.

[0223] FIG. 4: Survival and Self-renewal: Space / Ground RR cells before and after 2 weeks stroma co-culture recovery7: 2 paired Space (F) and Ground (G) ABM samples were cultured in BR for 6w and then subjected to Survival and Self-renewal assays to compare to the intact (before flight) original levels considered to be 100%. Each bar represents Mean+ / - SE for triplicate conditions. Statistical analysis included Student’s t-test and one-way Anova, including All Pairwise Multiple Comparison Procedures (Holm-Sidak method).

[0224] Fig 5, Nanostring analysis of DEgenes in hematopoietic cells recovered after 12d coculture with autologous primary or HS27a stroma.

[0225] (A-F) Gene expression (NANOSTRING™, Seattle, WA) differences between Flight and Ground nABM stem cells recovered with the stromal cell line HS27a. Volcano plot of gene expression. Log fold change (LogFC) is shown on the x-axis with the negative log of the nominal p-value of the Benjamini -Hochberg method on the y-axis. Red dots represent genes with nominal p-value < 0.05. (E-F) Boxplots of the top 12 significant genes expressed in flight-autologous stroma versus ground-autologous stroma. FIG. 5D: Volcano plot of gene expression. Log fold change (LogFC) is shown on the x-axis with the negative log of the nominal p-value of the Benjamini - Hochberg method on the y-axis. Red dots represent genes with nominal p-value < 0.05.

[0226] (G) Violin Plot of the distribution of expression of the 750 genes in the NANOSTRING™ panel across all samples. Dark Orange distributions represent nABM Stem Cells (Flight or Ground) recovered on individual matched Autologous Stroma while green distributions are the same nABM samples recovered on the stomal cell culture HS27a. Both HS27a and Autologous Stroma had similar effects on the flight samples while the recovery7of the Ground samples displayed a trend of lower overall gene expression.

[0227] (H-M) Gene expression (NANOSTRING™, Seattle, WA) differences between autologous nABM stroma and HS27a. (L) Volcano plot of gene expression. Log fold change (LogFC) is shown on the x-axis with the negative log of the nominal p-value of the Benjamini -Hochberg method on the y-axis. Red dots represent genes with PATENT 0321.158068PCT / SD2024-024PCT nominal p-value < 0.05. (M) Boxplot of the top 12 significant genes expressed in flight-autologous stroma versus ground-autologous stroma.

[0228] Example 2: Space Associated Stem Cell Hallmarks of Aging (SASHA) in Astronauts Hematopoietic stem and progenitor cell functionally organized multi-omics aging (HSPC-FOMA) analyses of 7 astronauts before, during and after ISS missions revealed hallmarks of accelerated aging including reduced self-renewal and telomere maintenance, mitochondrial dysfunction, genomic instability with clonal mutations together with cytokine and retrotransposon deregulation.

[0229] Highlights

[0230] • HSPC-FOMA astronaut analyses were performed for 10- and 21-day ISS missions

[0231] • Spaceflight altered HSPC cell cycle, self-renewal and immune repertoires

[0232] • Spaceflight altered HSPC telomere, mitochondrial and cytokine regulation

[0233] • Spaceflight altered HSPC mobilization and mechanoreceptor gene expression

[0234] • Space induced HSPC mutations, base deaminase and retrotransposon deregulation

[0235] Summary

[0236] Previous reports revealed immune dysfunction, chromosomal abnormalities, cytokine deregulation, and telomere length dynamics following prolonged spaceflight. However, the stress of space on hematopoietic stem and progenitor cells (HSPCs) that maintain lifelong hematopoiesis and immune responses was not studied. We performed HSPC functionally organized multi-omics aging (HSPC-FOMA) analyses in 7 astronauts before, during, and after short-duration ISS missions. Specifically, whole genome sequencing with telomere length, mitochondrial and clonal mutational profiling; whole transcriptome sequencing with RNA editing and retrotransposon analyses; single-cell RNA sequencing; cytokine arrays; and FACS-analyses were performed to assess HSPC and immune subpopulation survival dynamics. Overall, the observed space-associated stem cell hallmarks of aging, including spaceflightdependent alterations in HSPC survival and self-renewal, adenosine deaminase associated with RNAI (AD ARI), telomere maintenance, mobilization and cell cycle gene expression combined with space-associated clonal hematopoietic mutations, apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC3C) activation PATENT 0321.158068PCT / SD2024-024PCT and retrotransposon deregulation warrant countermeasure development to enable long-duration spaceflight.

[0237] Life-long blood cell production and effective immune responses are predicated on the maintenance of healthy hematopoietic stem cells (HSCs) that can reside dormant in protective niches; mobilize following a “fight or flight” response; selfrenew (regenerate); and differentiate into all blood cell types1'8. Hallmarks of stem cell aging include reduced self-renewal capacity, loss of dormancy, increased mobilization from bone marrow niches, telomere attrition and reduced telomere maintenance, genomic instability , mitochondrial stress, accumulation of clonal mutations9 10, and retrotransposon derepression11, which triggers interferon-related inflammation-associated aging (inflammaging)1 12 14. These hallmarks of stem cell aging can be acquired in response to chronic or acute macroenvironmental exposures or stressors1,4,15. fn addition to promoting hematopoietic stem and progenitor cell (HSPC) inflammaging, derepression of retrotransposons, which comprise approximately 35% of the human genome, can contribute to genomic and epitranscriptomic instability by activating DNA and RNA editing enzymes16. Under homeostatic conditions, base deaminases, including adenosine deaminase associated with RNA (AD ARI) and apolipoprotein B mRNA editing catalytic polypeptide 3 (APOBEC3) family enzymes, protect HSPC genomic integrity by restricting activation of retrotransposons. including human endogenous retroviruses (hERVs) with long terminal repeat (LTR) elements, Long INnterspersed Elements (LINEs) encoding reverse transcriptase, and the Short INnterspersed Elements (SINEs) that utilize LINE-encoded reverse transcriptase for retrotransposition, such as Alu sequences1,12’16. However, protracted activation of base deaminases has been linked to clonal APOBEC3C-induced cytosine-to-thymine (C-to-T) single base substitution (SBS) DNA mutations that fuel pre-malignant myeloproliferative neoplasm (MPN) HSPC proliferation and AD ARI - induced adenosine to inosine (A-to-I) RNA editing alterations that drive malignant regeneration in at least 20 malignancies1,13. Thus, early detection and reversal of microenvironmental and macroenvironmental stressors that accelerate HSPC aging are vital for preserving human health and longevity.

[0238] Pioneering astronaut research, including the National Aeronautics and Space Administration (NASA) Twins Study from a 1-year long mission and The Space Omics and Medical Atlas (SOMA) studies from short-term missions, has shown that PATENT 0321.158068PCT / SD2024-024PCT spaceflight serves as a profound stressor, capable of reshaping the immune system,17'19altering physiologic function20, and causing prolonged molecular and cellular changes in many tissues21. Moreover, NASA research utilizing animal models, simulated microgravity, and human-derived cell cultures has revealed changes in inflammatory cytokines and shifts in immune cell populations, including natural killer (NK) cells, monocytes, granulocytes, and T-cells typical of aging18,19,22'25. Previously, we investigated whether immune deficits arise from HSPC dysfunction in space in four month-long NASA-supported integrated space stem cell orbital research (ISSCOR) center (SpX-24, SpX-25, SpX-26 and SpX-27) missions to the International Space Station (ISS) that utilized novel Al-driven 3D nanobioreactors seeded with aged normal bone marrow-derived HSPCs and stromal cells. In these ISSCOR studies, we observed niche-dependent HSPC dysfunction following postspaceflight return compared with ground-based controls, which included reduced HSPC self-renewal capacity, base deaminase deregulation, reduced telomere maintenance gene expression, and dynamic retrotransposon derepression. Although we observed significant effects of spaceflight from in vitro nanobioreactor models of human HSPC aging, the temporal sequence and long-term impact of space associated stem cell hallmarks of aging in astronauts had not been addressed to date.

[0239] To investigate the direct functional and molecular impact of spaceflight as a macroenvironmental stressor, we performed the first sequential astronaut HSPC functionally organized multi-omics aging (HSPC-FOMA) analyses before, during, and after 10- and 21-day Axiom (Ax-2 and Ax-3) private crew missions to the ISS. Overall, HSPC-FOMA analyses revealed space-associated hallmarks of astronaut aging, including significant cell cycle alterations, decreased self-renewal capacity, genomic instability typified by clonal mutations together with HSPC mobilization gene, altered telomere maintenance, retrotransposon, inflammatory cytokine and AD ARI and APOBEC3C base deaminase deregulation. Mitigation strategies for these stem cell hallmarks of accelerated aging will need to be devised to enable long- duration spaceflight and may depend, at least in part, on targeted inflammatory cytokine, retrotransposon reverse transcriptase and base deaminase inhibition. As opportunities for human spaceflight expand to include iterative commercial manufacturing operations, space tourism and deep-space discovery missions, understanding the direct impact of space-associated stress on the functional capacity PATENT 0321.158068PCT / SD2024-024PCT of long-lived stem and progenitor cells to maintain tissue homeostasis, particularly in the hematopoietic system, has emerged as a pressing unmet need.

[0240] Results

[0241] Hematopoietic Stem and Progenitor Cell Functionally Organized Multi-omics Aging (HSPC-FOMA) Analyses of Astronauts

[0242] To determine the combined functional and molecular impact of 10- and 21- day missions to the ISS in four Ax-2 and three Ax-3 astronauts, we performed a longitudinal study involving sequential acquisition of 10 ml of peripheral blood, in accordance with NASA IRB approved protocols. Blood samples were collected for Ax-2 crew at launch -45 days (L-45 days), launch -2 days (L-2 days), inflight (prior to hatch closure), return +1 day (R+l day), return +42-55 days (R+42-55 days), and return +1 year (R+l year). For Ax-3 crew, samples were collected at launch -30 days (L-30 days), launch -1 day (L-l day), inflight (prior to hatch closure), return +0 days (R+0 days), return +21 days (R+21 days), and return +1 year (R+l year). (Fig. 6A, S1A-B). Upon arrival at the ISSCOR lab (UC San Diego Sanford Stem Cell Institute, CA) within 24-96 hours of venipuncture, peripheral blood mononuclear cells (PBMCs) were isolated by FICOLL-PAQUE™ followed by immunomagnetic bead purification of CD34+cells for HSPC functionally organized multi-omics aging (HSPC-FOMA) analyses.

[0243] For sequential functional analyses of isolated CD34 cells, we performed colony survival and replating (self-renewal) assays (Ax-2, n=24 and Ax-3, n=17 samples). For multi-omics analyses, we performed CD34+HSPC-enriched and CD34" cell whole genome sequencing (WGS; 90X coverage; Ax-2, n=47 and Ax-3, n=28 samples) telomere length and clonal mutation analyses; CD34+and CD34 whole transcriptome sequencing (RNA-Seq; Ax-2, n=8 samples and 3 timepoints) with unsupervised hierarchical clustering, gene set enrichment analyses (GSEA), RNA editing and retrotransposon analyses; and CD34+versus CD34" single-cell RNA sequencing (scRNA-Seq; Ax-2, n=39 and Ax-3. n=33 samples) with UMAP, GSEA, differential gene expression (DGE) analysis, single-cell transposable element (scTE), and pseudobulk RNA editing analyses. To assess HSPC and immune subpopulation dynamics, we performed FACS-based immunophenotyping (Ax-2, n=24 and Ax-3, n=15 samples). To assess inflammatory cytokine production, we performed cytokine arrays for Ax-2 and Ax-3 crew members from plasma at each time point (n=31) (Fig. S1A). PATENT 0321.158068PCT / SD2024-024PCT

[0244] To elucidate HSPC cellular and transcriptomic diversity, we performed scRNA-seq on CD34+enriched cells using the 10X GENOMICS™ 3?gene expression platform. Samples from all timepoints between Ax-2 and Ax-3 were integrated and followed by cluster identification using the SINGLER™ and SCTYPE™ algorithms. Uniform manifold approximation projection (UMAP) of 1,938 CD34+cells identified 4 distinct clusters and revealed an increase of CD34+cells during spaceflight (Fig 6B-C. S1G-H). Within the CD34+ population, particularly in 21 -day ISS mission duration Ax-3 crew members, we observed a transient population of cells with lower CD34 and higher expression of Protein C Receptor (PROCR), the gene that encodes the endothelial protein C receptor (EPCR), which is a marker of a highly regenerative stem cell population (Fig. 6B-C)24. Furthermore, scRNA-seq analysis of CD34+cells revealed alterations in G1 and S phases of the cell cycle during spaceflight in both missions (Fig. 6D-E). Notably, scRNA-seq revealed a significant reduction in G1 and increase in S phase gene expression inflight from 21 -day Ax-3 mission samples suggesting that spaceflight duration may reduce HSPC dormancy, which has been linked to loss of self-renewal potential26'29.

[0245] To evaluate spaceflight duration associated alterations in HSPC self-renewal and survival, fresh CD34+HSPCs were subjected to clonogenic survival and replating assays before, during and immediately after return from spaceflight. Long-term follow-up analyses were conducted to examine functional resilience over time. In keeping with spaceflight-associated stem cell aging, samples from Ax-2 and Ax-3 had significantly reduced multi-lineage colony formation inflight compared to all pre- and post-flight timepoints (Fig. 6F-G, SIC, E). While HSPC replating was similar to preflight in Ax-2 samples, there was a significant reduction inflight compared to L-30 days in Ax-3 HSPC samples (Fig. 6H-I, SID). Overall, astronaut HSPC self-renewal capacity after 21 -days of time spent in low earth orbit (LEO) was significantly reduced (Fig. 61) compared to L-45 days before launch (Fig. 6H). Notably, HSPCs derived from Ax-2 mission astronauts showed significantly increased self-renewal capacity immediately upon return (R+l day) compared to pre-mission timepoints and remained elevated at R+l year compared to baseline pre-flight samples (Fig. 6H, SID). Compared with Ax-2 samples, Ax-3 samples showed a significant reduction in self-renewal capacity inflight as well as upon immediate return from LEO but rebounded to similar replating levels as Ax-2 samples 30 days after return to earth PATENT 0321.158068PCT / SD2024-024PCT (Fig. S1F). Finally, scRNA-seq revealed differences in CD34low, CD34+CD38‘, CD34+CD38mid, CD34+CD38+HSPC subpopulations inflight and immediately upon return in Ax-3 compared with Ax-2 crew members (Fig. S1G-H). Overall, these data underscore the functional impact of spaceflight duration on stem and progenitor cell repertoires.

[0246] Space-associated Immune Repertoire Alterations

[0247] The landmark NASA Twins Study and SOMA studies have shown by multi- omics analyses that spaceflight induces immune alterations18,20. To characterize the impact of spaceflight duration on astronaut immune subpopulations, peripheral blood mononuclear cells (PBMCs) from 39 samples were stained and analyzed by fluorescence activated cell sorting (FACS). Upon Ax-2 crew member return from 10 days on the ISS, FACS analyses revealed a significant reduction in CD3+ T cells (R+l year) compared to pre-flight (L-45 days) (Fig. 7A). Moreover, FACS analysis revealed persistent significant reductions of CD 19+ B cells and CD56+ NK cells at R+l year (Fig. 7A). For Ax-3 crew members with an ISS mission duration of 21 days, FACS analysis showed significant reductions in CD19+ B cells and CD14+ monocytes inflight compared to pre-flight L-30 days and L-l day, followed by a significant increase at R+0 and R+21 days (Fig. 7B). To further gauge the temporal sequence and depth of immune alterations, CD4+ T-cell subset FACS analyses of Ax- 2 crew samples were performed and showed a slight reduction in CD4+ central memory T cells at R+42-55 days compared to L-45 days, and similarly of CD8+ naive T cells at R+42-55 days versus R+l day (Fig. 7C). Notably, parallel scRNA-seq analyses revealed a rebound by R+42-55 days and recovery to baseline at 1 year in most immune subsets following Ax-2 crew member return from space (Fig. 7D). In Ax-3 crew member samples, FACS analyses revealed a trend toward reduced CD4+ effector memory cells inflight compared with L-30 days (Fig. 7E) and scRNA-seq analyses showed a commensurate reduction in T cell gene expression inflight (Fig. 7F). Comparative scRNA-seq analyses of Ax-2 and Ax-3 samples, revealed significant differences in IL- lb inflight suggesting that inflammatory cytokine signaling deregulation may fuel immune repertoire differences (Fig. S2A-C). Together, these data demonstrate that longer exposure to the LEO environment detrimentally impacts immune repertoires. PATENT 0321.158068PCT / SD2024-024PCT

[0248] Space- Associated Hematopoietic Stem and Progenitor Cell Telomere. Mitochondrial and Cytokine Deregulation

[0249] To assess the impact of spaceflight duration on HSPC inflammaging, we performed WGS analyses of telomere length and scRNA-seq analyses of telomere maintenance gene expression changes in seven astronauts analyzing six timepoints for the Ax-2 mission and four for the Ax-3 mission (Fig. 8A-B. S3A-B). As expected, considering the long length of telomeres in HSPCs compared with differentiated cells, no significant telomere length changes were observed in the CD34+enriched HSPC fraction by WGS TELOMERECAT™ and TELSEQ™ analyses (Fig. 8A-B, S3A-B). However, scRNA-seq analyses revealed significant space-associated reductions in both telomerase and CST-POLA1 complex genes, which regulate telomere maintenance and also play a role in Type 1 interferon activation (Fig. 8C-D, S3D)30’31. Compared to inflight, POLD2, a component of the CST-POLA1 telomere maintenance complex, and TPP1, a shelterin protein that recruits telomerase to telomeres, remained significantly reduced in CD34+ HSPCs at R+l year for Ax-2 crew members (Fig. 8C-D). At R+21 days compared with inflight in the Ax-3 mission, CD34+cell scRNA-seq analyses showed a significant reduction in both POLD2-mediated telomere maintenance and the essential NHP2 telomerase component, which has been associated with the accelerated aging syndrome dyskeratosis congenita when deregulated (Fig. 8C-D)32.

[0250] Regarding other key hallmarks of aging15and previous reports linking telomere length changes and mitochondrial DNA amplification18-33, we next evaluated mitochondrial DNA copy number by WGS. Mitochondrial DNA WGS analyses revealed significant mitochondrial DNA copy number amplification in 21 -day mission duration Ax-3 astronauts upon return in contrast to 10-day mission duration Ax-2 astronauts (Fig. 8E-F, S3C). Interestingly, we observed increased mitochondrial DNA amplification upon return that reflected both the significant increase in the S phase proportion of cells inflight and a cellular stress response (Fig. 6D-E, 8G-H). In contrast to Ax-2 samples, gene set enrichment analyses (GSEA) of pseudobulk data showed significant alterations in mitochondrial translation gene expression that distinguished R+0 days from R+21 day Ax-3 samples (Fig. 81- J, S3E-F), indicative of sustained mitochondrial stress responses associated with the longer mission duration.

[0251] Space-associated hallmarks of stem cell aging in astronaut research also involved cytokine analysis of diluted plasma collected from all 7 Ax-2 and Ax-3 PATENT 0321.158068PCT / SD2024-024PCT mission astronauts across the various timepoints as well as whole transcriptome RNA- seq analyses of cytokine gene and cytokine receptor expression from 3 Ax-3 timepoints (Fig. S3G-J). These analyses demonstrated significantly increased levels of pro-inflammatory IL-23 expression inflight for both Ax-2 and Ax-3 crew members (Fig. S3G-H). Interestingly, an increase in IL-23 was already apparent pre-flight (L-2 days) for Ax-2 astronauts. In contrast to Ax-2 crew members, Ax-3 crew members showed a significant increase in IL- 10 and a decrease in IL- 15 inflight (Fig. S3G-H). Finally, whole transcriptome sequencing analysis of cytokine and cytokine receptor expression measured at L-45 days, L-2 days, and inflight for Ax-2 HSPCs showed overall deregulation (Fig. S3I-J) demonstrating that hallmarks of HSPC inflammaging are acquired in response to stressors associated with spaceflight. Space-associated Clonal Hematopoietic Stem and Progenitor Cell Mutations

[0252] To determine the contribution of space radiation-induced somatic mutagenesis in the CD34+fraction, we obtained NASA radiation logs from six locations on the ISS during these private astronaut missions (PAMs). Based on these NASA ISS dosimetry logs, we determined the average dose rate of exposure and human tissue dose equivalent during Ax-2 (May 22, 2023 - May 30, 2023) and Ax-3 (January 20, 2024 - February77, 2024)34. These measurements include Earth’s magnetic field, galactic cosmic rays (GCR), and solar particle events (SPE) for which there were 0 events detected during both Axiom missions. Specifically, the human tissue dose equivalents of radiation were 0.460 mGy / day and 0.521 mGy / day for Ax-2 and Ax-3, respectively (Fig. 9A). Interestingly, MUTATIONALP ATTERNS™ analyses showed that only a minority of C>T mutations were located in CpG dinucleotide sites thereby demonstrating that ionizing radiation was not the only contributor to mutagenesis and that whole genome sequencing analyses (WGS) would need to be employed sequentially to resolve space-specific mutagenesis patterns35(Fig. S4A, D).

[0253] To determine if HSPC mutations were acquired during spaceflight in Ax-2, CD34+ enriched cells were subjected to 90X WGS at L-2 days, inflight, R+l day, R+42-55 days, and R+l year. Results were compared to pre-flight L-45 days (Fig. 9B-D). A similar analysis was completed for Ax-3, with return timepoints at R+0 days and R+21 days and results normalized to inflight. For Ax-3 crew members, the mutational burden was detected for the 21 -day mission (Fig. 9F-H). While most somatic mutations acquired across the study (Fig. 9E, Fig. 91. S4B-C, S4K-L) were C-to-T single base substitutions (SBSs) with a few double base substitutions (DBSs), PATENT 0321.158068PCT / SD2024-024PCT a significant increase in insertions and deletions (indels) was detected at return + 21 days compared with return + 0 days in Ax-3 crew members (Fig. 9H).

[0254] With regard to space associated clonal hematopoiesis (SACH), single-sample mode analysis was performed with a cutoff of 10% variant allele frequency (VAF) and revealed that one Ax-2 astronaut had a pre-launch clonal CREBBP mutation (Fig. 9 J). Three of the four astronauts had detectable CH mutations at R+l year, including, KDM6A. NOTCH3. CSF3R, and SMC1A, suggesting that yearly follow-up will be important to track the persistence of these clones. In contrast to Ax-2 10-day ISS mission members, we observed increased acquisition of CH-associated mutations inflight for the 21 -day mission duration Ax-3 crew (Fig. 9K). Notably, one astronaut acquired a mutation in BCL1 IB inflight, which re-appeared at R+21 days. Like Ax-2, one Ax-3 astronaut acquired a mutation in RUNX1, a transcription factor important for regulating HSC self-renewal, at R+l year, thereby underscoring the importance of yearly CH analyses for astronauts. Additional non-synonymous and synonymous mutations acquired pre-flight, inflight, and post-flight normalized to L-45 days for Ax-2 and mutations acquired post-flight normalized to inflight for Ax-3 were observed and indicative of possible base deaminase deregulation (Fig. S4M-N). Notably, the observed increase in CH-associated mutations in Ax-3 suggests that the longer-duration missions may impact human HSPC fitness and predispose to precancer stem cell generation13in a spaceflight duration-dependent manner. Space-associated ADAR1 and APOBEC3C Base Deaminase Deregulation

[0255] Both AD ARI and APOBEC3C base deaminase deregulation has been linked to pre-cancer stem cell propagation and cancer stem cell generation in inflammatory microenvironments, particularly in myeloproliferative neoplasms (MPNs)13,36-38and acute myeloid leukemia (AML)39and thus were studied in Ax-2 and Ax-3 astronaut crew members before, during and after spaceflight to ascertain their respective roles in SACH (Fig. lOA-H)1 5,40,41. While scRNA-seq revealed that AD ARI expression increased during spaceflight in CD34+ cells in Ax-3 more than Ax-2 missions (Fig. 10A-F) corresponding with the observed increased G1 to S phase transition in Ax-3 (Fig. 6D-E), post-spaceflight analyses revealed a significant reduction in AD ARI base deaminase expression levels in both Ax-2 and Ax-3 crew members (Fig. 5C-D; Fig. S5A-B). This decline corresponds with the observed diminished replating capacity (Fig. 6H-I), consistent with AD ARl ’s well-established role in regulating HSC self-renewal38. To further investigate the role of AD ARI -mediated RNA editing PATENT 0321.158068PCT / SD2024-024PCT in HSPC functional decline following spaceflight, we developed a single-cell RNA editing (editome) analysis that revealed transient missense editing event in CDK13, a splicing and cell cycle regulatory gene previously found to be edited in MPNs (Fig. 10G-H)13. Moreover, AZINI, a well-known target of AD ARI editing, was found to be edited inflight during both space missions corresponding with AD ARI activation during spaceflight (Fig. 10G-H). Interestingly, while only one astronaut presented with an AZINI missense edit before spaceflight, multiple astronauts exhibited a missense edit in AZINI pre-flight, during spaceflight, or upon return (Fig. 10E-F). Finally, scRNA-seq pseudobulk analyses revealed significant upregulation of base deaminase APOBEC3C upon return from space (Fig. 10I-J, S5C-E) indicative of a base deaminase mediated mechanism of C-to-T mutagenesis in response to spaceflight that may fuel space associated clonal hematopoiesis (SACH). Space-associated Retrotransposon Deregulation

[0256] Both primate-specific APOBEC3 and AD ARI deaminases evolved to protect long-lived human stem cell populations from retroviral integration and genomic instability related to retrotransposon activation, including LINEs, Alu’s, and human endogenous retroviruses (HERVs)12'16'42. Previously, we observed that LINE element deregulation in post-spaceflight compared to ground HSPC niche nanobioreactors w as associated with functional HSPC aging. Therefore, we analyzed retrotransposon expression utilizing whole transcriptome sequencing available for Ax-2 mission timepoints (N=2 L-30 days, N=4 L-2 days and N=2 Inflight). In these analyses, we observed that retrotransposon expression changes occurred pre-flight, as part of a possible “fight or flight response”, and became more pronounced during spaceflight, with unsupervised hierarchical clustering clearly distinguishing the analyzed timepoints, specifically LINE elements and HERVs (Fig. HA, S6A-D). Furthermore, LINE-1 specific analyses revealed a significant reduction of LIME3A upon exposure to microgravity, which interestingly was already downregulated at L-2 days compared to L-45 days (Fig. S6A-B).

[0257] To assess the regulation of retrotransposons in response to aging and LEO exposure, we preformed single cell transposable element (scTE) analyses on young, middle aged and older peripheral blood and bone marrow derived HSPCs and compared these analyses to Ax-2 and Ax-3 crew- member HSPCs before, during and after spaceflight (Fig. 11B-E). These scRNA-seq analyses showed that LINEs were significantly upregulated upon return from the Ax-2 mission and continued to be PATENT 0321.158068PCT / SD2024-024PCT highly expressed at R+l year in a manner similar to aged HSPC samples (Fig. 11C). The LINE elements were highly expressed inflight and at R+0 days for Ax-3 crew but returned to pre-flight levels by R+21 days (Fig. 11 B, S6E). A temporal association between AD ARI upregulation and LINE overexpression was more prominent in Ax-3 compared with Ax-2 crew members by scRNA-seq TE analyses, suggesting a regulatory role of AD ARI. Interestingly, HERVs were highly expressed at R+lyear in Ax-2 (Fig. 11C, S6F). Similarly, AD AR I expression increased during spaceflight in Ax-3 crew members commensurate with HERV upregulation (Fig. 11C). Perhaps equally importantly, specific HERVs were activated in Ax-2 and Ax-3 HSCs in response to spaceflight and overall HERV expression varied in an astronaut dependent manner (Fig. 1 ID, E).

[0258] In addition to changes in retrotransposon activation, return to earth was associated with increased expression of mechanoreceptor, PIEZO1, gene expression as well as expression of nociceptive and “fight or flight'’ response genes, including RAMP1 and CALCRL, in both Ax-2 and Ax-3 crew member HSPCs (Fig. 11B- C)8’43. Remarkably, Calcium / Calmodulin-Dependent Protein Kinase II Alpha (CAMK2A), a gene central to the “fight or flight” response in response to stress44, was significantly changed inflight with the greatest log2FC in Ax-3 (Fig. S2B, Fig. 1 IB, D). Together, these human-specific retrotransposon and “fight or flight” response gene expression alterations provide valuable insights into drivers of accelerated aging under conditions of extreme stress and warrant long-term evaluation of the impact of spaceflight on HSPC fitness as well as targeted countermeasure development to enable future missions.

[0259] Discussion

[0260] Seminal research shows that spaceflight induces immune dysregulation18 1922'25, physiologic changes, and sustained molecular and cellular changes in tissues. However, these effects w ere not directly studied in purified hematopoietic stem and progenitor cell (HSPC) populations, which give rise to immune subpopulations that protect from latent virus reactivation and precancer evolution to cancer1. In this space- associated stem cell hallmarks of aging (SASHA) in astronauts study, our aim was to understand how spaceflight duration impacts HSPC function and contributes to immune system deregulation before, during, and after space missions. Using 39 fresh blood specimens collected sequentially from 7 astronauts during the Ax-2 10-day and Ax-3 21-day missions to the ISS, we profiled 1,938 HSPCs, 121,894 total cells, and PATENT 0321.158068PCT / SD2024-024PCT investigated the functional and molecular characteristics of HSPCs in the context of hallmarks of aging15by integrating single-cell RNA sequencing with HSPC-FOMA focused functional analyses and whole genome and transcriptome sequencing to assess HSPC clonogenicity and self-renewal capacity, evaluate mutational burden, identify mutational signatures, and enhance transcript coverage for retrotransposon analyses. We observed decreased HSPC self-renewal, reduced AD ARI expression and editing levels, elevated APOBEC3C expression, space-induced DNA mutagenesis, and dynamic retrotransposon changes.

[0261] While pioneering NASA studies in astronauts detected inflammatory cytokine changes, mitochondrial DNA amplification, and telomere length changes18-19-2l-24>25 45-47, these analyses focused on short-lived peripheral blood mononuclear cells (PBMCs)48,49rather than long-lived HSPC compartment18. Thus, we sought to understand the cumulative functional and molecular impact of variable duration (today versus 21 -day) exposure to the LEO-environment (microgravity and chronic radiation) on HSPCs. As expected, based on the long telomere length of HSPCs, telomere length was not significantly altered in CD34+cells. However, CST-POLA1 complex telomere maintenance and telomere genes were significantly changed as shown by scRNA-seq. Specifically, POLD2, a telomere maintenance gene, and TPP1, which recruits telomerase to telomeres, were significantly and persistently downregulated following a mission duration of 10 days in Ax-2 crew members. Moreover, Ax-3 crew members who had an ISS mission duration of + 21 days, also harbored a persistent decrease in the telomere maintenance gene POLD2 as well as significantly decreased expression of the telomerase gene, NHP2, which is vital component of the telomerase complex and can lead to telomere shortening and premature aging when deregulated.32These data suggest that longer mission duration has a more profound impact on both the telomerase complex and telomere maintenance genes.

[0262] Despite the observed variability among astronauts, these findings, along with indications of stem cell exhaustion based on HSPC-FOMA focused scRNA-seq based cell cycle assessments and self-renewal replating assays, support the observations that spaceflight accelerates HSPC aging. We did not see substantial changes in telomerase reverse transcriptase (TERT) expression. Instead, our WGS, whole transcriptome RNA-seq and scRNA-seq data suggest that HSPC population dynamics contribute to telomere maintenance changes during PATENT 0321.158068PCT / SD2024-024PCT spaceflight with the emergence of a highly regenerative PROCR and AD ARI -high HSPC population that coincides with upregulation of both LINE (long interspersed nuclear element) and HERV (human endogenous retrovirus) retrotransposon expression.

[0263] The self-renewal potential of HSCs is regulated, in part, by AD ARI activity'36. A decrease in AD ARI expression may be linked to the significantly reduced selfrenewal capacity observed throughout different timepoints of the missions. Additionally, reduced AD ARI expression also poses a risk for the reactivation of latent viruses50and the derepression of retrotransposons51. Specifically, upregulation of LINEs and HERVs expression closely aligned with increased AD ARI expression. Although these changes appear to be transient and the human body demonstrates resilience, differences between the Ax-2 and Ax-3 missions indicate that time in space is a significant contributor to genomic instability and inflammaging through retrotransposon deregulation.

[0264] A follow-up study to the NASA Twins Study found that spaceflight induced CH-associated mutations were acquired two decades sooner than the average age of detection40. Another study of 14 astronauts who participated in Shuttle missions from 1998 to 2001 identified nonsynonymous mutations with low variant allele fractions (VAF) in 17 CH-related genes, with TP53 and DNMT3A being the most frequently- mutated41. These studies detected CH mutations by targeted exome sequencing in purified lymphocytes40or bulk PBMCs40,41, which are primarily comprised of shortlived cells. In contrast, we analyzed WGS in single sample mode to ascertain the clonal mutations associated with spaceflight in peripheral blood derived long-lived HSPCs3’52’54Moreover, these private astronaut data suggest that accumulation of CH- associated mutations in HSPCs is spaceflight duration-dependent with 21-day mission astronauts having more persisting clonal mutations overtime. The clonal expansion of HSPCs has been linked to changes in the immune system's ability- to clear cells by retrotransposon activation resulting in modulation of the "don't eat-me" signal42. The derepression of retrotransposons may be linked to the transient nature of the observed Ax-2 and Ax-3 CH mutations42. In ongoing FOMA research, we will ascertain the competitive fitness of these clonally mutated HSPCs and whether they will be eradicated once immune fitness is restored post-spaceflight.

[0265] Our results demonstrate that HSPC-FOMA changes associated with launch occur in a spaceflight duration-dependent manner. In addition to the duration- PATENT 0321.158068PCT / SD2024-024PCT dependent effects seen during and after spaceflight, we noticed a pre-flight HSPC- FOMA response immediately before launch (Ax-2 L-2 days and Ax-3 L-l day). Preflight data suggest a ‘Tight or flight” associated response in anticipation of launch, characterized by modulation of a receptor activity modifying protein 1 (RAMP1), linked to nociceptive nerve regulation of HSC mobilization8. Interestingly, significantly differentially expressed CAMK2A in Ax-3, suggests that another “fight or flight” response occurred inflight immediately prior to hatch closure and return to Earth. Notably, changes in inflammatory cytokine levels, cell cycle dynamics, and retrotransposon expression were already observed pre-flight which might coincide with the “fight or flight” response seen in bone marrow under conditions of extreme stress8,55. Hematopoietic stress has also been suggested to trigger retrotransposon transcription in HSCs, causing HSCs to move out of their quiescent state16. Further, the differentiation of HSPCs to immune cells may be regulated by activation of retrotransposons, such as endogenous retroviruses, so repression or changes in retrotransposon expression may have a negative effect on the immune response42.

[0266] Changes to the innate immune response may tngger Type I interferon, which has been linked to AD ARI activation and has been shown to regulate retrotransposons after tissue injury13,16.

[0267] Additionally, PIEZO1, a mechanosensitive ion channel that responds to mechanical forces such as shear flow, was previously shown to be upregulated in response to simulated microgravity56. In our study, PIEZO1 , which has also been linked to T-cell activation57, was upregulated in the CD34+enriched samples upon return, indicating HSPCs may be sensing the change in gravitational forces that occurs upon return from flight. Moreover, calcitonin receptor like receptor (CALCRL), also linked to nociceptive nerve regulation8, was upregulated following spaceflight, indicative of a different cellular stress response related to return.

[0268] As the first comprehensive study of stem cell health in astronauts, our HSPC- FOMA study revealed nine significant hallmarks of accelerated aging in a time-in- space dependent manner including 1) loss of dormancy. 2) reduced self-renewal commensurate with reduced AD ARI self-renewal gene expression, 3) reduced multilineage colony formation, 4) reduced telomere maintenance, 5) increased mitochondrial copy number and mitochondrial stress-related gene expression, 6) genomic instability typified by increased indels. 7) space associated clonal hematopoiesis, 8) APOBEC3C base deaminases deregulation, and 9) cell-type and PATENT 0321.158068PCT / SD2024-024PCT context specific HERV and LINE retrotransposon activation1’15. Overall, these results enable prediction and ultimately inform the development of prevention strategies for accelerated stem cell aging in response to stress, including in space.

[0269] This study focuses on the temporal sequence of space-associated HSPC aging in two separate private astronaut missions (PAMs) to the ISS to demonstrate the acquisition of clonal hematopoietic (CH) mutations, alterations in telomere maintenance and inflammatory cytokines, deregulation of base deaminases and retrotransposons over time. As an active long-term study, return +yearly follow-up has not been concluded, and results are pending to determine whether current changes observed have prolonged effects. Due to the paucity of CD34+ HSPCs in peripheral blood, cell availability limited the number of downstream assays possible although we were able to study many functional and molecular hallmarks of stem cell aging. Combined with shifting launch and private crew schedules, comparisons between the 10- and 21 -day missions were done with as much standardization as possible. Additionally, due to the longitudinal nature of this study, there was some variability between reagent kits, specifically, library preparation kits and sequencing platforms, depending on chemistry and instrument advancements and availabilities that should be taken into consideration. Despite these differences, we performed robust analyses with statistically significant results that provide unique tools for predicting accelerated HSPC aging in response to stress, including in response to spaceflight.

[0270] Figure Legends Example 2

[0271] Figure 6, Hematopoietic Stem and Progenitor Cell Functionally Organized Multi- omics Aging (HSPC-FOMA) Analyses of Astronauts

[0272] (A) Experimental design of hematopoietic stem and progenitor cell (HSPC) Functionally Organized Multi-omics Aging Analyses (HSPC-FOMA) of Astronauts. In sequential studies, HSPCs were isolated from astronaut peripheral blood before, during, and after Axiom 2 and 3 missions to the International Space Station (ISS). Whole peripheral blood was returned within 96-hours of collection on ice and immediately processed by density centrifugation. Prior to CD34+ cell selection, the mononuclear cell fraction was stained for immunophenotyping analysis by FACS. After CD34+ selection, cells were divided for 90X whole genome sequencing (WGS), single cell RNA sequencing (scRNA-seq). whole transcriptome sequencing, and clonogenic survival and self-renewal assay. Diluted plasma was collected for cytokine array analysis. PATENT 0321.158068PCT / SD2024-024PCT

[0273] (B) Single cell RNA-sequencing (scRNA-seq) UMAP analysis of CD34 HSPCs from Axiom Mission 2 (Ax-2) over time (x-axis). Timepoints include L-2 days, n=4; inflight, n=4; R+l day, n=3; R+42-55 days, n=4; R+l year, n=4.

[0274] (C) scRNA-seq UMAP of CD34+HSPCs from Ax-3 over time (x-axis). Timepoints include L-30 days, n=3; L-l day, n=3; inflight, n=3; R+0 days, n=3; R+21 days, n=3; R+l year, n=2.

[0275] (D) Bar plots of cell cycle phases (Gl, S, G2M) analyzed by scRNA-seq over time of CD34+ cells normalized to total CD34+ cells captured from Ax-2 (N=4).

[0276] (E) Bar plots of cell cycle phases (Gl, S, G2M) analyzed by scRNA-seq over time of CD34+ cells normalized to total CD34+ cells captured from Ax-3 (N=3). Statistical analysis included Student’s two-tailed t-test.

[0277] (F-G) Summarized colony survival data of n=4 Ax-2 samples (F) and n=3 Ax-3 (G) CD34+ cells. Basal colony formation of original naive untreated cells preflight (F; Ax-2 L-45 days; G; Ax-3 L-30 days) were considered to be 100% and individual values were calculated as % of change. Data presented show the mean + / - SD for each sample. Statistical analysis included Student’s t-test and one-way Anova, including All Pairwise Multiple Comparison Procedures (Holm-Sidak method). (H-I) Summarized self-renewal data of n=4 Ax-2 samples (H) and n=3 Ax-3 (I) CD34+ cells. Basal colony formation of original naive untreated cells pre-flight (H; Ax-2 L-45 days; I; Ax-3 L-30 days) were considered to be 100% and individual values were calculated as % of change. Data presented show the mean + / -SD for each sample. Statistical analysis included Student’s t-test and one-way Anova, including All Pairwise Multiple Comparison Procedures (Holm-Sidak method).

[0278] Figure 7, Space-associated Immune Repertoire Alterations

[0279] (A-B) Immunophenotyping by FACS analysis of CD34+ cells (CD3-CD19- CD56-CD14-), CD3+ T cells, CD19+ B Cells, CD56+ NK cells, and CD14+ monocytes from bulk PBMCs isolated from Ax-2 (A) and Ax-3 (B) astronauts. Ax-2 mission includes timepoints up to R+42-55 days analyzed from cryopreserved bulk peripheral blood mononuclear cells (PBMCs) and fresh PBMCs at Return + 1 year. Ax-3 mission includes timepoints up to Return + 21 days measured in fresh PBMCs. Statistical significance was determined using a two-tailed t-test of unequal variance. P-values less than 0.05 were considered significant.

[0280] (C) Immunophenotyping by FACS analysis of T-cells from bulk peripheral blood mononuclear cells from cryopreserved samples from Ax-2. Analysis includes PATENT 0321.158068PCT / SD2024-024PCT

[0281] CD4+ (top) and CD8+ (bottom) T cells, naive T cells, activated T cells, central memory T cells, and effector memory T cells. Statistical significance was determined using a two-tailed t-test of unequal variance. P-values less than 0.05 were considered significant.

[0282] (D) Dot plot of T cell gene expression based on CD3+ T cells from Ax-2 scRNA-seq analysis. Statistical significance determined by Mann- Whitney U test with an FDR < 0.05. CD44. R+30 days vs R+l day, p-value = 0.029; adj. p = 0. 114.

[0283] (E) Immunophenotyping by FACS analysis of T-cells from bulk PBMCs from freshly processed samples from Ax-3 mission. Analysis includes CD4+ (top) and CD8+ (bottom) T cells, naive T cells, activated T cells, central memory T cells, and effector memory T cells. Statistical significance was determined using a two-tailed t- test of unequal variance. P-values less than 0.05 were considered significant.

[0284] (F) Dot plot of T cell gene expression based on CD3+ T cells from Ax-3 scRNA-seq analysis.

[0285] Figure 8, Space- Associated Hematopoietic Stem and Progenitor Cell Telomere, Mitochondrial and Inflammatory Cytokine Deregulation

[0286] (A) Bar graph illustrating whole genome sequencing analysis of the average TELOMERECAT™-estimated overall telomere length in base pairs (bp) of CD34+ cells derived from Ax-2 samples.

[0287] (B) Bar graph illustrating whole genome sequencing analysis of the average TELOMERECAT™-estimated overall telomere length in bp of CD34+ cells derived from Ax-3 samples. R+l year timepoint includes N=2 samples.

[0288] (C) Tables of significant differences between timepoints of telomerase maintenance gene expression in all CD34+ HSPCs in Ax-2 (left) and Ax-3 (right) by scRNA-seq. Statistics determined by Mann- Whitney U test with FDR < 0.05.

[0289] (D) Violin plots of mitochondrial stress-related gene expression in CD34+CD38- cells from normal control samples derived from normal aged-matched peripheral blood, young and aged bone marrow, and Ax-2 and Ax-3 astronauts. SSCI 8-21 are normal young and middle-aged donor PBMCs (PB) or bone marrow (BM) SAK527 and SAK528 are samples derived from normal aged BM. Telomerase pathway genes include TPP1, POLD2 , POLA1, NHP2, TERFI, and PRIME

[0290] (E) Bar graph of average mitochondrial copy number estimates (y-axis) from Ax-2 samples over time (x-axis) measured by WGS. Timepoints include L-45 days, n=4; L-2 days, n=4; inflight, n=4; R+l day, n=3; R+42-55 days, n=4; R+l year, n=4. PATENT 0321.158068PCT / SD2024-024PCT

[0291] Statistical significance determined by a two-way ANOVA with Tukey’s multiple comparisons test. No significant differences between any timepoints.

[0292] (F) Bar graph of average mitochondrial copy number estimates (y-axis) from Ax-3 samples over time (x-axis) measured by WGS. Timepoints include inflight, n=3; R+0 days, n=3; R+21 days, n=3; R+l year, n=2. Statistical significance determined by a two-way ANOVA with Tukey’s multiple comparisons test.

[0293] (G) Tables of significant differences between timepoints of mitochondrial stress-related gene expression in all CD34+ HSPCs in Ax-2 (top) and Ax-3 (bottom) by scRNA-seq. Statistics determined by Mann-Whitney U test with FDR < 0.05.

[0294] (H) Violin plots of mitochondrial stress-related gene expression in CD34+CD38- cells from normal control samples derived from normal aged-matched peripheral blood, young and aged bone marrow, and Ax-2 and Ax-3 astronauts. SSCI 8-21 are normal young and middle-aged donor PBMCs (PB) or bone marrow (BM) SAK527 and SAK528 are samples derived from normal aged BM. Telomerase pathway genes include PRKN. OPTN. and PINK1.

[0295] (I -J) For the sample groups R+l day and R+42-55 days from Ax-2 (K) and R+0 days and R+21 days from Ax-3 (L), pseudobulk counts were used to generate a heatmap for the expressed genes in the Mitochondrial translation category7. In the GSEA analysis for Ax-2, this category had a nominal p-value < 0.005 and the majority of genes had a positive logFC indicating expression greater in the R +42-55 days group. This is supported by the predominantly red cells associated with this group in the heatmap (K). In the GSEA analysis for Ax-3, this category' had an adjusted p-value < 0.05 and the majority of genes had a positive logFC indicating expression greater in the R+21 days group. This is supported by the hierarchical cluster at the top and the predominantly red cells associated with this group in the heatmap (L).

[0296] Figure 9, Space-associated Somatic Mutations and Clonal Hematopoietic Stem and Progenitor Cell Mutations

[0297] (A) Total radiation averaged from seven locations on the International Space Station for the duration of Ax-2 and Ax-3 missions. Average radiation based on galactic cosmic rays (GCRs), Trapped Particles (SAA), and Solar Particles (ESPE).

[0298] (B) Bar plot showing the total amount of somatic mutations (SBS and indels) from CD34+ cells acquired across the study timepoints in Ax-2, including L-2 days, inflight, R+l day, R+42-55 days, and R+l year. The data from each timepoint is PATENT 0321.158068PCT / SD2024-024PCT normalized to L-45 days. No significant differences between timepoints by two-way ANOVA with Tukey's multiple comparisons test.

[0299] (C) Bar plot showing the total number of somatic single base substitutions (SBS) from CD34+ cells acquired across the study timepoints in Ax-2, including L-2 days, inflight, R+l day, R+42-55 days, and R+l year. The data from each timepoint is normalized to L-45 days. No significant differences between timepoints by two-way ANOVA with Tukey’s multiple comparisons test.

[0300] (D) Bar plot showing the total number of somatic insertions and deletions (indels) from CD34+ cells acquired across the study timepoints in Ax-2, including L- 2 days, inflight, R+l day, R+42-55 days, and R+l year. The data from each timepoint is normalized to L-45 days. No significant differences between timepoints by two- way ANOVA with Tukey’s multiple comparisons test.

[0301] (E) Patterns of single base substitution (SBS) for the Ax-2 samples are shown using the SBS96 classification scheme on the x-axis. The y-axis is scaled differently in each plot to optimally show each mutational pattern with the y-axis reflecting the number of mutations for the respective mutational scheme. The data from each timepoint is normalized to L-45 days. Sample SSCI-004 at R+l day could not be collected for whole genome sequencing (WGS).

[0302] (F) Bar plot showing the total amount of somatic mutations (SBS and indels) from CD34+ cells acquired across the study timepoints in Ax-3, including R+0 days. R+21 days, and R+l year. The data from each timepoint is normalized to inflight. R+0 days and R+21 days include N=3 samples, and R+l year timepoint includes N=2 samples. No significant differences between timepoints by two-way ANOVA with Tukey’s multiple comparisons test.

[0303] (G) Bar plot showing the total number of somatic single base substitutions (SBS) from CD34+ cells acquired across the study timepoints in Ax-3, including R+0 days, R+21 days, and R+l year. The data from each timepoint is normalized to inflight. R+0 days and R+21 days include N=3 samples, and R+l year timepoint includes N=2 samples. No significant differences between timepoints by two-way ANOVA with Tukey’s multiple comparisons test.

[0304] (H) Bar plot showing the total number of somatic insertions and deletions (indels) from CD34+ cells acquired across the study timepoints in Ax-3, including R+0 days, R+21 days, and R+l year. The data from each timepoint is normalized to inflight. R+0 days and R+21 days include N=3 samples, and R+l year timepoint PATENT 0321.158068PCT / SD2024-024PCT includes N=2 samples. Statistical analysis determined by two-way ANOVA with Tukey’s multiple comparisons test.

[0305] (I) Patterns of single base substitution (SBS) for the Ax-3 samples are shown using the SBS96 classification scheme on the x-axis. The y-axis is scaled differently in each plot to optimally show each mutational pattern with the y-axis reflecting the number of mutations for the respective mutational scheme. The data from each timepoint is normalized to inflight. Sample SSCI-007 at R+l year has not been collected.

[0306] (J) Oncoplot displaying clonal hematopoiesis (CH) mutated genes acquired in CD34+ cells from each individual per timepoint. Sample SSCI-004 at R+l day could not be collected.

[0307] (K) Oncoplot displaying clonal hematopoiesis (CH) mutated genes acquired in CD34+ cells from each individual per timepoint. Sample SSCI-007 at R+l year has not been collected.

[0308] Figure 10. Space-associated AD ARI and APOBEC3C Base Deaminase Deregulation

[0309] (A) Dot plot of hematopoietic stem cell gene expression based on CD34+ cells from Ax-2 scRNA-seq analysis. Table of significant differences betw een timepoints of hematopoietic stem cell gene expression in all CD34+ HSPCs in Ax-2 by scRNA- seq. Statistics determined by Mann-Whitney U test with FDR < 0.05.

[0310] (B) Dot plot of hematopoietic stem cell gene expression based on CD34+ cells from Ax -3 scRNA-seq analysis. Table of significant differences between timepoints of hematopoietic stem cell gene expression in all CD34+ HSPCs in Ax-3 by scRNA- seq. Statistics determined by Mann-Whitney U test with FDR < 0.05.

[0311] (C-D) Violin plots showing key gene expression based on CD34+ cells from Ax-2 (C) and Ax-3 (D) by scRNA-seq analysis. Genes include CD34, CD38, PROCR, PIEZO 1, ADAR, and APOBEC3C. Statistics w as determined by Mann-Whitney U test w ith an FDR < 0.05.

[0312] (E-F) Log Expression of ADAR in pseudobulk generated counts from single cell RNA-Seq of CD34 positive cells derived from the peripheral blood of individuals at timepoints before, during, or after time spent in low' earth orbit (LEO). Data from Ax-2 mission (E) and Ax-3 mission (F). Statistics was determined by student’s t-test assuming equal variance. No significant differences between adjacent timepoints.

[0313] (G-H) Oncoplot visualization shows the top 20 edited genes across all time points in Ax-2 (G) and Ax-3 (H). The bar graph across the top shows total editing PATENT 0321.158068PCT / SD2024-024PCT events in each sample for the top 20 genes while bar graph on the right edge shows the percentage of samples with edits in the gene.

[0314] (I-J) Log Expression of APOBEC3C in pseudobulk generated counts from single cell RNA-Seq of CD34 positive cells derived from the peripheral blood of individuals at timepoints before, during, or after time spent in LEO. Data from Ax-2 mission (I) and Ax-3 mission (J). Statistics was determined by student's t-test assuming equal variance.

[0315] Figure 11. Space-associated Retrotransposon Deregulation

[0316] (A) Waterfall plots of the expression of the top 40 significant repeat elements (and L1ME3A) in each comparison of time points (L-2days vs L-45days, left; inflight vs L-2days, center; inflight vs L-45days, right). The y-axis represents the log fold change for the comparison while the x-axis indicates the repeat element with the bar colored by the family of the repeat.

[0317] (B) Dot plots generated from scTE analysis of LINEs expression of CD34+CD38- cells from Ax-2 and Ax-3. The top 50 significantly differentially expressed (adj. p value < 0.05) retrotransposons with the highest absolute log-fold change at any timepoint and control CD34+CD38- HSCs from healthy donor PBMCs.

[0318] (C) Violin plot of the LINEs total expression scores generated with AddModuleScore in all CD34+ HSPCs for each individual astronaut across timepoints and normal control samples derived from peripheral blood and bone marrow CD34+ HSPCs. SSCT 8-21 are normal young and middle-aged donor PBMCs (PB) or bone marrow (BM). SAK527 and SAK528 are samples derived from normal aged BM.

[0319] (D) Dot plots generated from scTE analysis of hERVs expression of CD34+CD38- cells from Ax -2 and Ax-3. The top 50 significantly differentially expressed (adj. p value < 0.05) retrotransposons with the highest absolute log-fold change at any timepoint and control CD34+CD38- HSCs from healthy donor PBMCs.

[0320] (E) Violin plot of HERV total expression scores generated with AddModuleScore in all CD34+ HSPCs for each individual astronaut across timepoints and normal control samples derived from peripheral blood and bone marrow CD34+ HSPCs. SSCI 8-21 are normal young and middle-aged donor PBMCs (PB) or bone marrow (BM). SAK527 and SAK528 are samples derived from normal aged BM. PATENT 0321.158068PCT / SD2024-024PCT

[0321] FIG. 12 (also called Figure SI): Hematopoietic Stem and Progenitor Cell Functionally Organized Multi-omics Aging (HSPC-FOMA) Analyses of Astronauts. related to Figure 6

[0322] (A) Schematic summarizing the types of cells and downstream analyses performed per individual per timepoint. Analyses included 90X whole genome sequencing, whole transcriptome sequencing, single cell RNA sequencing, functional clonogenic assay, and immunophenotyping. Circles indicate CD34+ HSPC fraction, squares indicate CD34- cell fraction, and triangles indicate bulk PBMCs. Half circle or squares indicate that cells were recombined after enrichment to ensure high capture efficiency on the 10X scRNA-seq platform.

[0323] (B) Table of hematopoietic stem and progenitor cell functionally organized multi-omics aging (HSPC-FOMA) study characteristics.

[0324] (C-D) Bar plots of n=4 Ax-2 and n=3 Ax-3 individual survival (C) and selfrenewal (D) capacity collected over time (x-axis). Survival (C) capacity measured as percent of multilineage colonies on the y-axis. Self-renewal (D) capacity measured as percent change from baseline set to 100% of first collection timepoint (Ax-2 L-45 days and Ax-3 L-30 days) on the y-axis.

[0325] (E-F) Bar plots comparing n=4 Ax -2 and n=3 Ax-3 survival (E) and selfrenewal (F) capacity collected at similar timepoints from each mission (L-45 days vs L-30 days; L-2 days vs L-l day; inflight day 10 vs inflight day 21; R+l day or R+0 days; R+42-55 days vs R+21 days). Basal colony formation of original naive untreated cells before the flight (L-45 d and L-30 d) were considered to be 100% and individual values were calculated as % of change. Data presented show the mean + / - SD for both samples. Statistical analysis included Student’s t-test and one-way Anova, including All Pairwise Multiple Comparison Procedures (Holm-Sidak method).

[0326] (G-H) Stacked bar plots of CD34+ labeled cells by scRNA-seq for Ax-2 (G) and Ax-3 (H). The y-axis indicates percent of cells from each labeled timepoint. Ax-2 timepoints include L-2 days. Inflight, R+l day, R+42-55 days, and R+l year. Ax-3 timepoints include L-30 days, L-l day. Inflight, R+0 days, and R+21 days.

[0327] FIG. 13 (also called Figure S2), Space-associated Immune Repertoire Alterations, related to Figure 7 PATENT 0321.158068PCT / SD2024-024PCT

[0328] (A) Table of top 15 significantly differentially expressed genes from all CD34+ cells in Ax-2 determined by scRNA-seq using MAST and FindAllMarkers. Timepoints include L-2 days, inflight, R+l day, R+42-55 days, and R+l year.

[0329] (B) Table of top 15 significantly differentially expressed genes from all CD34+ cells in Ax-3 determined by scRNA-seq using MAST and FindAllMarkers. Time points include L-30 days, L-l day. inflight, R+0 days. R+21 days, and R+l year.

[0330] (C) Table of top 25 significantly differentially expressed genes in all CD34+ cells between Ax-2 and Ax-3 missions ranked by log-fold change determined by scRNA-seq using MAST and FindAllMarkers (adj. p value < 0.05). Timepoints include preflight. Ax-2 L-2 days vs Ax-3 L-l day. Ax-2 vs Ax-3 inflight, early return, Ax-2 R+l day vs Ax-3 R+0 days, later return, Ax-2 R+42-55 days vs Ax-3 R+21 days, and 1-year return.

[0331] FIG. 14 (also called Figure S3): Space- Associated Hematopoietic Stem and Progenitor Cell Telomere. Mitochondrial and Cytokine Deregulation, related to Figure 8

[0332] (A) Line graph illustrating whole genome sequencing analysis of the average TELOMERECAT™-estimated overall telomere length in base pairs (bp) of CD34+ cells derived from individual Ax-2 (top) and Ax-3 (bottom) samples. Sample SSCI- 004 at R+l day could not be collected. Samples SSCI-007 at R+l year has not been collected.

[0333] (B) Line graph illustrating whole genome sequencing analysis of the relative telomere content on the y-axis of CD34+ cells derived from individual Ax-2 (top) and Ax-3 (bottom) samples. Sample SSCI-004 at R+l day could not be collected. Samples SSCI-007 at R+l year has not been collected.

[0334] (C) Line graph showing average mitochondrial copy number estimates from Ax-2 (N=4, top) and Ax-3 (N=3, bottom) samples. Sample SSCI-004 at R+l day could not be collected. Samples SSCI-007 at R+l year has not been collected.

[0335] (D) Violin plots of telomere maintenance gene expression in CD34+CD38- cells from normal control samples derived from normal aged-matched peripheral blood, young and aged bone marrow, and Ax-2 and Ax-3 astronauts. SSCI 8-21 are normal young and middle-aged donor PBMCs (PB) or bone marrow (BM) SAK527 and SAK.528 are samples derived from normal aged BM. Telomerase pathway genes include HIST2H2BE, RUVBL1, POLD3, CTC1, STM1, and POTI. PATENT 0321.158068PCT / SD2024-024PCT

[0336] (E-F) Gene set enrichment analysis (GSEA) was performed on pseudobulk data for the comparison of Launch-2 days vs Inflight and Retum+1 day vs Retum+42- 55 days during Ax-2 Mission (E), and Launch-1 day vs Inflight and Retum+0 days vs Retum+21 days during Ax-3 (F). Three categories of interest: Mitochondrial translation, Cell Cycle Checkpoints, and Interferon signaling are plotted to show the distribution of fold changes for genes in the category relative to all genes. The Mitochondrial translation category for Ax-2 has a nominal p-value < 0.005, which is consistent with the green line in the plot that decreases moving right on the x-axis. The Mitochondrial translation category for Ax-3 has an adjusted p-value < 0.05, which is consistent with the green line in the plot that decreases moving right on the x-axis.

[0337] (G-H) Bar plots of cytokine expression (IL- / ?, IL-10, IL-15, and IL-23) measured from diluted plasma in Ax-2 (G) and Ax-3 (H). Plots are averaged between N=4 samples for Ax-2 and N=3 samples for Ax-3. Statistical analysis included oneway ANOVA, including Tukey’s multiple comparisons test.

[0338] (I-J) Boxplots of inflammatory cytokine (I) and cytokine receptor (J) expression levels in CD34+ cells by whole transcriptome sequencing. Timepoints include L-45 days, L-2 days, and inflight.

[0339] FIG. 15 (also called Figure S4): Space-associated Clonal Hematopoietic Stem and Progenitor Cell Mutations, related to Figure 9:

[0340] (A) Bar plots presenting single base substitution mutations in their 6 possible substitution context and the cumulative C>T mutations at CpG sites and other sites in L-2 days, inflight, R+l day, and R+42-55 days versus L-45 days for Ax-2 samples.

[0341] (B) Patterns of small insertions and deletions (indels) for the Ax-2 samples are shown using the ID-83 classification scheme on the x-axis. The y-axis is scaled differently in each plot to optimally show each mutational pattern with the y-axis reflecting the number of mutations for the respective mutational scheme. The data from each timepoint is normalized to L-45 days. Sample SSCI-004 at R+l day could not be collected for WGS.

[0342] (C) Patterns of double base substitution (DBS) for the Ax-2 samples are shown using the DBS78 classification scheme on the x-axis. The y-axis is scaled differently in each plot to optimally show each mutational pattern with the y-axis reflecting the number of mutations for the respective mutational scheme. The data PATENT 0321.158068PCT / SD2024-024PCT from each timepoint is normalized to L-45 days. Sample SSCI-004 at R+l day could not be collected for whole genome sequencing (WGS).

[0343] (D) Line graph showing the total amount of somatic mutations (SBS and indels) from CD34+ cells acquired across the study timepoints in Ax-2, including R+l day, R+42-55 days, and R+l year. The data from each timepoint is normalized to inflight.

[0344] (E) Line graph showing the total number of somatic single base substitutions (SBS) from CD34+ cells acquired across the study timepoints in Ax-2, including R+l day, R+42-55 days, and R+l year. The data from each timepoint is normalized to inflight.

[0345] (F) Line graph showing the total number of somatic insertions and deletions (indels) from CD34+ cells acquired across the study timepoints in Ax-2, including R+l day, R+42-55 days, and R+l year. The data from each timepoint is normalized to inflight.

[0346] (G) Line graph showing the total amount of somatic mutations (SBS and indels) from CD34+ cells acquired across the study timepoints in Ax-3, including R+0 days, R+21 days, and R+l year. The data from each timepoint is normalized to inflight. R+0 days and R+21 days include N=3 samples, and R+l year timepoint includes N=2 samples. Sample SSCI-007 at R+l year has not been collected.

[0347] (H) Line graph showing the total number of somatic single base substitutions (SBS) from CD34+ cells acquired across the study timepoints in Ax-3, including R+0 days, R+21 days, and R+l year. The data from each timepoint is normalized to inflight. R+0 days and R+21 days include N=3 samples, and R+l year timepoint includes N=2 samples. Sample SSCI-007 at R+l year has not been collected.

[0348] (I) Line graph showing the total number of somatic insertions and deletions (indels) from CD34+ cells acquired across the study timepoints in Ax-3, including R+0 days, R+21 days, and R+l year. The data from each timepoint is normalized to inflight. R+0 days and R+21 days include N=3 samples, and R+l year timepoint includes N=2 samples. Sample SSCI-007 at R+l year has not been collected.

[0349] (J) Bar plots presenting single base substitution mutations in their 6 possible substitution context and the cumulative C>T mutations at CpG sites and other sites in R+0 days, R+21 days, and R+l year versus inflight for Ax-3 samples.

[0350] (K) Patters of small insertions and deletions (indels) for the Ax-3 samples are shown using the ID-83 classification scheme on the x-axis. The y-axis is scaled PATENT 0321.158068PCT / SD2024-024PCT differently in each plot to optimally show each mutational pattern with the y-axis reflecting the number of mutations for the respective mutational scheme. The data from each timepoint is normalized to inflight. Sample SSCI-007 at R+l year has not been collected.

[0351] (L) Patterns of double base substitution (DBS) for the Ax-3 samples are shown using the DBS78 classification scheme on the x-axis. The y-axis is scaled differently in each plot to optimally show each mutational pattern with the y-axis reflecting the number of mutations for the respective mutational scheme. The data from each timepoint is normalized to inflight. Sample SSCI-007 at R+l year has not been collected.

[0352] (M) Oncoplot displaying top 25 of the somatically mutated genes within Ax-2 samples. The data from each timepoint is normalized to L-45 days.

[0353] (N) Oncoplot displaying top 25 of the somatically mutated genes within Ax-3 samples. The data from each timepoint is normalized to inflight. Sample SSCI-007 at R+l year has not been collected.

[0354] FIG. 16 (also called Figure S5), Space-associated AD ARI and APOBEC3C Base Deaminase Deregulation, related to Figure 10:

[0355] (A) Boxplot of AD ARI isoform expression LogCPM values in L-45 days, L-2 days, and inflight from Ax-2 mission.

[0356] (B) Boxplot of AD ARI editing in L-45 days, L-2 days, and inflight from Ax-2 mission.

[0357] (C) Boxplot of APOBEC3 expression LogCPM values in L-45 days, L-2 days, and inflight from Ax-2 mission.

[0358] (D-E) Log Expression of APOBEC3C in pseudobulk generated counts from scRNA-Seq of CD34+ cells derived from the peripheral blood of individuals at timepoints before, during, or after time spent in LEO. Data from Ax-2 mission (D) and Ax-3 mission (E).

[0359] FIG. 17 (also called Figure S6), Space-associated Retrotransposon Deregulation, related to Figure 11

[0360] (A-B) Volcano plots of the expression of LINE elements (A) and HERV elements (B) in each comparison of time points (L-2days vs L-45days, left; inflight vs L-45days, center; inflight vs L-2days, right). The x-axis shows the log fold change and y-axis represents the negative log of the comparison p-value. Red dots indicate those elements with p-value < 0.05. PATENT 0321.158068PCT / SD2024-024PCT

[0361] (C) Heatmap of retrotransposons that are differentially expressed in any of the time point comparisons. The hierarchical cluster along the left edge shows how the expression patterns of the repeat element relate while the cluster across the top shows that samples within time points cluster together based on the retrotransposon.

[0362] (D) Table of all retrotransposons represented in Fig. 6A waterfall plots with adj. p value < 0.1. retrotransposons are ranked based on top positive and negative logfold change.

[0363] (E) Table of top 5 significantly differentially expressed LINEs in CD34+CD38- cells from Ax-2 and Ax-3 ranked by logFC (adj. p value < 0.05).

[0364] (F) Table of top 5 significantly differentially expressed HERVs in CD34+CD38- cells from Ax-2 and Ax-3 ranked by logFC (adj. p value < 0.05).

[0365] FIG. 18 illustrates a dot plot of hematopoietic stem cell gene expression based on CD34+ cells from Ax-2 scRNA-seq analysis. Table of significant differences between timepoints of hematopoietic stem cell gene expression in all CD34+ HSPCs in Ax-2 by scRNA-seq. Statistics determined by Mann- Whitney U test with FDR < 0.05.

[0366] FIG. 19 illustrates a dot plot of hematopoietic stem cell gene expression based on CD34+ cells from Ax-3 scRNA-seq analysis. Table of significant differences between timepoints of hematopoietic stem cell gene expression in all CD34+ HSPCs in Ax-3 by scRNA-seq. Statistics determined by Mann- Whitney U test with FDR < 0.05.

[0367] FIG. 20 illustrates Violin plots showing key gene expression based on CD34+ cells from Ax-2 by scRNA-seq analysis. Genes include CD34, CD38, PROCR, PIEZO 1, ADAR, and APOBEC3C. Statistics was determined by Mann-Whitney U test with an FDR < 0.05.

[0368] FIG. 21 illustrates Violin plots showing key gene expression based on CD34+ cells from Ax-3 by scRNA-seq analysis. Genes include CD34, CD38, PROCR, PIEZO 1, ADAR, and APOBEC3C. Statistics was determined by Mann-Whitney U test with an FDR < 0.05.

[0369] FIG. 22 illustrates Violin plot of the LINEs total expression scores generated with AddModuleScore in all CD34+ HSPCs for each individual astronaut across timepoints and normal control samples derived from peripheral blood and bone marrow CD34+ HSPCs. SSCI 8-21 are normal young and middle-aged donor PBMCs PATENT 0321.158068PCT / SD2024-024PCT

[0370] (PB) or bone marrow (BM). SAK527 and SAK528 are samples derived from normal aged BM.

[0371] FIG. 23 illustrates a Violin plot ofHERV total expression scores generated with ADDMODULESCORE™ in all CD34+ HSPCs for each individual astronaut across timepoints and normal control samples derived from peripheral blood and bone marrow CD34+ HSPCs. SSCI 8-21 are normal young and middle-aged donor PBMCs (PB) or bone marrow (BM). SAK527 and SAK528 are samples derived from normal aged BM.

[0372] Materials and Methods

[0373] Sample Collection and Processing

[0374] Peripheral blood (PB) from consenting individuals (N=7) as part of NASA IRB study 0547 was collected from 6 timepoints, L-30 ± 20 days, L-l ± 10 days, Inflight ± 10 days. R+l ± 10 days. R+30 ± 60 days, and R+l year. All PB samples were delivered to Sanford Consortium for Regenerative Medicine (2880 Torrey Pines Scenic Drive, La Jolla, CA 92037) and processed under BSL2 conditions within 24-96 hours of venipuncture. Depending on shipping duration, samples w ere either shipped at ambient temperature (within 24-hrs of vein contact), or with ice gel packs (>24-hrs of vein contact). Upon receipt of samples, peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll Paque (density 1.077 g / mL) density centrifugation. Samples were diluted 1 :3.5 in HBSS to a total volume of 35mL prior to underlaying Ficoll. Prepared samples were centrifuged for 20 minutes at 1500 RPM and 20°C without deceleration. The PBMCs were collected and washed twice with HBSS prior to proceeding with downstream experiments.

[0375] Hematopoietic Stem and Progenitor Cell Purification

[0376] Pelleted PBMCs were resuspended in 300uL of cold staining media (HBSS with 2% FBS). IOOUL of both CD34+microbeads (130-100-453, Miltenyi Biotec) and FcR Human Blocking Reagent (130-059-901, Miltenyi Biotec) were added to the resuspended cells and allowed to incubate in darkness for 30 minutes on ice. lOmL of staining media was added to the incubation mixture and spun at 11 OOrpm for 3 minutes at 4°C. After aspirating the supernatant, the pellet was resuspended in ImL of staining media and filtered through a Blue Top Round Bottom Falcon Tube (08-771- 23, Falcon). As the cells filtered through the Falcon tube, 500uL of staining media was used to w et an MS Column (130-042-201, Miltenyi Biotec). To separate the PATENT 0321.158068PCT / SD2024-024PCT

[0377] CD34+ population from its negative fraction, the cells were pipetted into the MS column, where the CD34- filtrate was collected. Once the filtrate ran through the column, the column was washed three times in 1 mL of staining media. When the staining media had fully eluted out of the MS column, the column was placed atop a new collection tube. ImL of staining media was then added to the column and plunged through the column with force and in quick succession to obtain the isolated CD34+ population.

[0378] HSPC and Immune Subpopulation FACS Analysis

[0379] All flow cytometry7stains were performed in HBSS / 2% FBS for 20 min on ice. FcR Blocking Reagent (Miltenyi, cat. 130-059-901) was added to all samples at a 1:50 dilution for 10 minutes on ice prior to antibody staining. 7-AAD viability staining solution (Biolegend, cat. 420404) was included in the antibody staining mixture at a 1 :50 dilution for all samples. Each sample was split in half and stained with two immunophenotyping panels. Panel 1 contained CD45 BV510, CD34 BV421, CD3 FITC, CD56 BUV737. CD19 APC-Cy7, CD14 PE-Cy7, CD16 APC, and 7-AAD viability staining solution. Panel 2 contained CD45 BV510. CD3 FITC, CD4 BUV737, CD8 BV711, CD25 PE-Cy7, CD127 BV605, CD69 BV650, CD45RA PE, CD45RO BV421, CD62L APC-Cy7, CD27 APC, CCR7, AF700, and 7-AAD viability staining solution. Analysis was performed on a LSRFortessa X-20 or LSR II instrument at the UC San Diego Human Embryonic Stem Cell Core and Salk Institute for Biological Studies Flow Cytometry Core Facility respectively. FlowJo software was used for analysis of cell populations. For all analyses 7-AAD+ cells were gated out. and single cells were gated based on FSC-H vs FSC-A. Statistical significance was determined using a two-tailed t-test of unequal variance. P-values less than 0.05 were considered significant.

[0380] HSPC Clonogenic and Survival Assays

[0381] Clonogenic experiments were performed as previously described58. Briefly, CD34+ cells were resuspended in fresh media and plated in methylcellulose (MC) in triplicate (Stemcell Technologies, catalog H4335). After 2 weeks, the primary colony (survival assay) clusters of more than 40 cells were counted and individual multilineage colonies were plucked, cells resuspended and re-plated again in fresh MC. Secondary' colonies (self-renewal assay) were counted after another 14 days. In survival assays, total number of colonies was considered to be 100% and results are presented as percent of change. In self-renewal assays, the data represents the percent of replated PATENT 0321.158068PCT / SD2024-024PCT colonies. Error bars indicate the SE of triplicate. Statistical analysis comprised Student's t-test and one-way Anova. including All Pairwise Multiple Comparison Procedures (Holm-Sidak method).

[0382] Single-Cell Library Preparation and Deep Sequencing

[0383] After selection, CD34-positive and CD34-negative fractions were either separately aliquoted or re-combined at a specific ratio determined by available cell number. Aliquots of 10.000 cells were resuspended at 1000 cells per uL in sterile filtered 0.04% BSA in PBS. For higher cell yields (CD34- fraction), 35,000 cells were resuspended at 1000 cells per uL in 0.04% BSA in PBS. Single-cell libraries were prepared using 10X-3’ v3.1 Gene Expression (10X Genomics) kit as per manufacturer's instruction. The libraries' quality and quantity were assessed using the TapeStation 4200 DNA High Sensitivity analysis and the Qubit 4 dsDNA high sensitivity assay, pooled and sequenced on the Illumina NovaSeq 6000 platform (Next Generation Sequencing Core, Salk Institute, La Jolla, CA, USA) using S4 flow cells (sequencing depth PE200) and 1% PhiX. For each sample, a custom calculation was done to assign a maximum number of reads based on the percentage of HSC present in the original sample resulting in range of 60 M to 500 M reads per sample.

[0384] HSPC single cell RNA-seq Analyses

[0385] Sequenced reads from each GEX library generated were aligned to the human reference genome GRCh38, downloaded from the 10X genomics website (10X Genomics, GRCh38-2020-A), and annotated with Ensembl release 98. Quality control, alignment, and quantification of reads were performed using the Cell Ranger v.7.1.0 (10X Genomics) ‘count’ pipeline, generating count matrices that filtered out empty droplets.

[0386] GEX count matrices were loaded with Seurat v.5. 1.0 (Satija Lab)39and individual datasets were bioinformatically cleaned for doublets using DoubletFinder v2.0.460. Then, count matrices were merged into a single object and filtered for cells with outlier UMI counts (low quali ty cells and doublets), high mitochondrial gene expression (due to cellular stress or loss of cytoplasmic RNA), and low number of sequenced genes. Molecular count data for each sample were individually log normalized and scaled in Seurat with the additional steps of regressing UMI counts, and gene counts during scaling to mitigate their effects on dimensionally reduced clustering. Highly variable genes were identified as the union of the top 3,000 genes from each sample after normalization, and then used to calculate principal PATENT 0321.158068PCT / SD2024-024PCT components using RunPCA. Normalized and scaled samples were integrated using the IntegrateLayers function using the “scVIIntegratiom method in Seurat59.

[0387] Visualization and clustering of the integrated object was performed by calculating the PCA for dimensionality reduction visualization using the UMAP function in Seurat (Satija Lab)59. After identified clusters via FindClusters in Seurat59, identities were assigned using SingleR61, scType62, and Azimuth (Butler A. Darby C, Hao Y. Hartman A, Hoffman P, Molla G, Satija R (2023). Azimuth: A Shiny App Demonstrating a Query-Reference Mapping Algorithm for Single-Cell Data. R package version 0.5.0, https: / / github.com / satiialab / azimuth.) using the Human PBMC reference (Integrated analysis of multimodal single-cell data Hao. Yuhan et al. Cell, Volume 184. Issue 13, 3573 - 3587. e29). Results were verified and corrected using differentially expressed genes calculated through FindAllMarkers in Seurat59and Enrichr63'65.

[0388] The CD34+ HSPCs were further classified into 5 clusters using the FindClusters function in Seurat2. These clusters were based on CD34 and CD38 gene expression profiles, removing one cluster that was negative for both CD34, CD38, and PTPRC (CD45). Clusters were named as follows: “CD34+CD38-”, “CD34+CD38mid”, “CD34+CD38+”, and “CD341ow”. Differentially expressed genes (DEGs) were calculated using the “MAST"’ algorithm66, which is tailored to scRNA-seq data DEG analysis using a model that parameterizes both stochastic dropout and characteristic bimodal expression distributions, for the FindMarkers function of Seurat (min.pct = 0.1, logfc.threshold = 0.25). DEGs from FindMarkers were used to generate ranked gene lists ordered by log-fold change for Gene Set Enrichment Analysis (GSEA) using the fgsea vl.22.0.67Fast gene set enrichment analysis67package and gene signatures were pulled from the Molecular Signatures Database (MSigDB) using msigdbr v7.5. 168'70Visualization of GSEA results was performed using the enrichplot v 1.16.2 package69. Visualization of functional enrichment result was performed using R package (version 4.3.2).

[0389] Pseudobulk analysis was performed by starting with the integrated object (above) and extracting the RNA counts and metadata to create a SingleCellExperiment object. The function aggregate Matrix from the Bioconductor71package DESeq2was used to create a counts matrix. These data w ere input into a DESeq2 object, normalized, and log transformed for visualization of specific genes. Single-cell Transposable Element (scTE) Analyses PATENT

[0390] 0321.158068PCT / SD2024-024PCT

[0391] The .bam files generated by CellRanger, were filtered using SAM tools72-73to remove reads lacking cell barcodes. After filtering, the scTE pipeline74was applied to realign reads to known transposable elements (TEs) using UCSC Repeatmasker73annotations. Once annotated, the scTE output was generated as a ,h5ad file for conversion to h5seurat form in R for further downstream single-cell analysis. AddModuleScore Function

[0392] To quantify the expression of predefined LINEs and hERVs lists across single cells, the AddModuleScore function in Seurat (version 5.0) was used to compute the average expression of each gene set (module) on a per-cell basis.

[0393] Nucleic Acid Extraction and Sequencing

[0394] For DNA, cells were pelleted and frozen at -80°C prior to extraction using the Qiagen DNeasy Blood and Tissue Kit according to manufacturers’ protocol (Qiagen, catalog 69504). DNA was eluted with Qiagen Buffer AE. Extracted DNA from these samples was sent to Novogene (Sacramento, CA) for 90x whole genome sequencing. DNA quality control was done using Qubit 4 Fluorometer (Invitrogen) and 4200 Tape Station system (Agilent). DNA sequencing library preparation for Ax-2 samples was performed using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (E7645L) following the manufacturer’s recommendations. DNA sequencing library' preparation for Ax-3 samples was performed using the Rapid Plus DNA Library Prep Kit (RK20208 ABclonal Technology) following the manufacturer’s recommendations. Qualified libraries were paired and sequenced on an Illumina NovaSeq 6000 platform using 150 PE sequencing chemistry to 90x coverage according to effective concentration and data volume. For RNA, cells were lysed in RLT buffer (Qiagen) and 10% beta-mercaptoethanol. Samples were extracted using Qiagen RNeasy Micro Kit according to manufacturers’ protocol (Qiagen, catalog 74004). RNA was eluted in RNase-free w ater and frozen at -80°C until assessed for quality. Samples with RNA integrity number (RIN) >7 were further processed for RNA-sequencing (The Scripps Research Institute Next Generation Sequencing Core). RNA-seq was performed on Illumina’s NextSeq 500 sequencer with 150bp paired-end reads.

[0395] Whole Genome Sequencing-based Identification of Somatic Mutations Raw sequence data w ere downloaded to the Triton Shared Compute Cluster (San Diego Supercomputer Center (2022): Triton Shared Computing Cluster. University of California, San Diego. Service. https: / / doi.org / 10.57873 / T34W2R) from ftp server PATENT

[0396] 0321.158068PCT / SD2024-024PCT link shared by Novogene (Sacramento, CA). All the post-sequencing analysis was performed within TSCC at UC San Diego. This methodology for identification of somatic mutations from bulk sequencing data follows established approaches from large genomics consortia76. Briefly, quality assurance of the raw FASTQ files were evaluated using FastQC (Version 0.12.0) and Mosdepth (Version 0.3.4)77,78. Raw sequence reads were aligned to the human reference genome GRCh38.dl.vdl. The aligned reads were marked duplicated using MarkDuplicates (Picard) from GATK (Version 4.0. 11.0)79. Concordance between paired samples were checked using Conpiar version 0.280. An ensemble variant calling pipeline (EVC) was used to identify single nucleotide variants (SNV) and short insertions and deletions (indels). EVC implements the SNV and indel variant calling from four variant callers (Mutect2, Strelka2, Varscan2, and MuSE2) and only mutations that are identified by any two variant callers were considered as bona fide mutations79,81'83.

[0397] Analysis of mutational profile

[0398] Analysis of mutational profiles was performed using our previously established methodology with the SIGPROFILER™ suite of tools used for summarization, simulation, visualization, and assignment of mutational profiles. Briefly, mutational matrixes for SBS, DBS and Indels were generated with SIGPROFILERMATRIXGENERATOR™ (Version 1.2. 16)84. Plotting of each mutational profile was done with SIGPROFILERPLOTTING™ (Version 1.3. 13). Annotation of the mutations

[0399] The mskcc / vcf2maf (Cyriac Kandoth. mskcc / vcf2maf: vcf2maf vl.6. (2020). doi: 10.5281 / zenodo.593251) was used to annotate each mutation with its genomic coordinates using ENSEMBL VARIANT EFFECT PREDICTOR™ (VEP. vl06) and the annotated files were saved as mutation annotation format (MAF). Plotting of the genetic annotation was done using MAFTOOLS™85(version 2.16.0) within R (v4.3.1) statistical language. Annotation of each C>T mutation to its CpG island is done using MUTATIONALP ATTERNS V3.10.0™ packages in R.

[0400] Whole Genome Sequencing-based Detection of Space-associated Clonal Hematopoiesis

[0401] To identify the mutation affecting clonal hematopoiesis associated drivers, mutations were called using Mutect2 single samples mode by using mark duplicated reads files from EVC pipeline. To identify the mutation affecting clonal hematopoiesis associated drivers, mutations were called using MUTECT2™ single samples mode by PATENT

[0402] 0321.158068PCT / SD2024-024PCT using mark duplicated reads files from EVC pipeline. Due to variations in the DNA library preparation kit and observed differences in allele frequency (AF) distribution from Mutect2 single samples mode call, low AF mutations were filtered out by applying a threshold of AF < 10%. Mutations were then filtered using 123 Clonal Hematopoiesis gene's genomic co-ordinates using BCFTOOLS VERSION 1.19™. Following annotating the mutation with mskcc / vcf2maf, they were filtered with the "ALPHAMISSENSE™" (Ref. Accurate proteome-wide missense variant effect prediction with ALPHAMIS SENSE™D) annotation for "likely pathogenic" mutations based on the pathogenicity score within the tool. " ALPHAMISSENSE™ " annotated matched samples were plotted using MAFTOOLS™85(version 2.16.0) within R (v4.3.1) statistical language.

[0403] Whole Genome Sequencing Telomere Length and Mitochondrial Copy Number Quantification

[0404] The mark duplicated reads were used for telomere length estimation using TELOMERECAT™86(Telomere Computational Analysis Tool). The bam21englh' parameters were used with simulation sets to 100 and output options set to details parameters. FASTMITOCALC™87tool was employed in order to determine the average mitochondrial DNA copy number by follow ing the parameter descried in the tools methods sections.

[0405] Whole Genome Somatic Mutation Sequencing Analyses

[0406] Somatic mutations in whole-genome sequencing data were identified using our ensemble variant calling pipeline, which is freely available under the permissive 2- clause BSD license at: https: / / github.com / AlexandrovLab / EnsembleVariantCallingPipeline. All other computational tools utilized in this publication have been mentioned in the methodology section and can be accessed through their respective publications.

[0407] Whole Transcriptome Sequencing (RNA-Seq) Analyses

[0408] Bulk RNA-Seq data were processed as previously reported18. Briefly, fastq files were processed by the FASTQC™ tool88to verity’ quality. Gene and isoform expression counts tables w ere generated with STAR RSEM™89’90using the human reference genome (GRCh38_pl3_v43) and GENCODE™ annotation

[0409] (gencode. v!9. annotation. gtf). Using the Bioconductor packages edgeR91and limma92samples were merged, filtered, normalized with the trimmed mean of M-values method91and transformed with limma-voom prior to limma differential expression PATENT 0321.158068PCT / SD2024-024PCT analysis comparing the various timepoints. Significance was defined as adjusted p- value < 0.05. Functional enrichment analysis was performed with the Bioconductor package fgsea67and the REACTOME™ database as the ontology93.

[0410] RNA Editome Analyses

[0411] RNA editing events within each sample were determined as described previously94. ADAR-driven events were the focus of this analysis, so positive strand A to G and negative strand T to C variants were retained for quantification of total edits within each sample group.

[0412] Retrotransposon Analyses

[0413] The .bam files generated by STAR alignment (described above), along with the repeatmasker.hg38.gtf file (downloaded from the UCSC genome browser), were used as input into the ‘TeatureCounts” function from the Subread R package95to generate counts of retrotransposons. Counts were aggregated for all samples, filtered, normalized, and converted to log counts per million (lepm). Retrotransposons were also annotated using the ’hg38Ta.out" file96. As above, differential expression of retrotransposons was determined using limma92Cytokine Arrays

[0414] Plasma was analyzed using the BioLegend LEGENDPLEX™ Human Hematopoietic Stem Cell Panel (IL-6, FLT3L, GM-CSF, IL-3, IL-34, IL-11, SCF, LIF, CXCL12 (SDF-1), IL-15, M-CSF. IL-7; Lot #: B387711) and LEGENDPLEX™ Human Inflammation Panel 1 (IL-lb, IFN-a2, IFN-g, TNF-a, MCP-1 , IL-6, IL-8, IL- 10, IL12p70, IL-17A, IL-18, IL-23, IL-33; Lot #: B408881) kits. Frozen plasma samples were thawed on ice and centrifuged prior to loading into sample wells. Samples were run according to the manufacturer's protocol. Samples were run on the MACSQUANT 10™ flow cytometer, and the FCS files were uploaded into LEGENDPLEX™’ s QOGNIT™ database for analysis. Concentrations are reported in pg / ml and bar plots done in GRAPHPAD PRISM™.

[0415] References Example 1 :

[0416] 1. B Engels, et al, Targeting stroma to treat cancers. Semin Cancer Biol. 2012 Feb; 22(1): 41-49.

[0417] 2. G. Pineda, et al, Tracking of Normal and Malignant Progenitor Cell Cycle Transit in a Defined Niche. J Sci Rep 6, 23885 (2016). PATENT 0321.158068PCT / SD2024-024PCT

[0418] 3. L Ladel, et al, Assessing hematopoietic stem cell fitness within a nanobioreactor in rmcrogravily. Cancer Res (2022) 82 (12_Supplement): 3154.

[0419] 4. Goff DJ, et al, A Pan-BCL2 Inhibitor Renders Bone-Marrow-Resident Human Leukemia Stem Cells Sensitive to Tyrosine Kinase Inhibition. Cell Stem Cell 12(3):316-28.

[0420] References Example 2:

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[0498] A number of embodiments of the invention have been described. Nevertheless, it can be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

PATENT0321.158068PCT / SD2024-024PCTWHAT IS CLAIMED IS:

1. A method for maintaining or extending the self-renewing capacity of hematopoietic stem cell (HSCs) in long term culture, or for regaining self-renewing capacity by hematopoietic stem cell (HSC) that that have lost their self-renewing capacity (so-called “exhausted HSCs) after long-term culture, the method comprising:(a) providing a plurality of HSCs that have been in long-term culture and / or are exhausted HSCs;(b) providing a three-dimensional (3D) nanobioreactor;(c) establishing or culturing in the 3D nanobioreactor: (i) an autologous primary stroma derived from a CD34-negative fraction of aged bone marrow (ABM) cells; (ii) an Hs27a cell line monolayer; or, (iii) both (i) and (ii);(d) plating or culturing in the 3D nanobioreactor on the autologous primary stroma and / or on the Hs27a cell line monolayer the plurality of HSCs that have been in long-term culture and / or are exhausted HSCs; and(e) culturing the plurality of HSCs that have been in long-term culture and / or are exhausted HSCs in the 3D nanobioreactor for at least 2 weeks, or between about 2 weeks and 4 weeks, or between one week and 5 weeks, or between 6 days and 6 weeks, wherein culturing the plurality of HSCs that have been in long-term culture and / or are exhausted HSCs in the 3D nanobioreactor for at least 2 weeks, or between about 2 weeks and 4 weeks, or between one and 5 weeks, maintains or extends the self-renewing capacity of the plurality of HSCs, or, results in the plurality of HSCs that that have lost their self-renewing capacity' (so-called “exhausted HSCs) to regain their self-renewing capacity or to substantially regain their self-renewing capacity'.

2. The method of claim 1, wherein the plurality7of HSCs are derived from or isolated from human aged bone marrow donors.

3. A hematopoietic stem cell (HSC) self-renewing capacity generating three-dimensional (3D) nanobioreactor made by a method comprising:(a) providing HSCs that have been in long-term culture and / or are exhausted HSCs;PATENT 0321.158068PCT / SD2024-024PCT(b) providing a three-dimensional (3D) nanobioreactor;(c) establishing or culturing in the three-dimensional (3D) nanobioreactor: (i) an autologous primary stroma derived from a CD34-negative fraction of aged bone marrow (ABM) cells; (ii) an Hs27a cell line monolayer; op (iii) both (i) and (ii), and(d) plating or culturing in the 3D nanobioreactor on the autologous primary stroma and / or on the Hs27a cell line monolayer HSCs that have been in long-term culture and / or are exhausted HSCs.

4. The HSC self-renewing capacity generating three-dimensional (3D) nanobioreactor of claim 3, wherein the HSCs are derived from or isolated from human aged bone marrow donors.

5. Use of a hematopoietic stem cell (HSC) self-renewing capacity generating three-dimensional (3D) nanobioreactor of claim 3 or claim 4 to: maintain or extend the self-renewing capacity of a plurality of HSCs, or, have or convert HSCs that that have lost their self-renewing capacity (so-called ‘"exhausted HSCs) to regain their self-renewing capacity or to substantially regain their self-renewing capacity.

6. A hematopoietic stem cell (HSC) self-renewing capacity generating three-dimensional (3D) nanobioreactor of claim 3 or claim 4 for use in: maintaining or extending the self-renewing capacity of a plurality of HSCs, or, for use in converting HSCs that that have lost their self-renewing capacity (so-called “exhausted HSCs) HSCs that have regained their self-renewing capacity or to substantially regain their self-ren ewing capacity.

7. A product of manufacture made by a method comprising:(a) providing HSCs that have been in long-term culture and / or are exhausted HSCs;(b) providing a three-dimensional (3D) bioreactor; and(c) establishing or culturing in the three-dimensional (3D) bioreactor: (i) an autologous primary’ stroma derived from a CD34-negative fraction of aged bone marrow’ (ABM) cells; (ii) an Hs27a cell line monolayer; or, (iii) both (i) and (ii).PATENT 0321.158068PCT / SD2024-024PCT(d) plating or culturing in the 3D bioreactor on the autologous primary' stroma and / or on the Hs27a cell line monolayer HSCs that have been in long-term culture and / or are exhausted HSCs.

8. A product of manufacture made by a method comprising:(a) providing HSCs that have been in long-term culture and / or are exhausted HSCs;(b) providing a three-dimensional (3D) bioreactor; and(c) establishing or culturing in the 3D bioreactor: (i) an autologous primary stroma derived from a CD34-negative fraction of aged bone marrow (ABM) cells; (ii) an Hs27a cell line monolayer; or, (hi) both (i) and (ii).

9. A method for maintaining or extending the self-renewing capacity of hematopoietic stem cell (HSCs) in long term culture, or for regaining self-renewing capacity by hematopoietic stem cell (HSC) that that have lost their self-renewing capacity (so-called "‘exhausted HSCs) after long-term culture, the method comprising:(a) providing the product of manufacture of claim 8;(b) plating or culturing in the 3D nanobioreactor on the autologous primary stroma and / or on the Hs27a cell line monolayer a plurality of HSCs that have been in long-term culture and / or are exhausted HSCs, and(e) culturing the plurality of HSCs that have been in long-term culture and / or are exhausted HSCs in the 3D nanobioreactor for at least 2 weeks, or between about 2 weeks and 4 weeks, or between one week and 5 weeks, or between 6 days and 6 weeks, wherein culturing the plurality of HSCs that have been in long-term culture and / or are exhausted HSCs in the 3D nanobioreactor for at least 2 weeks, or between about 2 weeks and 4 weeks, or between one and 5 weeks, maintains or extends the self-renewing capacity of the plurality of HSCs, or, results in the plurality of HSCs that that have lost their self-renewing capacity' (so-called “exhausted HSCs) to regain their self-renewing capacity or to substantially regain their self-renewing capacity.

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