WO2023183555A1

WO2023183555A1 - Method of promoting thymic recovery by administering hematopoietic stem cells with low c-kit expression

Info

Publication number: WO2023183555A1
Application number: PCT/US2023/016201
Authority: WO
Inventors: Marcel Van Den Brink; Harold ELIAS
Original assignee: Memorial Sloan-Kettering Cancer Center; Sloan-Kettering Institute For Cancer Research; Memorial Hospital For Cancer And Allied Diseases
Priority date: 2022-03-25
Filing date: 2023-03-24
Publication date: 2023-09-28

Abstract

A method of promoting thymic recovery in a subject following myeloablative conditioning for a hematopoietic stem cell (HSC) transplant is provided, including obtaining or having obtained an enriched population of c-Kit-expressing hematopoietic stem cells (HSC) from a population of donor c-Kit-expressing HSC wherein the enriched population of c-Kit-expressing HSC is enriched for HSC expressing a low level of c-Kit, and administering the enriched population of c-Kit-expressing HSC to the subject. Also provided is a method of treating cancer in a subject, including obtaining or having obtained an enriched population of c-Kit-expressing hematopoietic stem cells (HSC) from a population of donor c-Kit-expressing HSC wherein the enriched population of c-Kit-expressing HSC is enriched for HSC expressing a low level of c-Kit, and administering the enriched population of c-Kit-expressing HSC to the subject. In an example, the donor is not the subject or an identical sibling of the subject. In another example, the donor is a human of at least 60 years of age. In another example, the subject is a human of at least 40 years of age.

Description

METHOD OF PROMOTING THYMIC RECOVERY BY ADMINISTERING

HEMATOPOIETIC STEM CELLS WITH LOW C-KIT EXPRESSION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority from U.S. Provisional Patent Application No. 63/323,582, filed March 25, 2022, the entire contents of which are incorporated herein by reference.

BACKGROUND

[0002] Hematopoietic stem cell transplantation (HCT), including allogenic HCT and autologous HCT, is a therapy for several different disorders, including malignant and non- malignant disorders. Generally, an myeloablative conditioning regimen is applied to a subject damaging or killing cells of the subject’s immune system, followed by replacement with hematopoietic stem cells (HSC) from a donor leading to reconstitution of immunological tissues of the recipient subject. Methods for harvesting HSC from a donor, such as from donor bone marrow or donor peripheral blood, and administering the harvested HSC to a patient in need thereof are well known and used. However, a major challenge of HCT is recipients’ posttransplant immunodeficiency, which can pose significant health risks. Thus, fully successful HCT requires T cell immune reconstitution. The thymus generates a broad T cell repertoire, including self-tolerant T cells, but remains sensitive to HCT- and post-HCT related insults, including pre-HCT regimens, corticosteroids, infections, and graft-versus-host disease.

Endogenous thymic repair is often suboptimal, and poor or incomplete thymic recovery is linked to increased infection, relapse, secondary malignancies, and overall mortality in HCT recipients. There is therefore a significant need for HCT methodologies that may permit or promote T cell and thymic regeneration.

[0003] A further difficulty in HCT relates to identification of a donor suitable for a recipient. Availability of allogenic HCT is often preferable and sometimes required over, for example, autologous HCT. However, differences between a donor and a recipient, such as genetic differences, can impair the success of allogenic HCT, including thymic recovery, such as if a donor is or is deemed unsuitable as a donor for a given recipient. A method for successful allogenic HCT is therefore desirable, though difficult to accomplish. Very often, examples or models considered for HCT that may include thymic recovery are based on syngeneic models, where a donor and recipient are substantially genetically identical. Skilled persons are well aware that apparent success of a syngeneic HCT model or method is not a predictor or indicator that a comparable model or method would be successful if translated to allogenic, non-syngeneic HCT. Syngeneic HCT (for example, where a donor and recipient are identical twins) is rarely an option clinically. Thus, there remains a need for successful allogenic HCT that promotes thymic recovery.

[0004] An additional difficulty in identification of an appropriate candidate donor for HCT is limitations relating to donor age. Generally, increased age corresponds to decreased suitability as a donor for HCT. Current recommendations exclude those over the age of 60 years from the pool of HCT donors, in part owing to reduced effectiveness of HCT and thymic recovery for HCT recipients of hematopoietic stem cells (HSC) from such donors. Thus, there is a need for a successful HCT regimen suitable for obtaining HSC from younger as well as older donors. The present disclosure is directed to overcoming these and other deficiencies.

SUMMARY

[0005] In an aspect, provided is a method of promoting thymic recovery in a subject following myeloablative conditioning, including obtaining or having obtained an enriched population of c-Kit-expressing HSC from a population of donor c-Kit-expressing HSC wherein the enriched population of c-Kit-expressing HSC is enriched for HSC expressing a low level of c-Kit, and administering the population of c-Kit-expressing HSC to the subject, wherein the donor is not the subject or an identical sibling of the subject.

[0006] In an example, the myeloablative conditioning may be selected from one or both of irradiation and chemotherapy. Another example further includes administering another population of HSC to the subject, wherein the other population of HSC is not enriched for HSC expressing a low level of c-Kit. In yet another example, the donor is a human of at least 60 years of age. In still another example, the population of c-Kit-expressing hematopoietic stem cells were harvested from one or both of bone marrow of the donor and blood of the donor.

[0007] In a further example, the obtaining or the having obtained includes sorting donor cells and sorting includes fluorescence-activated cell sorting. In yet a further example, the HSC expressing a low level of c-Kit include HSC of a percentage of said population of donor c-Kit expressing HSC having lowest levels of c-Kit expression of said population, wherein the percentage is about 30%. In yet another example, at least about 90% of HSC of the enriched population of c-Kit-expressing HSC express a low level of c-Kit.

[0008] In another example, the subject has a cancer. In an example, the cancer may be selected from acute myeloid leukemia, myelodysplastic syndrome, a myeloproliferative neoplasm, a myelodysplastic and myeloproliferative neoplasm, a lymphoma, a multiple myeloma, and any combination of two or more of the foregoing.

[0009] In still another example, the subject has a hematologic condition. In an example, the hematologic condition may be selected from aplastic anemia, an inherited bone marrow failure, an acquired bone marrow failure, an immunodeficiency, and any combination of two or more of the foregoing. In another example, the subject is a human of at least 40 years of age.

[0010] In another aspect, provided is a method of promoting thymic recovery in a subject following myeloablative conditioning, including obtaining or having obtained an enriched population of c-Kit-expressing HSC from a population of donor c-Kit-expressing HSC wherein the enriched population of c-Kit-expressing HSC is enriched for HSC expressing a low level of c-Kit, and administering the population of c-Kit-expressing HSC to the subject, wherein the donor is a human of at least 60 years of age.

[0011] In an example, the myeloablative conditioning may be selected from one or both of irradiation and chemotherapy. Another example further includes administering another population of HSC to the subject, wherein the other population of HSC is not enriched for HSC expressing a low level of c-Kit. In yet another example, the donor is not the subject or an identical sibling of the subject. In still another example, the population of c-Kit-expressing hematopoietic stem cells were harvested from one or both of bone marrow of the donor and blood of the donor.

[0012] In a further example, the obtaining or the having obtained comprises sorting donor cells and sorting includes fluorescence-activated cell sorting. In still another example, the HSC expressing a low level of c-Kit include HSC of a percentage of said population of donor c- Kit expressing HSC having lowest levels of c-Kit expression of said population, wherein the percentage is about 30%. In yet another example, at least about 90% of HSC of the enriched population of c-Kit-expressing HSC express a low level of c-Kit. [0013] In a further example, the subject has a cancer. Tn an example, the cancer may be selected from acute myeloid leukemia, myelodysplastic syndrome, a myeloproliferative neoplasm, a myelodysplastic and myeloproliferative neoplasm, a lymphoma, a multiple myeloma, and any combination of two or more of the foregoing.

[0014] In yet another example, the subject has a hematologic condition. In an example, the hematologic condition may be selected from aplastic anemia, an inherited bone marrow failure, an acquired bone marrow failure, an immunodeficiency, and any combination of two or more of the foregoing. In an example, the subject is a human of at least 40 years of age.

[0015] In still another aspect, provided is a method of treating cancer in a subject, including obtaining or having obtained an enriched population of c-Kit-expressing HSC from a population of donor c-Kit-expressing HSC wherein the enriched population of c-Kit-expressing HSC is enriched for HSC expressing a low level of c-Kit, and administering the population of c- Kit-expressing HSC to the subject, wherein the donor is not the subject or an identical sibling of the subject and the subject received myeloablative conditioning before the administering.

[0016] In an example, the myeloablative conditioning may be selected from one or both of irradiation and chemotherapy. Another example further includes administering another population of HSC to the subject, wherein the other population of HSC is not enriched for HSC expressing a low level of c-Kit. In yet another example, the donor is a human of at least 60 years of age. In still another example, the population of c-Kit-expressing hematopoietic stem cells were harvested from one or both of bone marrow of the donor and blood of the donor.

[0017] In a further example, the obtaining or the having obtained includes sorting donor cells and sorting includes fluorescence-activated cell sorting. In yet a further example, the HSC expressing a low level of c-Kit include HSC of a percentage of said population of donor c-Kit expressing HSC having lowest levels of c-Kit expression of said population, wherein the percentage is about 30%. In yet another example, at least about 90% of HSC of the enriched population of c-Kit-expressing HSC express a low level of c-Kit.

[0018] In an example, the cancer may be selected from acute myeloid leukemia, myelodysplastic syndrome, a myeloproliferative neoplasm, a myelodysplastic and myeloproliferative neoplasm, a lymphoma, a multiple myeloma, and any combination of two or more of the foregoing. In an example, the subject is a human of at least 40 years of age. [0019] In yet another aspect, provided is a method of treating cancer in a subject, including obtaining or having obtained an enriched population of c-Kit-expressing HSC from a population of donor c-Kit-expressing HSC wherein the enriched population of c-Kit-expressing HSC is enriched for HSC expressing a low level of c-Kit, and administering the population of c- Kit-expressing HSC to the subject, wherein the donor is at a human of least 60 years of age, and the subject received myeloablative conditioning before the administering.

[0020] In an example, the myeloablative conditioning may be selected from one or both of irradiation and chemotherapy. Another example further includes administering another population of HSC to the subject, wherein the other population of HSC is not enriched for HSC expressing a low level of c-Kit. In still another example, the donor is not the subject or an identical sibling of the subject. In yet another example, the population of c-Kit-expressing hematopoietic stem cells were harvested from one or both of bone marrow of the donor and blood of the donor.

[0021] In a further example, the obtaining or the having obtained includes sorting donor cells and sorting includes fluorescence-activated cell sorting. In yet a further example, the HSC expressing a low level of c-Kit include HSC of a percentage of said population of donor c-Kit expressing HSC having lowest levels of c-Kit expression of said population, wherein the percentage is about 30%. In yet another example, at least about 90% of HSC of the enriched population of c-Kit-expressing HSC express a low level of c-Kit.

[0022] In an example, the cancer may be selected from acute myeloid leukemia, myelodysplastic syndrome, a myeloproliferative neoplasm, a myelodysplastic and myeloproliferative neoplasm, a lymphoma, a multiple myeloma, and any combination of two or more of the foregoing. In an example, the subject is a human of at least 40 years of age.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] These and other features, aspects, and advantages of the present disclosure will become better understood when the following detailed description is read with reference to the accompanying drawings, wherein:

[0024] FIGs. 1A and IB show a representative example of obtaining a population of HSC is enriched for HSC expressing a low level of c-Kit. FIG. 1 A shows flow cytometry plots representing the gates used to double FACS-sort c-Kit¹¹¹ and c-Kit¹⁰ HSC from bone marrow magnetically enriched for c-Kit + cells. FIG IB, top, shows a frequency distribution of c-Kit expression levels in HSC, with number of cells represented logarithmically on the X-axis and number of cells represented on the Y-axis. In this example, c-Kit¹⁰ cells were selected as cells falling within the lowest 30.9% of the frequency distribution. The plot in FIG. IB, bottom, shows the top about 30% and bottom about 30% of c-Kit expressors were defined as c-Kit¹¹¹ and c-Kit¹⁰ HSCs, respectively.

[0025] FIGs. 2A and 2B show better T-cell reconstitution post HCT exhibited following transplantation with c-Kit¹⁰ HSCs.

[0026] FIGs. 3A and 3B show better reconstitution of BM lymphoid precursors post HCT exhibited following transplantation with c-Kit¹⁰ HSCs.

[0027] FIGs. 4A and 4B show better thymic recovery independent of donor age following transplantation with c-Kit¹⁰ HSCs. 750 c-Kit¹¹¹ and c-Kit¹⁰ HSCs were double FACS- sorted from either 8-10 wk (FIG. 4A) or 23-24 mo-old (FIG. 4B) C57BL/6 (CD45.2/H-2K^b) mice and competitively transplanted into lethally irradiated BALB/cJ (H-2K^d) recipients with 500,000 competitor bone marrow cells (CD45.1). Thymic cellularity of recipient mice was evaluated at 8 weeks post-transplant. Results are representative of at least two independent experiments and shown as mean ± SEM. n = 9-10 mice. *, P < 0.05; **, P < 0.01.

[0028] FIGs. 5A and 5B show better reconstitution of thymocyte precursors recovery post HCT exhibited following transplantation with c-Kit¹⁰ HSCs.

[0029] FIGs. 6A and 6B show better thymic epithelial cell (TEC) recovery post HCT exhibited following transplantation with c-Kit¹⁰ HSCs.

[0030] FIGs. 7A and 7B illustrate better reconstitution of secondary lymphoid organs (spleen in 9A, lymph nodes in 9B) post HCT following transplantation with c-Kit¹⁰ HSCs from donors (n = 10-11/group).

[0031] FIGs. 7C and 7D illustrate better reconstitution of secondary lymphoid organs (spleen in 11 A, lymph nodes in 1 IB) post HCT following transplantation with c-Kit¹⁰ HSCs from >24 week-old donors (n = 9-10/group).

[0032] FIG. 8 shows better reconstitution of recent thymic emigrants (RTEs) following transplantation with c-Kit¹⁰ HSCs.

[0033] FIG. 9 illustrates c-Kit¹⁰ HSCs have higher lymphoid progenitor potential than c- Kit¹¹¹ HSCs.. [0034] FIG. 10 illustrates better functionality of c-Kit¹⁰ HSC-derived T cells harvested from spleen after listeria inoculation.

[0035] FIG. 11 shows that Old c-Kit¹⁰ HSCs exhibit preserved lymphoid reconstitution potential in bone marrow of middle aged recipients.

[0036] FIG. 12 shows a non-limiting, hypothetical model for HSC subsets with improved thymic recovery and T cell reconstitution potential.

DETAILED DESCRIPTION

[0037] This disclosure relates to a method of promoting thymic recovery in an HCT recipient. HCT refers to a transplantation of multipotent hematopoietic cells, including stem cells, usually derived from bone marrow, peripheral blood, or umbilical cord blood. In HCT, a host patient, or recipient, receives blood infusions or marrow stem cell transplants from a donor. In an example, the subject received myeloablative conditioning before HCT. Myeloablative conditioning includes killing cellular compartments of the subject’s immune system. For example, before receiving HCT, a subject may receive radiation, chemotherapy, or both, in amounts or durations to kill cells of the immune system. Non-exhaustive examples of myeloablative conditioning include, without limitation, BEAM (carmustine, etoposide, cytosine arabinoside, melphalan (Mel)), Cy/TBI (cyclophosphamide/total body irradiation), busulfan at about 12.8 mg/kg (Bu4)/Cy, fludarabine (Flu)/Bu4, high dose Mel, and CBV (carmustine, etoposide, Cy). Different amounts of myeloablative conditioning may be applied depending on the medical needs of the subject, with more or less immunoablation being required for treatment of different conditions, as would be appreciated by skilled persons in the field. Immunoablation may result in prolonged deficiencies in immune responsiveness, even following HCT, including long-term thymus impairment. Disclosed herein are methods and compositions for promoting immunological recovery and thymic health following HCT such as after myeloablative conditioning.

[0038] In some examples, a reduced intensity regimen of myeloablative conditioning may be used. Non-limiting examples of such reduced intensity regimens of myeloablative conditioning include, without limitation, Flu/Mel, Flu/Bu at about 9.6 mg/kg (Bu3), Flu/Bu at about 6.4 mg/kg (Bu2), Flu/Bu3/thiotepa, and Flu/Cy. In some examples, a nonmyeloablative regimen may be substituted in place of a myeloablative conditioning regimen. Non-exhaustive examples of nonmyeloablative regimens that may be substituted for a myeloablative regimen include, without limitation, Flu/TBI, total lymphocyte irradiation (TLI)/ Anti -thymocyte globulin (ATG), and low dose TBI. Other substitutes for myeloablative conditioning include minimal intensity antibody conditioning (e.g., CD45 antibody, alemtuzumab, Flu and low-Cy).

[0039] In allogenic HCT, a patient receives bone marrow or blood stem cells from a tissue-matched or a close matched donor, i.e. matched at major HLA loci, who may or may not be a relative. Identical twin allogeneic transplants are called syngeneic transplants.

[0040] In allogeneic HCT, multipotent hematopoietic stem cells are transplanted from one individual to another, such as, but not exclusively, where host patients have certain cancers of the blood or bone marrow, such as multiple myeloma or leukemia, congenital immunodeficiencies and bone marrow failures or other hematologic disease. In such cases, as well as other HCT cases, the recipient's immune system is usually destroyed with radiation or chemotherapy before the transplantation Methods to promote thymic regeneration follow such transplants are provided herein.

[0041] A value described by the term about followed by a numeral means the value may vary from the numeral by up to ±10% of the numeral and still be considered as within the value described, and includes the value varying from the numeral by 0% of the numeral.

[0042] A subject may be any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. The terms subject and patient may be used interchangeably herein, particularly in reference to a human subject. For the purposes of the present inventions, a subject may be immunocompromised, i .e. not able to fight off infections or control abnormal cell growth. Examples of immunocompromised subjects include subjects that have any of the following conditions, chemotherapy, exposure to radiation, deliberate irradiation, human immunodeficiency virus infections, transplantation, etc.

[0043] The terms treatment, treating, and the like generally mean obtaining a desired pharmacological and/or physiological effect. In relation to a therapeutic treatment of a subject the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of partially or completely curing a disease and/or adverse effect attributed to the disease. Thus treatment refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Subjects in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.

[0044] Treatment covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e. arresting its development; or (c) relieving the disease, i.e. causing regression of the disease. The present invention is directed towards treating patients with medical conditions relating to a loss of immunocompetence from a treatment related to a disease such as irradiation, chemotherapy, immunosuppression, etc. Treatment may include to exposing a subject to a therapy directed towards treating a disease, such as irradiation, chemotherapy, and the like, and receiving HCT. Treatment may also include preventing possible complications or deleterious consequences of HCT per se or other interventions administered in connection with HCT such as radiation, chemotherapy, or other means that impair or destroy cells of the immune system.

[0045] Administering or administration refers to transplanting HSC to a subject, such as through intravenous injection or infusion or other route. HSC may be obtained from a donor, such as from a donor’s bone marrow or a donor’s blood. In an example, HCT may be allogenic HCT, wherein a donor may be another individual, such as an HLA-matched donor. In an example, HCT may be autologous HCT, where a recipient’s own HSC are harvested then administered back to the recipient. In an example, HCT may be syngeneic HCT, where a donor of HCS is genetically identical to a recipient, such as where a donor and recipient are identical twins. Absent statements to the contrary in connection with a given example, all of the foregoing examples of HCT are included as possible types of HCT for which methods are disclosed herein. [0046] An effective amount of a HSC administered by HCT is an amount sufficient to carry out a specifically stated purpose. An effective amount may be determined empirically and in a routine manner, in relation to the stated purpose. In an example, HSC from more than one donor may be administered to a recipient, whether infused together or at different times. In an example, HSC from multiple donors may be pooled to form a population of donor HSC, wherein the pool includes HSC from more than one donor. In another example, a population of donor HSC may include HSC harvested from only one donor.

[0047] Methods for isolating a population of donor HSC from bone marrow or blood harvested from a donor are known. For example, flow cytometry may be used to identify, and isolate or separate from other types of cells, cells that express immunophenotypic signatures characteristic of HSC, discarding cells from a harvested sample that lack such a signature. For example, HSC from mice may be CD34^io/”, SCA-1⁺, C-kit7CDl 17⁺, CD48', CD150⁺, and Lin”, and HSC from humans may be Lin”CD34”, Thyl/CD90⁺, CD38^k!, C-kit/CD117⁺,CD123^low/- ,CD45RA’ and CD49F. Other signatures may also be used for identifying and isolating HSC from within blood or bone marrow tissue harvested from a donor (e.g., LSK CD150⁺CD48‘ CD244-CD229'), as would be known and appreciated by skilled persons. Donor HSC may be stored in a manner appropriate for maintaining cell viability, such as by freezing, in advance of a transplant procedure such as when they are transfused to a recipient.

[0048] A population of donor HSC may be a heterogeneous population of cells, with different HSC within the population expressing different proteins or markers, or different levels of proteins or markers from each other. In some instances, such differences may reflect, cause, or signify different functional propensities for different cells within the population of donor HSC to promote thymic regeneration upon administration to a recipient. As disclosed herein, administering a population of HSC enriched for HSC with a low level of expression of c-Kit (a.k.a. CD117), which is expressed by HSC, surprisingly and significantly promotes recovery of the recipient’s thymus and immunological health.

[0049] Previous studies provided conflicting indications of what role c-Kit does or may have, if any, in promoting successful HCT and, perhaps, thymic and immunological recovery in HCT recipients. c-Kit is a type III tyrosine kinase receptor which binds to its cognate ligand Stem Cell Factor (SCF) by Ig-like extracellular domains (D1-D3). The extracellular D4 domain residue, critical for ligand-induced homodimerization, is conserved across species In some loss of function studies, loss of c-Kit expression or function decreases HSC number and impairs HSC functioning. Administration of anti-c-Kit antibodies to an HCT recipient pre-transplant also mediates in vivo clearance of resident HSCs and may serve as a non-myeloablative conditioning strategy to improve engraftment of donor HSC. In contrast, gain of function mutations in c-Kit promote formation of myeloproliferative neoplasms, indicating deleterious effects of high c-Kit activity. HSC with high levels of c-Kit expression have also been shown to be deficient in potential for self-renewal and to be biased towards development into megakaryocytes in a syngeneic HCT model, as opposed to other myeloid or lymphoid lineages that promote thymic recovery. Prior indications of what role, if any, HSC c-Kit expression and levels thereof may have in immunological and thymic recovery following myeloablative conditioning and HCT, such as allogenic have, therefore been equivocal, inconclusive, and contradictory.

[0050] Surprisingly, disclosed herein is a method of HCT including administering to a recipient, after myeloablative conditioning, a population of HSCs enriched for low c-Kit expression. This promotes thymic seeding, thymic cellularity, and immunological recovery in HCT recipients. Obtaining a population of HSC from a donor, and from said population an enriched population of c-Kit-expressing HSC enriched for HSC expressing low levels of c-Kit, and administering the enriched population via HCT, such as to a recipient who previously received myeloablative conditioning, significantly improves thymic recovery and immunological function in the recipient.

[0051] A level of c-Kit expression in HSC may be determined by identifying a level of c- Kit expression across a population of HSC, ranking HSC according to their levels of c-Kit expression relative to each other and setting a cut-off level of expression whereby a cell that expresses c-Kit at a level below the cut-off level is characterized as expressing low level of c-Kit (c-Kit¹⁰). For example, HSC may be separated by a cell-sorting process, such as fluorescence activated cell sorting (FACS). In an example of FACS, cells may be contacted with one or more fluorescently tagged antigen binding protein such as an antibody, which binds to cells that express a given protein. FACS may be used to separate cells into different populations based on characteristics including which of one or more proteins a cell is determined to express, or not express. For example, FACS may be used to separate bone marrow cells by interrogating individual cells to determine a relative level of each of one or more given antigens it expresses, according to an expression profde, to form a population of cells matching said expression profdes. For example, bone marrow or other cells may be sorted and cells within said population expressing antigens consistent with an expression profile of HSCs may be separated from other cells. Bone marrow or other cells may be harvested from a donor and sorted by FACS to separate HSCs (such as by interrogating bone marrow cells for whether they express the aforementioned positive and negative HSC markers) and isolate cells that fit an HSC expression profile from the remaining bone marrow or other cells to form a population of HSCs.

[0052] In some examples, a source of HSC, such as bone marrow or other HSC source harvested from a donor, may first be subjected to a process for selecting c-Kit expressing cells, such as by a magnetic activated cell sorting (MACS). For example, magnetic nanoparticles bound to c-Kit antibodies may be contacted with cells from a harvested source of HSC, to bind to c-Kit expressing cells within the source, then a magnet used to collect such cells while cells not bound to such magnetic nanoparticles are discarded. Other MACS enrichment processes may be used in other examples to enrich a sample for sorting with HSC, such as lineage depletion or Sea and CD34 enrichment.

[0053] Furthermore, a relative level of expression of c-Kit per isolated HSC in a population of sorted HSCs may be determined, such as during FACS processing. Protein expression profiles of cells in the population of HSC include a level of expression of c-Kit. The population of HSC may be composed of HSC with relatively lower levels of c-Kit expression and relatively higher levels of c-Kit expression. Cells’ expression profiles may reflect a continuum of level of expression of c-Kit per HSC across the population of HSC. As disclosed herein, HSC including a mix of cells with relatively low and relatively high c-Kit expression may be separated into HSC with relatively low levels of c-Kit expression and cells with a relatively high level of c-Kit expression.

[0054] For example, computerized files, generated during cell sorting such as by FACS, representing expression profiles of HSCs in the population may be used to determine a cut-off level of c-Kit expression for determining whether an HSC is a c-Kit¹⁰ cell, expressing a relatively low level of c-Kit, or a moderate or c-Kit¹¹¹ cell, expressing a relatively higher level of c-Kit. In an example, a frequency distribution may be created, indicating how many HSCs of the population of HSCs express a given level of c-Kit. A cut-off value may then be selected, separating c-Kit¹⁰ HSC from c-Kit¹¹¹ HSC. In an example, a cut-off value may be about 30%. That is, a subpopulation of HSC may be identified which includes the about 30% of HSC expressing the lowest level of c-Kit expression. In another example, a higher or lower cut-off that about 30% may be selected. The population of HSC may then be subjected to a round of sorting such as by FACS, setting a selection gate such that cells expressing up to the cut-off value of c-Kit expression are separated from cells expressing more than the cut-off level gating the HSC such that HSC and selecting cells up to the cut-off value as c-Kit¹⁰ cells.

[0055] An example of a frequency distribution used in selecting a cut-off value for separating c-Kit¹⁰ from a population of HSCs is shown in FIG. IB. FIG. IB shows a frequency distribution of a population of HSC expressing levels of c-Kit. Levels of expression of c-Kit are presented on the x-axis, logarithmically, and number of cells on the y-axis. In this example, cells in the about 30% (in this case, 30.9%) area under the curve representing cells with the lowest level of c-Kit expression of the frequency distribution were selected to define c-Kit¹⁰ expressing cells. FACS sorting subsequently was used to separate cells within this population to form a population of c-Kit¹⁰ HSCs, made up of cells represented in the lower 30.9% of the frequency distribution. Cells in the upper about 30% (in this case, upper 30.5%) of the area under the curve were selected to form a population of c-Kit¹¹¹ expressing HSCs. FIGs. 1A and IB further shows a gating strategy for isolating HSC from a population of bone marrow cells, and c-Kit¹⁰ and c-Kit¹¹¹ HSC for isolation therefrom.

[0056] Thus, when collecting a population of HSC from tissue harvested from a donor, FACS may be used to identify and collect HSC preferentially over other cell types. As discussed above, various cellular proteins and patterns of protein expression may signify that a cell within harvested tissue from a donor is a HSC. Fluorescent markers capable of selectively tagging different HSC proteins may be used to identify and separate out HSC from within the tissue sample. In some examples, rounds of FACS may be performed in series, such as when fewer than all proteins to be used to identify and sort HSC are tagged in each round of HSC. In such a case, multiple rounds of FACS may be used, with one protein or some proteins tagged for a first round of FACS, and another or others tagged during a second round of FACS, which round is performed on a population of cells separated during a previous round of FACS. In another example, multiple proteins may be tagged and used to obtain a population of HSC in a single round of FACS. In some cases, c-Kit expression may be used as part of one or more FACS rounds for separating HSC from tissue harvested from a donor. In another example, c-Kit expression might not be used as part of one or more FACS rounds for separating HSC from within tissue harvested from a donor.

[0057] Once a population of donor HSC have been obtained, they may be processed to form an enriched population of c-Kit¹⁰ HSC, enriched for HSC expressing a low level of c-Kit. A level of expression of c-Kit (i.e., level of fluorescence from the fluorescently tagged c-Kit marker used) per cell may be determined, and HSC with a level of c-Kit below a cutoff selected for inclusion in an enriched population. For example, as part of a round of sorting such as by FACS, cells may be contacted with an anti-c-Kit antibody and the antibody linked or otherwise bound to a tag such as a fluorescent tag. Numerous anti-c-Kit antibodies, with numerous fluorescent probes, are known to skilled persons for use in sorting such as FACS. A commercially available or custom-made anti-c-Kit antibody may be used, such as a monoclonal c-Kit antibody. Although different c-Kit antibodies may bind to c-Kit with different affinities from each other, using a given c-Kit antibody for a given sorting process obviates differences in fluorescence per cell that could otherwise be attributed to c-Kit affinities or epitope recognition of different anti-c-Kit antibodies. A skilled person would therefore appreciate that a given c-Kit antibody per se is not an essential requirement to selecting a population of c-Kit¹⁰ cells. Usage of any given c-Kit antibody permits head-to-head comparisons of cells’ c-Kit expression levels, which comparison is used to prepare a population enriched for c-Kit¹⁰ HSC.

[0058] A frequency distribution of c-Kit expressing HSC represents c-Kit expression levels according to a given anti-c-Kit antibody, tagged with a given fluorescent or other tag, and sorting, such as FACS-sorting, parameters used, such as voltage, laser, or other sorting machine settings, during cell sorting. c-Kit¹⁰ cells may be sorted from other c-Kit-expressing cells by comparing c-Kit expression among a population of cells assessed by a given set of measurement parameters applied across interrogation of all cells during sorting. Thus, sorting serves as an internal control, with a low level of c-Kit expression determined according to parameters employed during a sorting process for forming a population enriched for c-Kit¹⁰ HSC.

[0059] A cut-off level may be about 30%, meaning a level that does not exceed the level of c-Kit expression expressed by any cell that is among the sub-population of c-Kit expressing HSC whose level of c-Kit expression is within the about 30% of HSC with the lowest levels of c- Kit. During a sorting round to form an enriched population of c-Kit expressing HSC, cells with more than the cut-off levels, for example within the sub-population of c-Kit-expressing HSC whose expression is within the about 70% of HSC with the highest levels of c-Kit expression, may be excluded from the enriched population.

[0060] As skilled persons would appreciate, a cutoff level may be another level corresponding to a percentage of HSC with the lowest level of c-Kit expression. The cut-off level may be 30% as in the foregoing example. Or, the cut-off level may be lower or higher. In an example, the cut-off level may be about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, or about

70%.

[0061] In another example, an enriched population of c-Kit-expressing HSC may be drawn from a population of donor HSC wherein the population of donor HSC includes HSC from more than one donor. HSC may be collected from bone marrow and/r peripheral blood of one or more donor to form a population of HSC, from which an enriched population of c-Kit- expressing HSC, enriched for HSC with a low level of c-Kit expression, may be sorted to for the enriched population of c-Kit expressing HSC. In an example, a level of c-Kit expression may be determined for the c-Kit expressing HSC in the population of donor HSC, whether from one or pooled from more than one donor. A cut-off level of expression may then be based on said percell level of expression, or average level of expression of the population, in like manner as in the foregoing examples. An enriched population of c-Kit expressing HSC may be selected from the population of donor HSC, whether from one donor or pooled donor HSC based on a cut-off level of expression which cut-off level of expression may be based on c-Kit expression of cells of said population of donor HSC.

[0062] An enriched population of c-Kit-expressing HSC may include HSC with a low level of c-Kit expression, such as may be determined in accordance with the foregoing examples. A sample may be enriched from HSC with a low level of c-Kit expression when at least a certain percentage of cells within the enriched population express a low level of c-Kit. For example, at least about 90% of the cells in the enriched population of c-Kit-expressing HSC may express a low level of c-Kit. In another example, about 100%, at least about 99%, at least about 98%, at least about 97%, at least about 96%, at least about 95%, at least about 94%, at least about 93%, at least about 92%, at least about 91%, at least about 90%, at least about 89%, at least about 88%, at least about 87%, at least about 86%, at least about 85%, at least about 84%, at least about 83%, at least about 82%, at least about 81%, at least about 80%, at least about 79%, at least about 78%, at least about 77%, at least about 76%, or at least about 75% of the cells in an enriched population of c-Kit-expressing HSC may express a low level of c-Kit. [0063] As would be understood by skilled persons, in an example, whether a population of donor HSC is from one donor or pooled from more than one donor, any of the foregoing percentages of cells of an enriched population of c-Kit-expressing HSC may express any of the foregoing low levels of expression of c-Kit, as determined according to any of the foregoing examples for determining a low level of c-Kit expression. All possible permutations of all of the foregoing combinations are hereby expressly contemplated by and included in the present disclosure. In an example, about 90% or more of cells in an enriched population of c-Kit- expressing HSC may express a low level of c-Kit, and the low level may be up to but not more than a level of expression expressed by a c-Kit-expressing HSC of the population of donor HSC from which the enriched population was sorted at a level within the lowest about 30% of per- HSC c-Kit expression levels. In any other example, the minimum percentage of cells may be lower than or higher than about 90%, the low level of c-Kit expression may be lower than or higher than that corresponding to said about 30% cut-off, and any combination of the two parameters may be applied in combination to characterize an enriched population of c-Kit expressing HSC.

[0064] In an example, an HCT recipient may receive an enriched population of c-Kit- expressing HSC, enriched for cells expressing a low level of c-Kit. In some examples, said subject may also receive a transplant of cells, which may include but need not be limited to HSC, that are not enriched for low levels of c-Kit-expression. For example, a recipient may receive a transplant of a number or volume of cells of an enriched population of c-Kit-expressing HSC as disclosed herein, which transplant may provide to the recipient an effective amount of low c-Kit- expressing HSC to promote thymic reconstitution and immunologic health. The recipient may also receive a transplant of a population of, for example, CD34⁺ donor bone marrow cells not necessarily limited to HSC or, enriched for low levels of c-Kit expression, which additional cells may serve a supportive role to the enriched population of c-Kit expressing cells. The additional, e.g. CD34⁺ bone marrow cells may include or provide to the recipient an overall volume or number of HSC as may be required for optimal or a preferred level of engraftment of the donor tissue. For example, a HCT recipient may receive anywhere from, for example, 5-10 million CD34+ cells per kg of recipient’s body mass, in addition to a population enriched for c-Kit¹⁰ according to the present disclosure. In some examples, a recipient may receive fewer than 5 million, or more than 10 million, cells per kg of the recipient’s body mass in addition to a population enriched for c-Kit¹⁰ according to the present disclosure..

[0065] In accordance with the present disclosure, a subject, HCT recipient may receive a transplant including a number, within a range, of HSC enriched for c-Kit¹⁰ HSC, wherein the range may be anywhere from about 0.3 million HSC enriched for c-Kit¹⁰ HSC per kg body mass to about 1.5 million HSC enriched for c-Kit¹⁰ HSC per kg body mass. In another example, a recipient may receive up to more than about 1.5 million HSC enriched for c-Kit¹⁰ HSC per kg body mass, such as up to about 2 million HSC enriched for c-Kit¹⁰ HSC, or up to about 3 million HSC enriched for c-Kit¹⁰ HSC, or up to about 4 million HSC enriched for c-Kit¹⁰ HSC per kg body mass, or more. A recipient may also receive less than about 0.3 million HSC enriched for c- Kit¹⁰ HSC per kilogram body weight.

[0066] For example, a recipient may receive from about 0.3 million HSC enriched for c- Kit¹⁰ HSC per kg body mass to about 1 million HSC enriched for c-Kit¹⁰ HSC per kg body mass. In another example, a recipient may receive from about 0.3 million HSC enriched for c-Kit¹⁰ HSC per kg body mass to about 0.5 million HSC enriched for c-Kit¹⁰ HSC per kg body mass. In another example, a recipient may receive from about 0.5 million HSC enriched for c-Kit¹⁰ HSC per kg body mass to about 1 million HSC enriched for c-Kit¹⁰ HSC per kg body mass. In another example, a recipient may receive from about 1 million HSC enriched for c-Kit¹⁰ HSC per kg body mass to about 1.5 million HSC enriched for c-Kit¹⁰ HSC per kg body mass. In another example, a recipient may receive from about 1 million HSC enriched for c-Kit¹⁰ HSC per kg body mass to about 2 million HSC enriched for c-Kit¹⁰ HSC per kg body mass. In another example, a recipient may receive from about 1.5 million HSC enriched for c-Kit¹⁰ HSC per kg body mass to about 2 million HSC enriched for c-Kit¹⁰ HSC per kg body mass, or from about 2 million HSC enriched for c-Kit¹⁰ HSC per kg body mass to about 2.5 million HSC enriched for c- Kit¹⁰ HSC per kg body mass, or from about 2.5 million HSC enriched for c-Kit¹⁰ HSC per kg body mass to about 3.5 million HSC enriched for c-Kit¹⁰ HSC per kg body mass, or from about 3 million HSC enriched for c-Kit¹⁰ HSC per kg body mass to about 4 million HSC enriched for c- Kit¹⁰ HSC per kg body mass.

[0067] In an example, the enriched population of c-Kit-expressing HSC may be transplanted together or in a single transplant or infusion session along with a non-c-Kit¹⁰- enriched population of donor cells. Such populations of cells may be combined before transplant, in an example. Tn another example, two or more transplant or infusion sessions may occur, with an enriched population of c-Kit^lo-expressing HSC given at one session and a non-enriched population of other donor cells given a another session. In other examples, there may be none, one, or more than one session where only an enriched population of c-Kit^lo-expressing HSC are given to a recipient, and none, one, or more than session where only a non-enriched population of other donor cells is given, and none, one, ore more than one session where both populations are given to the recipient. Different sessions may include the administering of enriched populations of c-Kit^lo-expressing HSC where enriched populations have different minimum percentages of HSC with low levels of c-Kit expression, or cells with a low level of c-Kit expression in different enriched populations may have different maximum levels of c-Kit expression, or any combination of the foregoing, according to all of the aforementioned permutations of these parameters.

[0068] Conventionally, a pool of prospective HSC donors is limited by what has been considered an acceptable upper age limit for a donor. For example, conventionally a prospective human HSC donor may be excluded if the donor is 60 years of age or older. Older donor’s may conventionally have been considered as unsuitable because they may have been believed not to provide a therapeutically sufficient number or type of HSC for a therapeutically effective HCT treatment. As surprisingly disclosed herein, however, older donors may be included as HSC donors for an HCT recipient when an enriched population of c-Kit-expressing HSC, enriched for low-c-Kit-expressing HSC, is selected from a population of donor HSC harvested from an older donor. As disclosed herein, as with enriched populations from younger donors, enriched populations from older donors also promote thymic recovery and immunological health in HCT recipients having received myeloablative conditioning. For example, as disclosed herein, a human donor may be at least 50 years old, or at least 55 years old, or at least 60 years old, or at least 65 years old, or at least 70 years old, or at least 75 years old, or at least 80 years old, or at least 85 years old. Enriched populations of c-Kit expressing HSC, enriched for HSC with low levels of c-Kit expression, may include cells from donors of different ages, including from none, one, or more than one donor younger than the above age ranges and none, one, or more than one donor within any one or more of the above ranges, wherein at least two donors may be of different ages from each other within different age ranges from each other. [0069] Also as disclosed herein, a recipient of low-c-Kit-expressing HSC may be young or middle aged or old. Such transplants, in accordance with aspects of the present disclosure, promote thymic recovery and immunological health in HCT recipients of various ages having received myeloablative conditioning. Recipients may be young, or middle aged, or old, and receive the foregoing benefits of transplantation with low-c-Kit-expressing HSC. A young human recipient may be younger than 40 years of age, a middle aged human recipient may be between 40-60 years of age, and an old human recipient may be older than 60 years of age. For example, a young recipient may be 10 years old or younger, or from 10-15 years old or younger, or from 15-20 years old or younger, or from 20-25 years old or younger, or from 25-30 years old or younger, or from 30-35 years old or younger, or from 35-40 years old or younger, or any overlapping or sub-range within the foregoing ranges. A middle agreed human recipient may be from 40-45 years of age or from 45-50 years of age or from 50-55 years of age or from 55-60 years of age, or any overlapping or sub-range within the foregoing ranges. An old recipient may be from 60-65 years of age or older or from 65-70 years of age or older or from 70-75 years of age or older or from 75-80 years of age or older or from 80-85 years of age or older or from 85- 90 years of age or older, or any overlapping or sub-range within the foregoing ranges.

[0070] As further surprisingly disclosed herein, allogenic HCT may effectively promote thymic reconstitution and regeneration and immunological health when an enriched population of c-Kit-expressing HSC is administered to a recipient, enriched for HSC expressing low levels of c-KIT. For example, a donor may be other than an identical twin of a recipient, and other than the recipient, of HCT (i.e., the HCT may be allogenic, rather than syngeneic or autologous, respectively). Conventionally, thymic reconstitution and immunological recovery following allogenic HCT may be highly unpredictable, often resulting in incomplete or recovery of immune function or resulting in prolonged or long-term compromise of thymic function and immune competency. Surprisingly, as disclosed herein, administering an enriched population of c-Kit-expressing HSC, enriched for HSC with a low level of c-Kit expression, in an allogenic HCT promotes thymic recovery and immune function.

[0071] Combinations of the foregoing features of donors are expressly disclosed and included herein. For example, included herein are all of the foregoing examples of ages of HSC donors, whether young or old, in allogenic HCT. All aforementioned combinations of parameters of characterizing an enriched population of c-Kit-expressing HSC (i.e., what constitutes a low level of c-Kit expression for HSC of a given enriched population of c-Kit-expressing HSC, and what minimum percentage cells in a population of c-Kit-expressing HSC have low-c-Kit- expression in order for the population of cells to be an enriched population) are also expressly contemplated an included in all combinations of features of donors.

[0072] An HCT recipient, receiving an enriched population of c-Kit-expressing HSC, enriched for HSC with a low level of c-Kit expression, may be receiving HCT for treatment of any condition, disorder, or illness requiring HCT as a treatment or for which HCT may be an effective treatment, and myeloablative conditioning may precede HCT. For example, the subject may have cancer. For example, the cancer may be acute myeloid leukemia, myelodysplastic syndrome, a myeloproliferative neoplasm, a myelodysplastic and myeloproliferative neoplasm, a lymphoma, a multiple myeloma, and any combination of two or more of the foregoing. In another example, the subject may have a hematologic condition. For example, the subject may have aplastic anemia, an inherited bone marrow failure, an acquired bone marrow failure, an immunodeficiency, and any combination of two or more of the foregoing.

[0073]

WORKING EXAMPLES

[0074] The following examples are intended to illustrate particular embodiments of the present disclosure, but are by no means intended to limit the scope thereof.

[0075] MATERIAL AND METHODS

[0076] Animal use

[0077] Young mice (6-10 weeks of age): C57BL/6J (CD45.2/H-2Kb), B6.SJL- PtprcaPepcb/BoyJ (CD45.1), BALB/cJ (H-2Kd) mice were purchased from The Jackson Laboratory. Aged C57BL/6 (CD45.2/H-2Kb) mice (23-24 months of age) were obtained from National Institute of Aging (Baltimore, MD). Aged mice (ranging between 18-24 months of age) correlate with humans ranging between 56-69 years of age. Middle-aged BALB/cJ (H-2Kd) mice (ranging between 7-8 months of age) were obtained from were purchased from The Jackson Laboratory. Middle-aged mice (ranging between 7-8 months of age) correlate with humans ranging between 40-60 years of age. RAG2-EGFP-CD45.1 chimeric mice were generated by crossing FVB-Tg(RAG2-EGFP)lMnz/J (JAX 005688) and B6.SJL-PtprcaPepcb/BoyJ (CD45.1, JAX 002014). For consistency all experiments were carried out using only female mice. These mice were allowed to acclimatize in our vivarium for at least 2 weeks before experiments. Mice were euthanized with C0₂ gas inhalation and were maintained under pathogen-free conditions according to an NYU I ACUC -approved protocol.

[0078] Cell Preparation, Staining and FACS-based Purification of Hematopoietic Stem Cells

[0079] Femurs, tibias, pelvises, and the spine were dissected from euthanized mice and cleaned of muscles and connective tissue on ice in IX PBS. Bone marrow (BM) cells were collected by crushing all the bones with a sterile mortar and pestle in FACS buffer: IX PBS containing 2% Fetal Bovine Serum (Hyclone) and 2 mM EDTA. BM cells (young and aged CD45.2) were enriched for c-Kit⁺ cells using mouse CD117 magnetic microbeads

(Miltenyi Biotec) per manufacturer’s protocol, to eliminate c-Kif cells (i.e., mature cells) from the harvested bone marrow, which facilitates downstream cell sorting. A method of eliminating mature cells may also be carried out using Lineage cocktail beads, wherein Lin" cells (immature fraction containing HSCs) is collected for downstream FACS-based sorting. For humans, there is a similar Lineage cocktail which is routinely used to enrich for cells from the stem cell compartment, prior to sorting.

[0080] Collected cells were then stained in PBS/2.5% fetal calf serum with antibodies against lineage (Lin) markers (CD3s, CD4, CD8a, B220, Gr-1, Mac-1 and Teri 19) (PE-Cy7), Sca-1 (BV421), c-Kit (APC), CD150/ SLAM (PE), CD48 (BV510) and CD34 (FITC) for 2 hrs on ice followed by wash in in PBS/2.5% fetal calf serum. Finally, propidium iodide (Molecular Probes) was added as a viability dye before acquiring the data. All monoclonal antibodies were purchased from Biolegend and eBiosciences. Cells were sorted using a FACS Aria II cell sorter (BD Biosciences). The c-Kit/CD117 antibody used was clone 2B8. For all experiments in which c-Kit^hi, c-Kit¹⁰ HSCs were purified, they were double-sorted to ensure >95% purity (meaning such that at least 95% of cells in the population of HSC enriched for c-Kit¹⁰ HSC were c-Kit¹⁰ HSC and at least 95% of cells in the population of HSC enriched for c-Kit¹¹¹ HSC were c- Kit¹¹¹ HSC). Cells were sorted for HSC by FACS according to a gating strategy illustrated in FIGs. 1 A and IB. c-Kit-expressing BM cells were first magnetically enriched for using magnetic activated cell sorting (MACS). The c-Kit-expressing BM cells were stained with 12 antibodies against various cell surface markers to isolate HSCs (Lin~c-Kit⁺Scal’^r [L"S⁺K⁺]CD150’^r CD34' CD48"). Almost all cells in the CDI50⁺CD34‘ gate were collected for HSCs A histogram showing distribution of c-Kit MFI (Mean Fluorescence Intensity) across HSCs (CD150⁺ CD34‘ CD48'LSK) is shown in FIG. IB. Based on MFI, the top about 30% and bottom about 30% c-Kit expressors defined c-Kit¹¹¹ and c-Kit¹⁰ HSCs, respectively, which were sorted based on the indicated gates. The HSC population with the identified subsets (c-Kit¹¹¹ and c-Kit¹⁰ HSCs) is shown in the c-Kit Sca-1 flowplot in FIG. IB, bottom.

[0081] The c-Kit/CDl 17 antibody used here was clone 2B8. Other anti-c-Kit/CDl 17 antibodies are known and may also be used to sort cells based on c-Kit expression within the context of the present disclosure, as would be understood by skilled persons, and any such anti-c- Kit/CDl 17 antibody is explicitly contemplated for inclusion in the method disclosed herein. For example, an antibody directed to one or more extracellular domain of c-Kit (e.g., D1-D5) may be used, for sorting cells that express c-Kit. A non-limiting list of examples of applicable anti-c- Kit/CDl 17 antibodies for detecting c-Kit expression includes monoclonal antibodies to human c- Kit/CD117, such as 104D2, A3C6E2, S18022G, W18195C,YB5.B8, and monoclonal antibodies to mouse c-Kit/CDl 17, such as 2B8, 3C11, ACK2, QA17A09, S18020H.

[0082] Transplantation experiments

[0083] For competitive transplantation assays, double-FACS sorted c-Kit¹¹¹, c-

Kit¹⁰ HSCs cells from young or aged CD45.2 mice were mixed with unfractionated BMMCs from young CD45.1 mice. For RTE experiments, 750 double-FACS sorted c-Kit¹¹¹, c- Kit¹⁰ HSCs cells from aged RAG2-GFP (CD45.1) mice were mixed with 5xl0⁵ unfractionated BMMCs from young CD45.2 mice. Cells were transplanted via retro-orbital sinus injections into lethally irradiated young (6-10 week old) or middle aged (7-8 month old) BALB/cI recipient mice (9 Gy, single dose, using a X-Ray source) under isoflurane anesthesia, within Ih postirradiation.

[0084] Bone Marrow analysis of Hematopoietic Stem and Progenitor Cells

[0085] To calculate bone marrow cellularity and the frequency of hematopoietic precursors, two femurs and two tibias were flushed into FACS buffer. Collected cells were then incubated with red blood cell lysis buffer (ACK lysis buffer, Thermo Fisher Scientific) for 8 minutes and then washed twice with PBS/2.5% FBS. Cells were resuspended and then stained in PBS/2.5% fetal calf serum with fluorochrome-conjugated antibodies

(Biolegend and eBiosciences) against lineage (Lin) markers (CD3s, CD4, CD8a, B220, Gr-1, Mac-1 and Teri 19) (PE-Cy5), Sca-1 (BV711), c-Kit (APC-Cyanine7), CD150/ SLAM (BV605), CD135/Flt3 (BV421 or PE-Cy5), CD127/IL7Ra (APC), CD48 (BV510), CD16/32 (AF-700), CD34 (FITC), CD45.2 (BUV395), CD45.1 (PE-Cy7 or AF-700), H-2K^d (PE) and H-2K^b (BV650 or BV786). For additional thymic adhesion molecule analyses, antibodies against CCR7 (BV421), CCR9 (FITC) and PSGL1 (PE) were used for chimerism studies. Following a 30-45 incubation on ice, cells were washed in in PBS/2.5% fetal calf serum. Finally, propidium iodide (Molecular Probes) was added as a viability dye before acquiring the data. Cells were analyzed or sorted using a FACS LSR II UV (BD Biosciences).

[0086] Peripheral blood analysis

[0087] Peripheral blood samples were collected in 50 mM EDTA solution (Thermo Fisher Scientific) via retro-orbital sinus bleeds. A mixture of 1.2% dextran/0.9% NaCl was then added to the cells and incubated for 45 min at 37°C. Collected cells were then incubated with red blood cell lysis buffer (ACK lysis buffer, Thermo Fisher Scientific) for 8 minutes and then washed twice with PBS/2.5% fetal bovine serum. Cells were resuspended and then stained in PBS/2.5% fetal calf serum with antibodies against CD3 (AF700), B220 (PE-Cy7), Gr-1 (APC), Mac-1 (APC), NK1-1 (BV605), CD45.2 (BUV395), CD45.1 (BV421), H-2K^d (PE) and H-2K^b (FITC) for chimerism studies for 30 mins followed by wash in in PBS/2.5% FBS. Finally, propidium iodide (Molecular Probes) was added as a viability dye before acquiring the data.

Cells were analyzed using a FACS LSR II UV (BD Biosciences).

[0088] Thymus digestion

[0089] All steps were performed at 4 C unless indicated. Primary recipient (BALB/cJ) thymi at 8 wks post-transplant were excised and enzymatically digested following an adapted protocol (Dudakov et al., Science 2012). Briefly, thymi were mechanically dissociated into ca. 2 mm pieces. Tissue pieces were incubated with a digestion buffer (RPMI, 10% FCS, 62.5 um/mL liberase TM, 0.4 mg/ml DNase I) twice for 30 min at 37 C. Between incubation steps, supernatant containing dissociated cells was transferred to 50 mL conical tubes equipped with 100 um fdter.

[0090] Thymus analysis of CD45+ and CD45- compartment

[0091] For thymic immunophenotypic analysis, cells were first incubated with Fc block solution (anti-CD16/CD32 antibody) for 10 min on ice. Discard solution and stain in PBS/2.5% fetal calf serum with fluorochrome-conjugated antibodies against lineage (Lin) markers (CD19, CDl lb, NK1-1, TCRyS, Gr-1, Teri 19) (Biotin), Streptavidin (PE-TexasRed), CD4 (PE-Cy7), CD8 (BV71 1), CD25 (BV510), CD44 (AF700), c-Kit (APC), CD135/Flt3 (BV421), CD45.2 (BUV395), CD45.1 (BV605), H-2K^d (PE) and H-2K^b (FITC). for chimerism studies. To characterize the CD45- compartment, thymic cells were stained with antibodies against UEA-1 (FITC), 6C3 (PE), EPCAM (BV605), PDGFRa (BV421), MHC-II (APC), CD31 (PE-Cy7), CD45 (BUV395) and Ter-119 (PE-Cy5.5) for 30 mins followed by wash in in PBS/2.5% FBS. Antibodies were purchased from either (Biolegend and eBiosciences), except Ulex europaeus agglutinin 1 (UEA-1), conjugated to FITC, was purchased from Vector Laboratories (Burlingame, CA). Finally, 7-AAD (Molecular Probes) was added as a viability dye before acquiring the data. Flow Cytometric analysis was performed on FACS LSR II UV (BD Biosciences).

[0092] Lymphoid Progenitor Assay

[0093] Lymphoid progenitor production from young and old B6 bone marrow was measured by mixing double-FACS sorted 200 c-Kit¹¹¹ and c-Kit¹⁰ HSCs with 50,000 S17 stromal cells in 1.5 mL of methylcellulose (MC) medium. MC medium was prepared by supplementing a-MEM with 30% heat-inactivated FCS, 1% methylcellulose (STEMCELL Technologies), 5x10" ⁵ M 2ME, 2 mM L-glutamine, 50 pg/mL gentamicin, 100 U/mL streptomycin, 100 pg/mL penicillin, 0.1 mM MEM vitamins, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 20 ng/mL stem cell factor, 20 ng/mL Flt3L ligand, and 50 ng/mL IL-7 (all from Biosource). The mixture was plated in non-tissue-culture treated 3.5-cm² dishes (Becton Dickinson). Following 12 days of culture, the contents of the plates were harvested, cells were enumerated, and examined for production of CD45+ CD 127+ CD 135+ CD 19 lymphoid progenitors by flow cytometry. All cultures were placed at 37C, 5% CO2 humidified incubators until processing.

[0094] Statistical Analysis

[0095] Unless otherwise stated, statistical differences between two groups were determined using unpaired Student’s t-tests. Statistical differences between more than two groups were determined by one-way or two-way ANOVA with a Bonferroni post hoc test. P- values between indicated groups are shown in each figure.

[0096] EXAMPLE 1 : c-Kit¹⁰ HSCs Exhibit Better Peripheral Blood T Cell Reconstitution Post HCT.

[0097] 750 c-Kit¹¹¹ and c-Kit¹⁰ HSCs were double FACS-sorted from either 8-10 wk (FIG.

2A) or 23-24 mo-old (FIG. 2B) C57BL/6 (CD45.2/H-2K^b) mice and competitively transplanted into lethally irradiated BALB/cJ (H-2K^d) recipients with 500,000 competitor bone marrow cells (CD45.1). Donor-derived (H2-K^b) chimerism levels of CD45+, Myeloid cells (Grl+CDl lb+), B cells (B220+), and CD3+ T cells were evaluated in the peripheral blood (PB) of primary recipients at 8 weeks post-transplant (BMT). Results are representative of at least two independent experiments and shown as mean ± SEM. n = 9-10 mice. *, P < 0.05; **, P < 0.01. Recipients of c-Kit¹⁰ HSC from 8-10 wk-old or 23-24 mo-old donors exhibited significantly higher percentages of donor-derived CD45+ cells, B cells (B220+), and CD3+ T cells than did recipients of c-Kit¹¹¹ HSC from 8-10 wk-old or 23-24 mo-old donors, respectively. This demonstrates that recipients of c-Kit¹⁰ HSCs exhibit increased T-cell reconstitution which is indicative of better thymic recovery and function.

[0098] EXAMPLE 2: c-Kit¹⁰ HSCs Show Better Reconstitution of BM Lymphoid Precursors Post HCT.

[0099] 750 c-Kit¹¹¹ and c-Kit¹⁰ HSCs were double FACS-sorted from either 8-10 wk (FIG.

3A) or 23-24 mo-old (FIG. 3B) C57BL/6 (CD45.2/H-2K^b) mice and competitively transplanted into lethally irradiated young (6-10 week old) BALB/cJ (H-2K^d) recipients with 500,000 competitor bone marrow cells (CD45.1). Donor-derived (H2-K^b) chimerism of LT HSC (Lineage'Sca-l⁺cKit⁺ CD34'CD48'Flt3'CD150⁺), Myeloid Progenitor cells (Lineage'Sca-l'cKit⁺), Lymphoid-Primed Multipotent Progenitor cells (LMPP: Lineage'Sca-l⁺cKit⁺ Flt3⁺CD150") and Common Lymphoid Progenitors cells (CLP, LineageTL7RaH4t3⁺Sca^mid/10wKit¹⁰) were evaluated in the Bone Marrow (BM) of primary recipients at 8 weeks post-transplant (BMT). Results are representative of at least two independent experiments and shown as mean ± SEM. n = 9-10 mice. *, P < 0.05; **, P < 0.01. Recipients of c-Kit¹⁰ HSC from 8-10 wk-old or 23-24 mo-old donors exhibited significantly higher numbers of donor-derived LT HSC, LMPP, CLP cells than did recipients of c-Kit^lu HSC from 8-10 wk-old or 23-24 mo-old donors, respectively. This demonstrates that recipients of c-Kit¹⁰ HSCs exhibit robust reconstitution of the stem cell compartment, with increased generation of BM lymphoid committed progenitors which repopulate the thymus and promotes recovery.

[0100] EXAMPLE 3 : c-Kit¹⁰ HSCs Exhibit Better Thymic Recovery Independent of Donor Age.

[0101] 750 c-Kit¹¹¹ and c-Kit¹⁰ HSCs were double FACS-sorted from either 8-10 wk (FIG.

4A) or 23-24 mo-old (FIG. 4B) C57BL/6 (CD45.2/H-2K^b) mice and competitively transplanted into lethally irradiated young (6-10 week old) BALB/cJ (H-2K^d) recipients with 500,000 competitor bone marrow cells (CD45. 1). Thymic cellularity of recipient mice was evaluated at 8 weeks post-transplant (BMT). Results are representative of at least two independent experiments and shown as mean ± SEM. n = 9-10 mice. *, P < 0.05; **, P < 0.01. Recipients of c-Kit¹⁰ HSC from 8-10 wk-old or 23-24 mo-old donors exhibited significantly higher levels of thymic cellularity than did recipients of c-Kit¹¹¹ HSC from 8-10 wk-old or 23-24 mo-old donors, respectively. This demonstrates that c-Kit¹⁰ HSCs support better thymic recovery following HCT. [0102] EXAMPLE 4: c-Kit¹⁰ HSCs Exhibit Better Reconstitution of Thymocyte Precursors Recovery Post HCT.

[0103] 750 c-Kit¹¹¹ and c-Kit¹⁰ HSCs were double FACS-sorted from either 8-10 wk (FIG.

5A) or 23-24 mo-old (FIG. 5B) C57BL/6 (CD45.2/H-2K^b) mice and competitively transplanted into lethally irradiated BALB/cJ (H-2K^d) recipients with 500,000 competitor bone marrow cells (CD45.1). Donor-derived (H2-K^b) chimerism of Early Thymic Progenitors (ETP: Lineage’ CD4’ CD8’CD44⁺ CD25’Flt3⁺ c-Kit⁺), DN1 (Lineage CD4’ CD8’CD44⁺ CD25’), DN2 (Lineage CD4’ CD8’CD44⁺ CD25⁺), DN3 (Lineage’ CD4’ CD8’CD44’ CD25⁺), and DN4 (Lineage CD4’ CD8’ CD44’ CD25’) were evaluated in the thymi of primary recipients at 8 weeks post-transplant (BMT). Results are representative of at least two independent experiments and shown as mean ± SEM. n = 9-10 mice. *, P < 0.05; **, P < 0.01. Recipients of c-Kit¹⁰ HSC from 8-10 wk-old or 23-24 mo-old donors exhibited significantly higher numbers of donor-derived ETP, DN1, DN2, DN3, and DN4 cells than did recipients of c-Kit¹¹¹ HSC from 8-10 wk-old or 23-24 mo-old donors, respectively. This demonstrates that increased c-Kit¹⁰ HSC derived thymocyte precursors in c-Kit¹⁰ HSC recipients is indicative of better thymic recovery and function.

[0104] EXAMPLE 5: c-Kit¹⁰ HSCs Recipients Exhibit Better Thymic Epithelial Cells (TEC) Recovery Post HCT.

[0105] 750 c-Kit¹¹¹ and c-Kit¹⁰ HSCs were double FACS-sorted from either 8-10 wk (FIG.

6A) or 23-24 mo-old (FIG. 6B) C57BL/6 (CD45.2/H-2K^b) mice and competitively transplanted into lethally irradiated BALB/cI (H-2K^d) recipients with 500,000 competitor bone marrow cells (CD45.1). Populations within the CD45- compartment, specifically, Endothelial cells (CD45‘ EpCAM’Ter-119 PDGFRa’ CD31⁺), Fibroblasts (CD45’ EpCAM’Ter-l 19’CD3 LPDGFRa⁺), Thymic Epithelial Cells (TEC: CD45’EpCAM⁺), Cortical TEC (cTEC: CD45’ EpCAM⁺UEA-l¹⁰ 6C3¹¹¹ MHCII¹¹¹¹⁰), and Medullary TEC (mTEC: CD45’ EpCAIVEUEA- 1¹¹¹ 6C3¹⁰ MHCII^I1I/Ic”) were evaluated in the thymi of primary recipients at 8 weeks post-transplant (BMT). Results are representative of at least two independent experiments and shown as mean ± SEM. n = 9-10 mice. *, P < 0.05; **, P < 0.01. Recipients of c-Kit¹⁰ HSC from 8-10 wk-old or 23-24 mo-old donors exhibited significantly higher numbers of TEC and mTEC cells than did recipients of c- Kit¹¹¹ HSC from 8-10 wk-old or 23-24 mo-old donors, respectively. Recipients of c-Kit¹⁰ HSC from 8-10 wk-old donors also exhibited significantly higher numbers of endothelial cells (p = 0.05), fibroblasts (p = 0.05), and cTEC cells than did recipients of c-Kit¹¹¹ HSC from 8-10 wk-old donors. This demonstrates that. c-Kit¹⁰ HSCs promote thymic recovery by increased repopulation of thymic epithelial cells of the thymic stromal compartment.

[0106] EXAMPLE 6: Young c-Kit¹⁰ HSCs Exhibit Better Reconstitution of Secondary Lymphoid Organs

[0107] 750 c-Kit¹¹¹ and c-Kit¹⁰ HSCs were double FACS-sorted from young 8-10 wk

(FIGs. 7A and 7B) or 23-24 mo-old (FIG. 7C and 7D) C57BL/6 (CD45.2/H-2K^b) mice and competitively transplanted into lethally irradiated young (6-8 week old) BALB/cJ (H-2K^d) recipients with 500,000 competitor bone marrow cells (CD45.1). Donor-derived (H2-K^b) chimerism levels of CD4+ subsets: CD4+ naive (CD4+CD62L+CD44-), CD4+ Central Memory (CD4+CD62L+CD44+), and CD4+ Effector Memory (CD4+CD62L-CD44+), and of CD8+ subset: CD8+ naive (CD8+CD62L+CD44-), CD8+ Central Memory (CD8+CD62L+CD44+), and CD+ Effector Memory (CD8+CD62L-CD44+) cells from spleen (FIGs. 7A and 7C) and lymph node (FIGs. 7B and 7D) were determined 8 weeks after transplant and % of each cell type that was donor-derived was calculated. Mean ± SEM. n = 10-11 mice. Asterisks indicate p < 0.05 or lower a. Recipients of c-Kit¹⁰ HSC from young donors exhibited significantly higher percentages of donor-derived CD4+, CD4+ naive, and CD8+ naive cells in spleen, and CD4+, CD4+ Central Memory, CD8+, CD8+ naive, and CD8+ Central Memory in lymph node, than did recipients of c-Kit¹¹¹ HSC. This demonstrates that c-Kit¹⁰ HSCs exhibit better reconstitution of secondary lymphoid organs than do c-Kit¹¹¹ HSCs independent of age.

[0108] EXAMPLE 7: c-Kit¹⁰ HSCs Exhibit Better Reconstitution of Recent Thymic Emigrants (RTEs).

[0109] 750 c-Kit¹¹¹ and c-Kit¹⁰ HSCs were double FACS-sorted from 24 mo-old RAG2-

GFP mice and competitively transplanted into lethally irradiated young (6-8 week old) BALB/cJ (H-2K^d) recipients with 500,000 competitor bone marrow cells (CD45.1). Donor-derived (CD45 1 -GFP) chimerism of RTEs (CD3+ T cells) were evaluated in the peripheral blood (PB) of primary recipients at 8 weeks post-transplant (BMT). Results are shown in FIG. 8 as mean ± SEM. n = 5-6 mice. *, P < 0.05; **, P < 0.0E By 8 weeks post-BMT, a higher percentage of T cells (PB) of recipients of c-Kit¹⁰ HSC than of T cells of recipients of c-Kit¹¹¹ HSC were CD3+ T cells. This demonstrates that increased c-Kit¹⁰ HSCs derived recent thymic emigrants (RTEs), or de novo generated T cells, is secondary to better thymic recovery and function.

[0110] EXAMPLE 8: c-Kit¹⁰ HSCs Have Higher Lymphoid Progenitor Potential. [0111] 200 c-Kit¹¹¹ and c-Kit¹⁰ HSCs from young (10-12wk) or old (23-24mo) C57BL/6 were double FACS sorted into cytokine supplemented methylcellulose media, and lymphoid progenitor (CD45⁺CD127⁺CD135⁺CD19‘) forming potential was assessed by flow cytometry after 12d of in vitro culture. Results as shown in FIG. 9 are representative of at least four independent experiments and shown as mean ± SEM. n = 5-7. *, P < 0.05; **, P < 0 01. A higher percentage of live cells of recipients of c-Kit¹⁰ HSC than of live cells of recipients of c-Kit¹¹¹ HSC were lymphoid progenitors, whether the donors were 8-10 wk-old or 23-24 mo-old. This demonstrates that c-Kit¹⁰ HSCs exhibit higher generation of lymphoid progenitors, similar to in- vivo findings disclosed herein, supporting thymic recovery.

[0112] EXAMPLE 9: c-Kit¹⁰ HSCs Derived T cells are Better Functionally [0113] 750 c-Kit¹¹¹ and c-Kit¹⁰ HSCs were double FACS-sorted from either 8-10-week- old OT-1 (Va2+Vb5+/CD45.2) mice (FIG. 10) and competitively transplanted into lethally irradiated young (6-8 week old) BALB/cJ (H-2K^d) recipients with 500,000 competitor bone marrow cells (CD45.1). 8-week post-transplant (BMT), 10000 recipient splenocytes were adoptively transferred into young (10-12wk) C57BL/6 secondary recipients. 24 hours posttransfer, mice were injected with the bacteria L. monocytogenes with the OVA construct (LVOVA i to evaluate functional CD8+ T cell response to primary infection. OT1 -derived (Va2+Vb5/H2-K^b) chimerism of CD8+ T cells was evaluated in the Spleen of secondary recipients 7 days post-infection. 8 weeks post-transplant (BMT). Results as shown in FIG. 10 represent at least two independent experiments and are shown as mean ± SEM. n = 5 mice. *, P < 0.05; **, P < 0.01. Secondary recipients of c-Kit¹⁰ HSC- derived T cells showed a significantly robust CD8 response to primary infection. This demonstrates that recipients of c-Kit¹⁰ HSCs exhibit functionally better T cells. [01 14] EXAMPLE 10: Old c-Kit¹⁰ ETSCs Exhibit Preserved Lymphoid Reconstitution Potential in Bone Marrow of Middle Aged Recipients

[0115] 750 c-Kit¹¹¹ and c-Kit¹⁰ HSCs were double FACS-sorted from 23-24 mo-old

C57BL/6 (CD45.2/H-2K^b) mice were competitively transplanted into lethally irradiated BALB/cJ (H-2K^d) recipients with 500,000 competitor bone marrow cells (CD45.1). Populations within the CD45- compartment, specifically, Endothelial cells (CD45'EpCAM'Ter-l 19 PDGFRa CD31⁺), Fibroblasts (CD45'EpCAM'Ter-119'CD3 rPDGFRa⁺), Thymic Epithelial Cells (TEC: CD45'EpCAM⁺), Cortical TEC (cTEC: CD45'EpCAM⁺UEA-l¹⁰ 6C3^hi MHCII¹¹' ¹⁰), Age-associated TEC (aaTEC: CD45'EpCAM⁺UEA-L 6C3' MHCII¹¹¹⁷¹⁰) and Medullary TEC (mTEC: CD45" EpC AM^UEA-1¹¹¹ 6C3¹⁰ MHCII¹¹¹⁷¹⁰') were evaluated in the thymi of primary recipients at 8 weeks post-transplant (BMT). Results as shown in FIG.11 are representative of at least two independent experiments and shown as mean ± SEM. n = 5-6 mice. *, P < 0.05; **, P < 0.01. Recipients of c-Kit¹⁰ HSC from 23-24 mo-old donors exhibited significantly higher numbers of cTEC with reduction in age-associated TECs. This demonstrates that. c-Kit¹⁰ HSCs promote rejuvenation in aged thymi by robust recovery of the thymic stromal compartment.

[0116] FIG. 12 shows a non-limiting, hypothetical model for HSC subsets with improved thymic recovery and T cell reconstitution potential. Varying c-Kit expressing HSC subsets have differential thymic recovery and T cell reconstituting capacity. c-Kit¹⁰ HSCs exhibit higher lymphoid progenitor potential which support better thymic recovery and T cell reconstitution post-transplant. In contrast, c-Kit¹¹¹ HSCs have lower lymphoid progenitor potential with decreased thymic recovery and T cell reconstitution post-transplant.

[0117] FURTHER EXAMPLES

[0118] In an additional aspect, provided is an enriched population of c-Kit-expressing HSC for use in promoting thymic recovery in a subject following myeloablative conditioning, wherein the enriched population of c-Kit-expressing HSC was obtained from a population of donor c-Kit-expressing HSC and the enriched population of c-Kit-expressing HSC is enriched for HSC expressing a low level of c-Kit, wherein the donor is not the subject or an identical sibling of the subject.

[0119] In an example, the myeloablative conditioning is selected from one or both of irradiation and chemotherapy. In another example, the subject is further administered another population of HSC, wherein the other population of HSC is not enriched for HSC expressing a low level of c-Kit. Tn yet another example, the donor is a human of at least 60 years of age. Tn still another example, the population of c-Kit-expressing hematopoietic stem cells were harvested from one or both of bone marrow of the donor and blood of the donor.

[0T20] In a further example, the enriched population of c-Kit-expressing HSC was obtained by sorting donor cells and sorting includes fluorescence-activated cell sorting. In yet a further example, the HSC expressing a low level of c-Kit include HSC of a percentage of said population of donor c-Kit expressing HSC having lowest levels of c-Kit expression of said population, wherein the percentage is about 30%. In yet another example, at least about90% of HSC of the enriched population of c-Kit-expressing HSC express a low level of c-Kit.

[0121] In another example, the subject has a cancer. In an example, the cancer is selected from acute myeloid leukemia, myelodysplastic syndrome, a myeloproliferative neoplasm, a myelodysplastic and myeloproliferative neoplasm, a lymphoma, a multiple myeloma, and any combination of two or more of the foregoing.

[0122] In still another example, the subject has a hematologic condition. In an example, the hematologic condition is selected from aplastic anemia, an inherited bone marrow failure, an acquired bone marrow failure, an immunodeficiency, and any combination of two or more of the foregoing. In an example, the subject is a human of at least 40 years of age.

[0123] In an additional aspect, provided is an enriched population of c-Kit-expressing HSC for use in promoting thymic recovery in a subject following myeloablative conditioning, wherein the enriched population of c-Kit-expressing HSC was obtained from a population of donor c-Kit-expressing HSC and the enriched population of c-Kit-expressing HSC is enriched for HSC expressing a low level of c-Kit, wherein the donor is a human of at least 60 years of age. [0124] In an example, the myeloablative conditioning is selected from one or both of irradiation and chemotherapy. In another example, the subject is further administered another population of HSC, wherein the other population of HSC is not enriched for HSC expressing a low level of c-Kit. In yet another example, the donor is not the subject or an identical sibling of the subject. In still another example, the population of c-Kit-expressing hematopoietic stem cells were harvested from one or both of bone marrow of the donor and blood of the donor.

[0125] In a further example, the enriched population of c-Kit-expressing HSC was obtained by sorting donor cells and sorting includes fluorescence-activated cell sorting. In yet a further example, the HSC expressing a low level of c-Kit include HSC of a percentage of said population of donor c-Kit expressing HSC having lowest levels of c-Kit expression of said population, wherein the percentage is about 30%. In yet another example, at least about 90% of HSC of the enriched population of c-Kit-expressing HSC express a low level of c-Kit.

[0126] In another example, the subject has a cancer. In an example, the cancer is selected from acute myeloid leukemia, myelodysplastic syndrome, a myeloproliferative neoplasm, a myelodysplastic and myeloproliferative neoplasm, a lymphoma, a multiple myeloma, and any combination of two or more of the foregoing.

[0127] In still another example, the subject has a hematologic condition. In an example, the hematologic condition is selected from aplastic anemia, an inherited bone marrow failure, an acquired bone marrow failure, an immunodeficiency, and any combination of two or more of the foregoing. In an example, the subject is a human of at least 40 years of age.

[0128] In an additional aspect, provided is an enriched population of c-Kit-expressing HSC for use in treating cancer in a subject, wherein the enriched population of c-Kit-expressing HSC was obtained from a population of donor c-Kit-expressing HSC and the enriched population of c-Kit-expressing HSC is enriched for HSC expressing a low level of c-Kit, wherein the donor is not the subject or an identical sibling of the subject and the subject received myeloablative conditioning before the administering.

[0129] In an example, the myeloablative conditioning is selected from one or both of irradiation and chemotherapy. In another example, the subject is further administered another population of HSC, wherein the other population of HSC is not enriched for HSC expressing a low level of c-Kit. In yet another example, the donor is a human of at least 60 years of age. In still another example, the population of c-Kit-expressing hematopoietic stem cells were harvested from one or both of bone marrow of the donor and blood of the donor.

[0130] In a further example, the enriched population of c-Kit-expressing HSC was obtained by fluorescence-activated cell sorting. In yet a further example, the HSC expressing a low level of c-Kit include HSC of a percentage of said population of donor c-Kit expressing HSC having lowest levels of c-Kit expression of said population, wherein the percentage is about 30%. In yet another example, at least about 90% of HSC of the enriched population of c-Kit- expressing HSC express a low level of c-Kit.

[0131] In an example, the cancer is selected from acute myeloid leukemia, myelodysplastic syndrome, a myeloproliferative neoplasm, a myelodysplastic and myeloproliferative neoplasm, a lymphoma, a multiple myeloma, and any combination of two or more of the foregoing. In an example, the subject is a human of at least 40 years of age.

[0132] In an additional aspect, provided is an enriched population of c-Kit-expressing HSC for use in treating cancer in a subject, wherein the enriched population of c-Kit-expressing HSC was obtained from a population of donor c-Kit-expressing HSC and the enriched population of c-Kit-expressing HSC is enriched for HSC expressing a low level of c-Kit, wherein the donor is a human of at least 6-0 years of age and the subject received myeloablative conditioning before the administering.

[0133] In an example, the myeloablative conditioning is selected from one or both of irradiation and chemotherapy. In another example, the subject is further administered another population of HSC, wherein the other population of HSC is not enriched for HSC expressing a low level of c-Kit. In yet another example, the donor is not the subject or an identical sibling of the subject. In still another example, the population of c-Kit-expressing hematopoietic stem cells were harvested from one or both of bone marrow of the donor and blood of the donor.

[0134] In a further example, the enriched population of c-Kit-expressing HSC was obtained by fluorescence-activated cell sorting. In yet a further example, the HSC expressing a low level of c-Kit include HSC of a percentage of said population of donor c-Kit expressing HSC having lowest levels of c-Kit expression of said population, wherein the percentage is about 30%. In yet another example, at least about 90% of HSC of the enriched population of c-Kit- expressing HSC express a low level of c-Kit.

[0135] In an example, the cancer is selected from acute myeloid leukemia, myelodysplastic syndrome, a myeloproliferative neoplasm, a myelodysplastic and myeloproliferative neoplasm, a lymphoma, a multiple myeloma, and any combination of two or more of the foregoing. In an example, the subject is a human of at least 40 years of age.

[0136] Although some non-limiting examples have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the present disclosure and these are therefore considered to be within the scope of the present disclosure as defined in the claims that follow.

[0137] It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail herein (provided such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein. Tn particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as being part of the inventive subject matter disclosed herein and may be used to achieve the benefits and advantages described herein.

Claims

WHAT IS CLAIMED IS:

1. A method of promoting thymic recovery in a subject following myeloablative conditioning, comprising obtaining or having obtained an enriched population of c-Kit-expressing hematopoietic stem cells (HSC) from a population of donor c-Kit-expressing HSC wherein the enriched population of c-Kit-expressing HSC is enriched for HSC expressing a low level of c-Kit, and administering the enriched population of c-Kit-expressing HSC to the subject, wherein the donor is not the subject or an identical sibling of the subject.

2. The method of claim 1, wherein the myeloablative conditioning is selected from one or both of irradiation and chemotherapy.

3. The method of claim 1 or 2, further comprising administering another population of HSC to the subject, wherein the other population of HSC is not enriched for HSC expressing a low level of c-Kit.

4. The method of any one of claims 1 through 3, wherein the donor is a human of at least 60 years of age.

5. The method of any one of claims 1 through 4, wherein the population of donor c- Kit-expressing HSC was harvested from one or both of bone marrow of the donor and blood of the donor.

6. The method of any one of claims 1 through 5, wherein the obtaining or the having obtained comprises sorting donor cells and sorting comprises fluorescence-activated cell sorting.

7. The method of any one of claims 1 through 6, wherein the HSC expressing a low level of c-Kit comprise HSC of a percentage of said population of donor c-Kit expressing HSC having lowest levels of c-Kit expression of said population, wherein the percentage is about 30%.

8. The method of any one of claims 1 through 7, wherein at least about 90% of HSC of the enriched population of c-Kit-expressing HSC express a low level of c-Kit.

9. The method of any one of claims 1 through 8, wherein the subject has a cancer.

10. The method of claim 9, wherein the cancer is selected from acute myeloid leukemia, myelodysplastic syndrome, a myeloproliferative neoplasm, a myelodysplastic and myeloproliferative neoplasm, a lymphoma, a multiple myeloma, and any combination of two or more of the foregoing.

1 1 . The method of any one of claims 1 through 8, wherein for the subject has a hematologic condition.

12. The method of claim 11, wherein the hematologic condition is selected from aplastic anemia, an inherited bone marrow failure, an acquired bone marrow failure, an immunodeficiency, and any combination of two or more of the foregoing.

13. The method of any one of claims 1 through 12, wherein the subject is a human of at least 40 years of age.

14. A method of promoting thymic recovery in a subject following myeloablative conditioning, comprising obtaining or having obtained an enriched population of c-Kit-expressing hematopoietic stem cells (HSC) from a population of donor c-Kit-expressing HSC wherein the enriched population of c-Kit-expressing HSC is enriched for HSC expressing a low level of c-Kit, and administering the enriched population of c-Kit-expressing HSC to the subject, wherein the donor is a human of at least 60 years of age.

15. The method of claim 14, wherein the myeloablative conditioning is selected from one or both of irradiation and chemotherapy.

16. The method of claim 14 or 15, further comprising administering another population of HSC to the subject, wherein the other population of HSC is not enriched for HSC expressing a low level of c-Kit.

17. The method of any one of claims 14 through 16, wherein the donor is not the subject or an identical sibling of the subject.

18. The method of any one of claims 14 through 17, wherein the population of donor c-Kit-expressing HSC were harvested from one or both of bone marrow of the donor and blood of the donor.

19. The method of any one of claims 14 through 18, wherein the obtaining or the having obtained comprises sorting donor cells and sorting comprises fluorescence-activated cell sorting.

20. The method of any one of claims 14 through 19, wherein the HSC expressing a low level of c-Kit comprise HSC of a percentage of said population of donor c-Kit expressing HSC having lowest levels of c-Kit expression of said population, wherein the percentage is about 30%.

21 . The method of any one of claims 14 through 20, wherein at least about 90% of HSC of the enriched population of c-Kit-expressing HSC express a low level of c-Kit.

22. The method of any one of claims 14 through 21, wherein the subject has a cancer.

23. The method of claim 22, wherein the cancer is selected from acute myeloid leukemia, myelodysplastic syndrome, a myeloproliferative neoplasm, a myelodysplastic and myeloproliferative neoplasm, a lymphoma, a multiple myeloma, and any combination of two or more of the foregoing.

24. The method of any one of claims 14 through 21, wherein for the subject has a hematologic condition.

25. The method of claim 24, wherein the hematologic condition is selected from aplastic anemia, an inherited bone marrow failure, an acquired bone marrow failure, an immunodeficiency, and any combination of two or more of the foregoing.

26. The method of any one of claims 1 through 12, wherein the subject is a human of at least 40 years of age.

27. A method of treating cancer in a subject, comprising obtaining or having obtained an enriched population of c-Kit-expressing hematopoietic stem cells (HSC) from a population of donor c-Kit-expressing HSC wherein the enriched population of c-Kit-expressing HSC is enriched for HSC expressing a low level of c-Kit, and administering the enriched population of c-Kit-expressing HSC to the subject, wherein the donor is not the subject or an identical sibling of the subject and the subject received myeloablative conditioning before the administering.

28. The method of claim 27, wherein the myeloablative conditioning is selected from one or both of irradiation and chemotherapy.

29. The method of claim 27 or 28, further comprising administering another population of HSC to the subject, wherein the other population of HSC is not enriched for HSC expressing a low level of c-Kit.

30. The method of any one of claims 27 through 29, wherein the donor is a human of at least 60 years of age.

31. The method of any one of claims 27 through 30, wherein the population of donor c-Kit-expressing HSC were harvested from one or both of bone marrow of the donor and blood of the donor.

32. The method of any one of claims 27 through 31 , wherein the obtaining or the having obtained comprises sorting donor cells and sorting comprises fluorescence-activated cell sorting.

33. The method of any one of claims 27 through 32, wherein the HSC expressing a low level of c-Kit comprise HSC of a percentage of said population of donor c-Kit expressing HSC having lowest levels of c-Kit expression of said population, wherein the percentage is about 30%.

34. The method of any one of claims 27 through 33, wherein at least about 90% of HSC of the enriched population of c-Kit-expressing HSC express a low level of c-Kit.

35. The method of any one of claims 27 through 34, wherein the cancer is selected from acute myeloid leukemia, myelodysplastic syndrome, a myeloproliferative neoplasm, a myelodysplastic and myeloproliferative neoplasm, a lymphoma, a multiple myeloma, and any combination of two or more of the foregoing.

36. The method of any one of claims 27 through 35, wherein the subject is a human of at least 40 years of age.

37. A method of treating cancer in a subject, comprising obtaining or having obtained an enriched population of c-Kit-expressing hematopoietic stem cells (HSC) from a population of donor c-Kit-expressing HSC wherein the enriched population of c-Kit-expressing HSC is enriched for HSC expressing a low level of c-Kit, and administering the enriched population of c-Kit-expressing HSC to the subject, wherein the donor is a human of at least 60 years of age, and the subject received myeloablative conditioning before the administering.

38. The method of claim 37, wherein the myeloablative conditioning is selected from one or both of irradiation and chemotherapy.

39. The method of claim 37 or 38, further comprising administering another population of HSC to the subject, wherein the other population of HSC is not enriched for HSC expressing a low level of c-Kit.

40. The method of any one of claims 37 through 39, wherein the donor is not the subject or an identical sibling of the subject.

41 . The method of any one of claims 37 through 40, wherein the population of donor c-Kit-expressing HSC were harvested from one or both of bone marrow of the donor and blood of the donor.

42. The method of any one of claims 37 through 41, wherein the obtaining or the having obtained comprises sorting donor cells and sorting comprises fluorescence-activated cell sorting.

43. The method of any one of claims 37 through 42, wherein the HSC expressing a low level of c-Kit comprise HSC of a percentage of said population of donor c-Kit expressing HSC having lowest levels of c-Kit expression of said population, wherein the percentage is about 30%.

44. The method of any one of claims 37 through 43, wherein at least about 90% of HSC of the enriched population of c-Kit-expressing HSC express a low level of c-Kit.

45. The method of claims 37 through 44, wherein the cancer is selected from acute myeloid leukemia, myelodysplastic syndrome, a myeloproliferative neoplasm, a myelodysplastic and myeloproliferative neoplasm, a lymphoma, a multiple myeloma, and any combination of two or more of the foregoing.

46. The method of any one of claims 37 through 45, wherein the subject is a human of at least 40 years of age.