Compositions and methods for hematopoietic stem and progenitor cell expansion

Abstract

The invention provides compositions and methods for the ex vivo expansion and maintenance of mammalian hematopoietic stem and progenitor cells (HSPCs), including human HSPCs.

Description

COMPOSITIONS AND METHODS FOR HEMATOPOIETIC STEM AND PROGENITORCELL EXPANSION

[0001] This invention was made with Government support under contract DK116944 awarded by the National Institutes of Health. The Government has certain rights in the invention.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. provisional application No. 63 / 610,125 filed December 14, 2023, which is incorporated herein by reference in its entirety.FIELD

[0003] The invention provides compositions and methods for the ex vivo expansion and maintenance of mammalian hematopoietic stem and progenitor cells (HSPCs), including human HSPCs.BACKGROUND

[0004] Hematopoietic stem and progenitor cells, collectively “HSPCs”, constitute a population of cells with the ability to differentiate into all peripheral blood cells, including cells of both myeloid and lymphoid lineage. HSPCs comprise three subtypes of cells, referred to as the long-term hematopoietic stem cell (LT-HSC), short-term hematopoietic stem cell (ST- HSPC), and the multipotcnt progenitor cell (MPP). The MPP population in turn gives rise to the common lymphoid progenitors (CLPs) and common myeloid progenitors (CMPs). CLPs differentiate into T cells, natural killer cells, and B cells while CMPs give rise to erythrocytes, platelets, neutrophils, and macrophages. Both CLPs and CMPs may give rise to dendritic cells.

[0005] Due to their ability to differentiate into myriad hematopoietic cell types, HSPCs are sought after as cell therapy in the treatment of various diseases and disorders where they may be used in the therapeutic replacement or reconstitution of particular types of hematopoietic cells or even a patient's entire hematopoietic system, e.g., via bone marrow transplantation procedures.

[0006] Unfortunately, human HSPCs represent a small proportion of cells in the body and difficulties in their ex vivo expansion and maintenance has limited clinical translation. Moreefficient methods for culturing HSPCs are needed in order to realize the clinical potential of these rare cells. The present invention address this need.BRIEF SUMMARY

[0007] The present invention relates to compositions and methods for hematopoietic stem cell and hematopoietic progenitor cell (HSPC) expansion and maintenance in long-term culture.

[0008] Provided is a defined hematopoietic stem and progenitor cell (HSPC) culture medium comprising a basal medium and an amount of lusutrombopag. In aspects, the amount of lusutrombopag is from about 20-100 nM. In aspects, the medium comprises stem cell factor, optionally wherein the amount of stem cell factor is from about 2.5-25 ng / ml. In aspects, the medium comprises one or more of insulin, transferrin, selenium, ethanolamine, penicillin, streptomycin, and L-glutamine. In aspects, the medium comprises a human hematopoietic stem cell self-renewal agonist. In aspects, the medium comprises a solubilizing agent, optionally wherein the solubilizing agent is an amphiphilic polymer. In aspects, the medium comprises the solubilizing agent such as polyvinyl caprolactam-polyvinyl acetate-polyethylene or polyvinyl alcohol.

[0009] Also provided is a defined hematopoietic stem and progenitor cell (HSPC) culture medium comprising a basal medium and from about 20-40 nM or 25-35 nM lusutrombopag, from about 5-15 ng / ml or 8-12 ng / ml stem cell factor, and one or more of insulin, transferrin, sodium selenite, ethanolamine, penicillin, streptomycin, and L-glutamine. In aspects, the medium comprises a human hematopoietic stem cell self-renewal agonist and / or a solubilizing agent. In aspects, the medium comprises a solubilizing agent such as polyvinyl caprolactampolyvinyl acctatc-polycthylcnc or polyvinyl alcohol.

[0010] In accordance with any of the foregoing aspects, the basal medium may be minimal essential medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), or Iscove's Modified Dulbecco's Medium (IMDM).

[0011] In aspects of any of the foregoing, the medium is substantially free of one or more of albumin, animal serum, polyvinyl alcohol, butyzamide, and / or a phosphatidylinositol 3-kinase (PI3K) activator.

[0012] Also provided are methods for culturing mammalian hematopoietic stem and progenitor cells (HSPCs), the method comprising culturing the HSPC in a chemically defined basal medium comprising an amount of lusutrombopag. In aspects, culturing the HSPCscomprises maintaining the HSPCs in a controlled environment of about 37 °C in an atmosphere of about 5-10 % CO2 and about 3-20 % O2. In aspects, the amount of lusutrombopag is from about 20-100 nM. In aspects, the medium comprises stem cell factor, optionally wherein the amount of stem cell factor is from about 2.5-25 ng / ml. In aspects, the medium comprises one or more of insulin, transferrin, selenium, ethanolamine, penicillin, streptomycin, and L-glutamine. In aspects, the medium comprises a human hematopoietic stem cell self-renewal agonist. In aspects, the medium comprises a solubilizing agent, optionally wherein the solubilizing agent is an amphiphilic polymer. In aspects, the medium comprises a solubilizing agent such as polyvinyl caprolactam-polyvinyl acetate-polyethylene or polyvinyl alcohol. In aspects, the medium comprises from about 20-40 nM or 25-35 nM lusutrombopag, from about 5-15 ng / ml or 8-12 ng / ml stem cell factor, and one or more of insulin, transferrin, sodium selenite, ethanolamine, penicillin, streptomycin, and L-glutamine.

[0013] In accordance with any of the foregoing methods, culturing the HSPC results in an increase in the number of HSPCs over a period of time. In aspects, the period of time is 7 days, 14 days, or 28 days. In aspects, the period of time is 28 days.

[0014] Also provided is a method for long term culture of mammalian hematopoietic stem and progenitor cells (HSPCs), the method comprising culturing the HSPCs in a chemically defined basal medium comprising an amount of lusutrombopag for at least 28 days. In aspects, culturing the HSPC results in an increase in the number of HSPCs. In aspects, culturing the HSPCs comprises maintaining the HSPCs in a controlled environment of about 37 °C in an atmosphere of about 5-10 % CO2 and about 3-20 % O2. In aspects, the amount of lusutrombopag is from about 20-100 nM. In aspects, the medium comprises stem cell factor, optionally wherein the amount of stem cell factor is from about 2.5-25 ng / ml. In aspects, the medium comprises one or more of insulin, transferrin, selenium, ethanolamine, penicillin, streptomycin, and L-glutamine. In aspects, the medium comprises a human hematopoietic stem cell self-renewal agonist. In aspects, the medium comprises a solubilizing agent, optionally wherein the solubilizing agent is an amphiphilic polymer. In aspects, the medium comprises the solubilizing agent such as polyvinyl caprolactam-polyvinyl acetate-polyethylene or polyvinyl alcohol. In aspects, the medium comprises from about 20-40 nM or 25-35 nM lusutrombopag, from about 5-15 ng / ml or 8-12 ng / ml stem cell factor, and one or more of insulin, transferrin, sodium selenite, ethanolamine, penicillin, streptomycin, and L-glutamine.

[0015] In accordance with any of the foregoing methods, the basal medium may be minimal essential medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), or Iscove's Modified Dulbecco's Medium (IMDM).

[0016] In accordance with any of the foregoing methods, the medium is substantially free of one or more of albumin, animal serum, polyvinyl alcohol, butyzamide, and / or a phosphatidylinositol 3-kinase (PI3K) activator.

[0017] Also provided is a method for ex vivo production of lymphoid cells, the method comprising culturing mammalian hematopoietic stem and progenitor cells (HSPCs) in a chemically defined basal medium comprising an amount of lusutrombopag and an amount of stem cell factor. In aspects, the lymphoid cells are selected from one or more of T cells, natural killer cells, B cells, and dendritic cells.

[0018] Also provided is a method for ex vivo production of myeloid cells, the method comprising culturing mammalian hematopoietic stem and progenitor cells (HSPCs) in a chemically defined basal medium comprising an amount of lusutrombopag and an amount of stem cell factor. In aspects, the myeloid cells are selected from one or more of erythrocytes, platelets, neutrophils, macrophages, and dendritic cells.

[0019] Also provided is a method for expanding a population of mammalian hematopoietic stem and progenitor cells (HSPCs) ex vivo, the method comprising culturing the HSPCs in a chemically defined basal medium comprising an amount of lusutrombopag and an amount of stem cell factor. In aspects, the culturing is performed for a period of from 7-28 days, preferably at least 14-28 days. In aspects, the mammalian HSPCs are human HSPCs.

[0020] Other technical features may be readily apparent to one skilled in the art from the following figures, descriptions, and claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1A is a line graph showing total viable cells (cell number) on day 7 of culture with the indicated amounts of TPO agonist (nM), either butyzamide (circles) or lusutrombopag (squares).

[0022] FIG. IB is a line graph showing number of CD34+ cells on day 7 of culture with the indicated amounts of TPO agonist (nM), either butyzamide (circles) or lusutrombopag (squares).

[0023] FIG. 2 is a bar graph showing number of CD34+ cells on day 28 of culture in the presence of either butyzamide (number too low to measure, ND) or lusutrombopag. Shown is mean number of HSPCs derived from 10,000 human cord blood CD34+ cell after 28-day culture at 20% O2 in medium supplemented with either 100 nM butyzamide or 33 nM lusutrombopag. N.D. not detected, (n = 2 cell cultures from single human cord blood)

[0024] FIG. 3 is a bar graph showing number of CD34+ cells on day 7 of culture in the presence of either avatrombopag, eltrombopag or lusutrombopag. Shown is mean number of HSPCs derived from 5,000 human cord blood CD34+ cell after 7-day culture at 20% O2 (n = 3 cell cultures from single human cord blood) in medium supplemented with either 3 pM avatrombopag, 7 pM eltrombopag, or 33 nM lusutrombopag. N.D., not detected.

[0025] FIG. 4A is a bar graph showing total viable cells (cell number) on day 7 of culture in the presence of lusutrombopag (33 nM) and varying amounts of oxygen, from left 20%, 5%, 3%, or 1%. Shown is mean number of total viable cells derived from 10,000 human cord blood CD34+ cells (n = 5 cell cultures from single human cord blood). Statistical significance was calculated using ANOVA. ****P < 0.0001; ns, not significant.

[0026] FIG. 4B is a bar graph showing number of CD34+ cells on day 7 of culture in the presence of lusutrombopag (33 nM) and varying amounts of oxygen, from left 20%, 5%, 3%, or 1%. Shown is mean number of HSPCs derived from 10,000 human cord blood CD34+ cells (n = 5 cell cultures from single human cord blood). Statistical significance was calculated using ANOVA. ****P < 0.0001; ns, not significant.

[0027] FIG. 5A is a bar graph showing total viable cells (cell number) on day 7 of culture in 5% O2 and in the presence of lusutrombopag (33 nM) and varying amounts of SCF (ng / ml), from left 0, 1, 10, 100. Shown is mean number of total viable cells derived from 10,000 human cord blood CD34+ cells (n = 4 cell cultures from single human cord blood). Statistical significance was calculated using ANOVA. *P < 0.5, ***P < 0.01, ****P < 0.0001.

[0028] FIG. 5B is a bar graph showing number of CD34+ cells on day 7 of culture in 5% O2 and in the presence of lusutrombopag (33 nM) and varying amounts of SCF (ng / ml), from left 0, 1, 10, 100. Shown is mean number of HSPCs derived from 10,000 human cord blood CD34+ cells (n = 4 cell cultures from single human cord blood). Statistical significance was calculated using ANOVA. *P < 0.5, ***P < 0.01, ****P < 0.0001.

[0029] FIG. 6 A is a line graph showing total viable cells (cell number) over time in culture (days) supplemented with butyzamide 100 nM, 20% O2 (circles), lusutrombopag 33 nM, 20%O2 (squares), lusutrombopag 33 nM, 5% O2 (triangles) or lusutrombopag 33 nM and 10 ng / ml SCF 5% O2 (inverted triangles). Shown is mean number of total viable cells derived from 10,000 human cord blood CD34+ cell during 28-day culture under the indicated conditions (n = 4 cell cultures from single human cord blood). Statistical significance was calculated using ANOVA. *P < 0.5, ****P < 0.0001.

[0030] FIG. 6B is a bar graph showing number of CD34+ cells on day 14 of culture in media supplemented with (from left) butyzamide 100 nM, 20% O2, lusutrombopag 33 nM, 20% O2, lusutrombopag 33 nM, 5% O2 or lusutrombopag 33 nM and 10 ng / ml SCF, 5% O2. Shown is mean number of HSPCs derived from 10,000 human cord blood CD34+ cell after 14-day culture under the indicated conditions (n = 4 cell cultures from single human cord blood).Statistical significance was calculated using ANOVA. *P < 0.5, ****P < 0.0001.

[0031] FIG. 7 is a bar graph showing mean percentage of phenotypic LTN-CD41 -, HSPC (LIN-CD41- / CD34+) and HSC (LIN-CD41- / CD34+ / EPCR+) derived from 50,000 human cord blood CD34+ cells during 28-day optimized lusutrombopag-supplemented culture (n = 3 independent healthy donors).

[0032] FIG. 8 A is a line graph showing mean number of phenotypic LIN-CD41- cells in lusutrombopag-supplemented culture at the indicated time (days). Shown is mean number of phenotypic cells derived from 50,000 human cord blood CD34+ cell during 28-day optimized lusutrombopag-supplemented culture (n = 3 independent healthy donors).

[0033] FIG. 8B is a line graph showing mean number of phenotypic HSPC (LIN-CD41- / CD34+) cells in lusutrombopag-supplemented culture at the indicated time (days). Shown is mean number of phenotypic cells derived from 50,000 human cord blood CD34+ cell during 28-day optimized lusutrombopag-supplemented culture (n = 3 independent healthy donors).

[0034] FIG. 8C is a line graph showing mean number of phenotypic HSC (LIN-CD41- / CD34+ / EPCR+) cells in lusutrombopag-supplemented culture at the indicated time (days). Shown is mean number of phenotypic cells derived from 50,000 human cord blood CD34+ cell during 28-day optimized lusutrombopag-supplemented culture (n = 3 independent healthy donors).

[0035] FIG. 9 A is a bar graph showing mean percentage of human bone-marrow chimerism at week 16 in primary recipient NSG mice transplanted with 2,000 human cord blood CD34+- derived cells (n = 7 mice in total; 3-4 replicate mice each with two independent healthy donors). Statistical significance was calculated using an unpaired t-test. *P < 0.5.

[0036] FIG. 9B is a bar graph showing mean percentage of human bone-marrow chimerism at week 12 in secondary recipient NSG mice transplanted with 3 x 106 whole bone marrow from primary recipient (n = 4 mice, 4 replicate mice with single healthy donor). Statistical significance was calculated using an unpaired t-test. *P < 0.5.

[0037] FIG. 10 is a bar graph illustrating mean number of types of colonies generated with 500 unmanipulated human cord blood CD34+ cells or 500 28-day expanded human cord blood CD34+ cells at day 14 in Methocult 4434 (n = 3 cell cultures from single human cord blood). CFU-G: colony forming unit-granulocyte; CFU-M: colony forming unit-macrophage; CFU- GM: colony forming unit-granulocyte / macrophage; CFU-GEMM; colony forming unit granulocyte / erythrocyte / macrophage / megakaryocyte; CFU-E: colony forming unit erythroid; BFU-E: burst forming unit-erythroid. Error bars denote s.d.

[0038] FIG. 1 1 A is a line graph showing mean number of total viable cells derived from 20,000 human mobilized peripheral blood CD34+ cell during 14-day optimized lusutrombopag- supplemented culture (n = 3 cell cultures from single human mobilized peripheral blood).

[0039] FIG. 1 IB is a line graph showing mean number of phenotypic HSPC (LIN-CD41- / CD34+) cells derived from 20,000 human mobilized peripheral blood CD34+ cell during 14- day optimized lusutrombopag- supplemented culture (n = 3 cell cultures from single human mobilized peripheral blood).

[0040] FIG. 12A-E shows lusutrombopag- supplemented HSPC culture stably expands multiple types of HPCs and engraftable HSC for 28 days. (A) Mean ± S.D. of fold changes in total viable cells during 28-day HSPC culture, started with 50,000 UCB-HSPCs. N = 2 experimental replicates from single donor. (B) Colony assay with 500 unmanipulated or 500 expanded umbilical cord blood (UCB)-HSPCs. Mean number ± S.D. of colony forming unit-Granulocyte (CFU-G), CFU-macrophagc (CFU-M), CFU-granulocytc, macrophage (CFU-GM), CFU-granulocytc, erythroid, macrophage, and megakaryocyte (CFU-GEMM), burst forming unit-erythroid (BFU-E), and CFU-erythroid (CFU-E) on day 14 was shown. Red indicates erythroid-committed progenitors, and blue indicates myeloid-committed progenitors. N = 2 experimental replicates from single donor. (C) Mean ± S.D. fold change of CFU-G, CFU-M, CFU-GM, CFU-GEMM, CFU-E, and BFU-E on day 14 in total expanded UCB-HSPCs. N = 2 experimental replicates from single donor. (D) Mean human chimerism in bone marrow from primary recipient NSG mice transplanted with UCB- HSPCs. On primary BMT, 5,000 unmanipulated CD34+UCB or 50,00028-day expanded UCB- HSPCs were transplanted. N = 4 mice from single donor. (E) Lineage contribution of transplantedHSPCs. % CD19+B cells and CD33+myeloid cells in human cells (human CD45+HLA-ABC+) are shown. N = 4 mice from single donor.

[0041] FIG. 13A-D shows lusutrombopag-supplemented HSPC culture provides a platform for genetic engineering, targeting functional HPCs and HSCs. (A) Representative flowcytometry of genetically engineered HSPC that expands in a lusutrombopag-supplimented culture. Lentiviral infection was performed to insert EF1-GFP cassette into HPCs and HSCs. Before lentivaral infection, HSPCs were expanded for 7 days in a lusutrombopag-supplimented culture. 7 days post infection, GFP expression was mesured by FACS in bulk viable cell population (left: PF), HPC subpopulation (middle: Lineage"CD41"CD34+EPCR"), and HSC subpopulation (right: Lineage" CD41"CD34+EPCR"). (B) Mean ± S.D. of %GFP+in each subpopulation were shown. N = 2 experimental replicates from single donor. (C) Mean GFP+human chimerism in bone marrow from primary recipient NSG mice transplanted with GFP+UCB -HSPCs. On primary BMT, FACS-purified 15,000 GFP+expanded HSPC were transplanted in 2Gy-irradiated NSG mice. N = 4 mice from single donor. (D) Lineage contribution of transplanted GFP+HSPCs. % CD19+B cells and CD33+myeloid cells in GFP+human cells (GFP+human CD45+HLA-ABC+) were shown. N = 4 mice from single donor.DETAILED DESCRIPTION

[0042] Ex vivo expansion and maintenance of hematopoietic stem cells and hematopoietic progenitor cells, collectively referred to as “HSPC” or “HSPC cells”, is essential for realizing the clinical potential of these cells. The present inventors unexpectedly found that lustrombopag supplementation both promotes expansion of HSPC and provides for their maintenance in long-term culture.

[0043] Lustrombopag is a thrombopoietin receptor agonist and FDA-approved for the treatment of thrombocytopenia in patents with chronic liver disease. Other thrombopoietin receptor agonists include butyzamide, avatrombopag and eltrombopag. However, as discussed herein, the present inventors unexpectedly found that neither avatrombopag nor eltrombopag was effective to maintain HSPC in short-term culture and none of butyzamide, avatrombopag or eltrombopag was effective to maintain HSPC in long-term culture. In this context, “maintain in culture” refers to the ability to keep cells alive and proliferating ex vivo and in vitro. The terms “cultured” and “culturing” refer to the process of maintaining cells in vitro or ex vivo in a suitable culture medium and under environmental conditions suitable to maintain cell viability and promote cell proliferation. For example, in the case of HSPCs cultured in accordance withthe methods described here, suitable environmental conditions include a temperature of about 37 °C and an atmosphere of about 5-10 % CO2 and about 3-20 % 02- Suitable environmental conditions may be maintained for example in an incubator or bioreactor. HSPCs may be cultured, for example, in a suspension culture. Suitable culture systems are known in the art and may include, for example, continuous or batch perfusion systems.

[0044] A "culture medium” refers to a medium used to culture cells. In accordance with the compositions and methods described here, a culture medium for expanding HSPCs and maintaining HSPCs in long term culture, e.g., for at least 28 days, is provided. The culture medium comprises a basal medium, lusutrombopag, and one or more additional components.

[0045] The term “basal media” refers to a chemically defined medium containing a buffering system, such as a phosphate based buffer, a citrate based buffer, or a HEPES (4-(2- hydroxyethyl)-! -piperazineethanesulfonic acid) based buffer, and other components essential for cell survival and proliferation including amino acids, a carbon source such as glucose that can be metabolized by the cells and isotonic salts, e.g., calcium, magnesium, potassium, sodium, and phosphate. In aspects, the basal medium is free of undefined proteins, hydrolysates, or other components of unknown composition. In aspects, the basal medium is free of animal serums such as fetal bovine serum (FBS) and fetal calf serum (FCS). In aspects, the basal medium is free of albumin.

[0046] An HSPC culture medium in accordance with the compositions and methods described here includes a basal medium supplemented with from about 20-100 nM lusutrombopag, or from about 20-50 nM lusutrombopag, or from about 20-40 nM lusutrombopag, or from about 25-35 nM lusutrombopag. or from about 30-35 nM lusutrombopag. In aspects, the HSPC culture medium contains about 20 nM, about 25 nM, about 35 nM, about 45 nM, about 55 nM, about 65 nM, about 75 nM, about 80 nM, about 85 nM, about 90 nM, or about 100 nM lusutrombopag.

[0047] In aspects, the HSPC culture medium also contains a hematopoietic cell growth factor, for example, stem cell factor or “SCF”. In aspects, the HSPC culture medium contains from about 2.5-25 ng / ml SCF. In aspects, the HSPC culture medium contains about 2.5 ng / ml, about 5 ng / ml, about 10 ng / ml, about 15 ng / ml, about 20 ng / ml, or about 25 ng / ml SCF.

[0048] In aspects, the HSPC culture medium contains about 20-40 nM lusutrombopag and about 5-15 ng / ml SCF. In aspects, the HSPC culture medium contains about 25-35 nMlusutrombopag and about 8-12 ng / ml SCF. In aspects, the HSPC culture medium contains about 30-35 nM lusutrombopag and about 8-12 ng / ml SCF.

[0049] Additional components that may be added to the basal medium to form an HSPC culture medium in accordance with the compositions and methods described here include one or more of insulin, transferrin, selenium, e.g., in the form of sodium selenite, ethanolamine, penicillin, streptomycin, L-glutamine, and a human hematopoietic stem cell self-renewal agonist. Accordingly, an HSPC culture medium in accordance with the compositions and methods described here includes a basal medium supplemented with lusutrombopag and / or SCF and one or more of insulin, transferrin, selenium, ethanolamine, penicillin, streptomycin, L- glutamine, a human hematopoietic stem cell self-renewal agonist such as a pyrimidoindole derivative, (?.g.,UM729. Other pyrimidoindole derivative agonists of human hematopoietic stem cell self-renewal are described in Fares et al. Science. 2014 Sep 19; 345(6203): 1509-1512.

[0050] In aspects, the HSPC culture medium may also contain a solubilizing agent, for example, an amphiphilic polymer. In aspects, the solubilizing agent is polyvinyl alcohol or polyvinyl caprolactam-polyvinyl acetate-polyethylene, also referred to as “PCL-PVAc-PEG”, available from Sigma- Aldrich and sold under the tradename, Soluplus®.

[0051] In aspects, an HSPC culture medium in accordance with the compositions and methods described here contains from about 20-40 nM or 25-35 nM lusutrombopag, from about 5-15 ng / ml or 8-12 ng / ml SCF, and one or more of insulin, transferrin, sodium selenite, ethanolamine, penicillin, streptomycin, L-glutamine, UM729, and PCL-PVAc-PEG.

[0052] In aspects, an HSPC culture medium in accordance with the compositions and methods described here may contain from about 0.01 mg / ml insulin, about 5.5 mg / ml transferrin, about 6.7 pg / ml sodium selenite, about 2 mg / ml ethanolamine, and about 2 mM L-glutamine.

[0053] Suitable basal media for use in the compositions and methods described here include Minimal Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), and Iscove's Modified Dulbecco's Medium (IMDM), all commercially available, e.g., from Thermo Fisher Scientific (Invitrogen, Gibco). Other suitable basal media include Opti-MEM, Ham’s Nutrient Mixture F-12, and RPM11640 also available from Thermo Fisher Scientific brands. Basal media adapted for stem cell culture may also be used, for example, StemSpan™ and MyeloCult™ available from StemCell Technologies Inc., and Hematopoietic Progenitor Growth Medium (HPGM™) available from the Lonza brand of Thermo Fisher Scientific, and StemPro™34 (Thermo Fisher Scientific).

[0054] In aspects of the methods described here, the HSPC may be characterized, sorted, and / or purified according to cell lineage using affinity reagents, including antibodies, against cell surface markers of lineage. Such markers include the transmembrane phosphoglycoprotein CD34, which serves as a marker for hematopoietic stem and progenitor cells and endothelial protein C receptor (EPCR, also known as CD201), which serves as a marker for HSC. Lineage markers for differentiated blood cells include CD3, CD4, and CD8, which serve as markers for T cells; CD19 and CD20, which serve as markers for B cells; CD2, CD7, and CD10 which serve as markers for lymphoid cells; CDl lb, CD14, and CD16, which serve as markers for myeloid cells; CD41, which serves as a marker for megakaryocytic cells; CD56 which serves as a marker for NK cells; and CD235, which serves as a marker for erythroid cells.

[0055] The HSPC obtained by the methods described here may be used to produce immune cells for immunotherapy for use e.g., in cancer and other indications. Any suitable differentiation protocol may be used. An exemplary macrophage differentiation protocol includes expanding the HSCPs for about for 21 days in medium supplemented with 10 ng / ml TPO, 50 ng / ml SCF, 50 ng / ml FLT3-L, 20 ng / ml GM-CSF, and 100 ng / ml M-CSF. For terminal macrophage differentiation, expanded HSPCs are subsequently cultured in medium supplemented with 20 ng / ml GM-CSF and 100 ng / ml M-CSF. Differentiated macrophages are characterized by the expression of surface protein including CDl lb, CD14 and CD68.

[0056] The HSPC obtained by the methods described here may be used to produce blood cells such as platelets for use in the treatment of a disease or disorder where platelet depletion is present. For example, the disease or disorder may include thrombocytopenia, platelet function disorder, certain autoimmune diseases, such as immune thrombocytopenia (ITP), lupus, and rheumatoid arthritis, and certain cancers, such as leukemias and lymphomas.

[0057] The HSPC obtained by the methods described here may be used to produce hematopoietic stem cells and hematopoietic progenitor cells for transplantation.

[0058] In aspects, provided is a method for culturing mammalian hematopoietic stem and progenitor cells (HSPCs), the method comprising culturing the HSPC in a chemically defined basal medium comprising an amount of lusutrombopag and, optionally, an amount of stem cell factor..

[0059] In aspects, provided is a method for ex vivo production of lymphoid cells, where the method includes culturing mammalian hematopoietic stem and progenitor cells (HSPCs) in a chemically defined basal medium that includes an amount of lusutrombopag and an amount ofstem cell factor. In aspects, the lymphoid cells are selected from one or more of T cells, natural killer cells, B cells, and dendritic cells.

[0060] In another aspect, provided is a method for ex vivo production of myeloid cells, where the method includes culturing mammalian hematopoietic stem and progenitor cells (HSPCs) in a chemically defined basal medium that includes an amount of lusutrombopag and an amount of stem cell factor. In aspects, the myeloid cells are selected from one or more of erythrocytes, platelets, neutrophils, macrophages, and dendritic cells.

[0061] In one aspect, provided is a method for expanding a population of mammalian hematopoietic stem and progenitor cells (HSPCs) ex vivo, where the method includes culturing the HSPCs in a chemically defined basal medium that includes an amount of lusutrombopag and an amount of stem cell factor. In aspects, the culturing is performed for a period of from 7- 28 days, preferably at least 14-28 days.

[0062] In aspects, provided is a method for producing hematopoietic stem cells or hematopoietic progenitor cells for transplantation, where the method includes culturing the HSPCs in a chemically defined basal medium that includes an amount of lusutrombopag and an amount of stem cell factor. In aspects, the HSPCs obtained by the methods described here are superior to cells obtained by conventional methods in terms of their rate of survival posttransplantation.Example 1

[0063] Provided below is evidence showing that human HSPCs derived from cord blood or peripheral blood can be stably and reproducibly expanded using the compositions and methods described here. The expansion of human HSPC is confirmed using both in vitro and in vivo functional assays.

[0064] Human HSPC culture was performed as follows. Human cord blood CD34+ cell cultures were performed using IMDM, 1% ITSX, 1% P / S / G, 1 pM 740Y-P, 1 pM UM729, 0.1% PCL-PVAc-PEG (Soluplus; BASF) and one of butyzamide, lusutrombopag, avatrombopag, or eltrombopag at 37 °C with 5% CO2. For long-term cultures, medium changes were made every two to three days by collecting all cell suspension. The cell suspension was washed with PBS and centrifuged with 300 g at 24 °C for 5 mins. The supernatant was aspirated, and the cell pellet was suspended in fresh medium. After the optimization of human HSPC culture, 33 nM Lusutrombopag and 10 ng / ml stem cell factor (SCF) at 37 °C with 5 % CO2 and 5 % O2 were used, otherwise indicated.

[0065] US 20220298479 describes a serum-free, albumin-free culture medium comprising polyvinyl alcohol, a PI3K activator such as 740Y-P, and thrombopoietin (TPO) or a TPO receptor agonist, particularly butyzamide, for culturing human hematopoietic stem cells. Unexpectedly, the present inventors found that the TPO lusutrombopag supports human cord blood HSPCs better than Butyzamide in a short-term 7-day culture.

[0066] FIG. 1A shows total number of viable cells on day 7 of culture with the indicated amounts (nM) of either butyzamide or lusutrombopag.

[0067] FIG. IB shows total number of lineage HSPCs, defined as CD34+ cells, on day 7 of culture with the indicated amounts (nM) of either butyzamide or lusutrombopag. The data indicate that the EC50 of lusutrombopag is lower than butyzamide.

[0068] However, as shown in FIG. 2, lusutrombopag is able to maintain human cord blood HSPCs in a long-term culture out to 28 days, where butyzamide cannot.

[0069] In short term ( 7 day) culture, two other TPO agonists, avatrombopag and eltrombopag, failed to support human cord blood HSPCs.

[0070] FIG. 4A and FIG. 4B show that physiological 5% O2 concentration is optimal for human cord blood HSPC expansion in a lusutrombopag- supplemented culture.

[0071] FIG. 5A and FIG. 5B show that 10 ng / ml stem cell factor (SCF) is optimal for human cord blood HSPC expansion in a lusutrombopag-supplemented culture at 5% 02.

[0072] FIG. 6A and FIG. 6B show that 33 nM lusutrombopag and 10 ng / ml SCF- supplemented culture in 5% O2 performs best for long-term human cord blood HSPC expansion.

[0073] FIG. 7 shows that 33 nM lusutrombopag and 10 ng / ml SCF-supplemented culture in 5% O2 stably and reproducibly supports immunophenotypic human cord blood HSPCs and HSCs in long-term culture out to at least 28 days.

[0074] FIG. 8A, FIG. 8B, and FIG. 8C show that 33 nM lusutrombopag and 10 ng / ml SCF- supplemented culture in 5% O2 stably and reproducibly expands human cord blood HSPCs and HSCs in long-term culture out to at least 28 days.

[0075] FIG. 9A and FIG. 9B show that 33 nM lusutrombopag and 10 ng / ml SCF- supplemented culture in 5% O2 expands human functional HSCs that are engraftable in immunodeficient mice.

[0076] FIG. 10 shows that 33 nM lusutrombopag and 10 ng / ml SCF-supplemented culture in 5% O2 expands functional human hematopoietic progenitors that generate myeloid and erythroid blood cells.

[0077] FIG. 11A and FIG. 11B show that 33 nM lusutrombopag and 10 ng / ml SCF- supplemented culture in 5% O expands immunophenotypic human PB-HSPCs.Example 2

[0078] To demonstrate lusutrombopag can maintain and expand human HSCs and HPCs under conditions without adding PI3K activator (PI3Ka), consistent with in vitro surface marker profiling and in vivo transplantation data, an experiment was performed adding a different common cytokine, FLT3 ligand (FLT3-L). For cytokine-free expansion culture, human cord blood CD34+cell cultures were performed using IMDM, 1% ITSX, 1% P / S / G, 1 pM 740Y-P, 0.1%31.6 nM lusutrombopag and PCL-PVAc-PEG (Soluplus; BASF) at 37 °C with 5% CO2 and 5% O2, unless specifed. For cytokine-supplemented expansion culture, 10 ng ml-1recombinant human SCF (PeproTech) and 10 ng ml-1recombinant mouse THPO (PeproTech) were used. The addition of FLT3-L achieved >600-fold change of total viable cells derived from human HSPCs during a 28-day culture (FIG. 12A). A colony-forming capacity assay revealed that 28-day expanded human HSPCs were able to generate granulocyte-, macrophage-, erythroid- and megakaryocyte-containing colonies (FIG. 12B) when defined numbers of fresh or cultured cells were subjected to CFU assays using Methocult H4434 (Stemcell Technologies). Cells were incubated in a humidified atmosphere at 37 °C with 5% CO2. After two weeks, the number of colonies was counted using a Nucleocounter NC-3000 and types of colonies were validated according to manufacturer’s protocol. Notably, estimated fold-change of colony forming units in total expanded HSPCs was > 1000 in colony forming unit-granulocyte (CFU-G) and CFU- macrophage (CFU-M), >800 in CFU-granulocyte / macrophage (CFU-GM), and about 100 in CFU-granulocyte / erythroid / macrophage / megakaryocyte (CFU-GEMM), CFU-erythroid (CFU- E) and burst forming unit-erythroid (BFU-E) (FIG. 12C). Lusutrombopag-supplemented HSPC culture robustly expands myeloid-committed progenitor as well as erythroid-committed and megakaryocyte-committed progenitors. Furthermore, an HSC transplantation assay in immunodeficient NSG mice showed more than double human chimerism in 28-day expanded human HSPCs derived from 75-equivalent human HSPCs, compared to 5,000 unmanipulated human HSPC. Both unmanipulated and 28-day expanded HSPC reconstituted CD19+B cell and CD33+myeloid cell in vivo (FIG. 12D,E). Lusutrombopag-supplemented HSPC culture withoutPI3Ka expanded engraftable human HSC with an estimated fold-change of 133.4 during a 28- day culture.

[0079] To demonstrate the utility of lusutrombopag- supplemented HSPC cultures for gene therapy, lentiviral transduction of GFP fluorescent protein cassettes was carried out in the expanded HSPCs. To obtain lentiviral supernatants, HEK293T cells were transiently transfected with lentiviral vector plasmids containing the GFP cassettes, MD2G packaging plasmid (Invitrogen) and PAX2 envelope plasmid (Invitrogen). Forty-eight hours later, the viral supernatant was collected and utilized for transduction (MOI of 5) of UCB-HSPCs that had been expanded for 7 days in lusutrombopag-supplemented HSPC culture. Seven days post- lentiviral infection, flow cytometric analysis was carried out. Cells were stained with a lineage cocktail (biotinylated CD3, CD4, CD8, CD10, CDl lb, CD14, CD16, CD19, CD20, CD56, and CD235a) and then with antibodies (APC-labelled anti-human CD34, BV421-labelled antihuman CD49c, FTTC-labelled anti-human CD90, PE-labelled anti-human CD201 , APC-Cy7- labcllcd anti-human CD41a, BV711-labclcd anti-Strcptavidin). Following a wash step, flow cytometric analysis was performed using a FACS Ariall (BD), or FACSymphony (BD) using propidium iodide as a dead stain. Flowcytometry revealed clear GFP expression in either HPC subpopulation (Lineage‘CD41'CD34+EPCR‘) or HSC subpopulation (Lineage‘CD41‘ CD34+EPCR+) among expanded HSPC (FIG. 13A,B). These findings are contemplated to show that lusutrombopag-supplemented HSPC culture can be utilized for successful gene engineering of HPCs and HSCs.

[0080] To show the engineered HSPCs in lusutrombopag-supplemented HSPC culture were functional HPCs and HSCs, FACS-purified GFP+expanded HSPCs were transplanted in 2Gy irradiated immunodeficient mice. The animal experimental protocol was approved by Stanford University’s Administrative Panel on Laboratory Animal Care. Immunodeficient NOD.Cg- PrkdcscldIl2rgtmlWil / SzJ mice were purchased from Jackson Laboratory and eight- to twelve- week-old female NSG mice were irradiated using 200 rad, 12-24 h before transplantation. 5,000 unmanipulated or 50,000 exapnded HSPCs were injected via retro-tail vein injection per mouse. Sixteen weeks post transplantation, mice were euthanized and bone marrow was harvested from tibia and femur, using a pestle and mortar. After red blood lysis, the samples were then stained for 30 min at 4 °C with the following antibodies: PE-labeled anti-human CD33, PE-Cy7-labelled antihuman HLA-ABC, APC-labelled anti-human CD19, APC-Cy7-labelled anti-mouse CD45.1, PE- labelled anti-human CD33, and BV450-labelled anti-human CD45. Multilineage engraftment wasestablished by the presence of myeloid cells (CD33+) and B cells (CD19+) in engrafted human cells (mCD45‘, human CD45+HLA-ABC+cells)., Human myloid and lymphoid reconstitution was detected in one mouse, indicating that successful gene engineering and engraftment (FIG. 13C,D). Moreover, only human myeloid reconstitution was detected among three mice (FIG. 13C,D).

[0081] Taken together, these experiments demonstrate lusutrombopag-supplemented HSPC cultures achieve stable HSPC expansion without need for PI3Ka, highlighting the significant stability and utility benefits of the methods provided herein for HSPC-based gene therapy.

[0082] While the invention has been described by means of specific embodiments and applications thereof, modifications and variations could be made thereto by those skilled in the art without departing from the scope set forth in the claims.

[0083] The present invention is set forth in various levels of detail. In certain instances, details not necessary for one of ordinary skill in the art to understand the invention may have been omitted.

[0084] Section headings are for organizational purposes only and are not to be construed as limiting the subject matter described.Other Terminology

[0085] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting beyond the scope of the appended claims. An “embodiment” may refer to an illustrative representation of a method or article in which a disclosed concept or feature may be provided or embodied, or a representation of a manner in which a concept or feature may be provided or embodied. Such illustrated embodiments are to be understood as examples (unless otherwise stated), and other manners of embodying the described concepts or features, such as may be understood by one of ordinary skill in the art upon learning the concepts or features from the present disclosure, are within the scope of the disclosure. Accordingly, disclosed embodiments are to be considered in all respects as illustrative and not restrictive, the scope of the claimed subject matter being indicated by the appended claims, and not limited to the foregoing description or particular embodiments or arrangements described or illustrated herein. It is intended that the present subject matter covers such modifications and variations as come within the scope of the appended claims and their equivalents.

[0086] Unless defined otherwise, technical terms used herein are to be understood as commonly understood by one of ordinary skill in the art to which the disclosure belongs.

[0087] The phrases “at least one”, “one or more”, and “and / or”, as used herein, are open- ended expressions that are both conjunctive and disjunctive in operation. The terms “a”, “an”, “the”, “first”, “second”, etc., do not preclude a plurality. For example, the term “a” or “an” entity, as used herein, refers to one or more of that entity. As such, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein.

[0088] The term “about” when used before a numerical designation, e.g., temperature, time, amount, concentration, and such other, including a range, indicates approximations which may vary by ( + ) or ( - ) 10%, 5%, 1%, or any subrange or subvalue there between. Preferably, the term “about” means that the value may vary by + / - 10%.

[0089] The term “comprises / comprising” does not exclude the presence of other elements, components, features, regions, integers, steps, operations, etc. Additionally, although individual features may be included in different claims, these may possibly advantageously be combined, and the inclusion in different claims does not imply that a combination of features is not feasible and / or advantageous. By contrast, the transitional phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention.

[0090] A “cell line” refers to a population of cells that can be propagated in culture, that is in vitro, through at least 10 passages while remaining phenotypically homogenous.

Claims

CLAIMSWhat is claimed is:

1. A defined hematopoietic stem and progenitor cell (HSPC) culture medium comprising a basal medium and an amount of lusutrombopag.

2. The HSPC culture medium of claim 1, wherein the amount of lusutrombopag is from about 20-100 nM.

3. The HSPC culture medium of claim 2, wherein the medium comprises stem cell factor, optionally wherein the amount of stem cell factor is from about 2.5-25 ng / ml.

4. The HSPC culture medium of any one of claims 1 to 3, wherein the medium comprises one or more of insulin, transferrin, selenium, ethanolamine, penicillin, streptomycin, and L- glutamine.

5. The HSPC culture medium of claim 4, wherein the medium comprises a human hematopoietic stem cell self-renewal agonist.

6. The HSPC culture medium of claim 4 or 5, wherein the medium comprises a solubilizing agent, optionally wherein the solubilizing agent is an amphiphilic polymer.

7. A defined hematopoietic stem and progenitor cell (HSPC) culture medium comprising a basal medium and from about 20-40 nM or 25-35 nM lusutrombopag, from about 5-15 ng / ml or 8-12 ng / ml stem cell factor, and one or more of insulin, transferrin, sodium selenite, ethanolamine, penicillin, streptomycin, and L-glutamine.

8. The HSPC culture medium of claim 7, wherein the medium comprises a human hematopoietic stem cell self-renewal agonist and / or a solubilizing agent.

9. The HSPC culture medium of any one of claims 1 to 8, wherein the basal medium is selected from minimal essential medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), and Iscove's Modified Dulbecco's Medium (IMDM).

10. The HSPC culture medium of any one of claims 1 to 9, wherein the medium is substantially free of albumin, animal serum, polyvinyl alcohol, butyzamide, and a phosphatidylinositol 3- kinase (PI3K) activator.

11. A method for culturing mammalian hematopoietic stem and progenitor cells (HSPCs), the method comprising culturing the HSPC in a chemically defined basal medium comprising an amount of lusutrombopag.

12. The method of claim 11, wherein culturing the HSPCs comprises maintaining the HSPCs in a controlled environment of about 37 °C in an atmosphere of about 5-10 % CO2 and about 3-20 % O2.

13. The method of claim 11 or 12, wherein the amount of lusutrombopag is from about 20-100 nM.

14. The method of any one of claims 11 to 13, wherein the medium comprises stem cell factor, optionally wherein the amount of stem cell factor is from about 2.5-25 ng / ml.

15. The method of any one of claims 11 to 14, wherein the medium comprises one or more of insulin, transferrin, selenium, ethanolamine, penicillin, streptomycin, and L-glutamine.

16. The method of any one of claims 1 1 to 15, wherein the medium comprises a human hematopoietic stem cell self-renewal agonist.

17. The method of any one of claims 11 to 16, wherein the medium comprises a solubilizing agent, optionally wherein the solubilizing agent is an amphiphilic polymer.

18. The method of any one of claims 11 to 17, wherein the medium comprises from about 20- 40 nM or 25-35 nM lusutrombopag, from about 5-15 ng / ml or 8-12 ng / ml stem cell factor, and one or more of insulin, transferrin, sodium selenite, ethanolamine, penicillin, streptomycin, and L-glutamine.

19. The method of any one of claims 11 to 18, wherein the basal medium is selected from minimal essential medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), and Iscove's Modified Dulbecco's Medium (IMDM).

20. The method of any one of claims 11 to 19, wherein the medium is substantially free of albumin, animal serum, polyvinyl alcohol, butyzamide, and a phosphatidylinositol 3-kinase (PI3K) activator.

21. The method of any one of claims 11 to 20, wherein culturing the HSPC results in an increase in the number of HSPCs over a period of time.

22. The method of claim 21, wherein the period of time is 7 days, 14 days, or 28 days.

23. The method of claim 21, wherein the period of time is 28 days.

24. A method for long term culture of mammalian hematopoietic stem and progenitor cells (HSPCs), the method comprising culturing the HSPCs in a chemically defined basal medium comprising an amount of lusutrombopag for at least 28 days.

25. The method of claim 24, wherein culturing the HSPC results in an increase in the number of HSPCs.

26. The method of claim 24 or 25, wherein culturing the HSPCs comprises maintaining the HSPCs in a controlled environment of about 37 °C in an atmosphere of about 5-10 % CO2 and about 3-20 % O2.

27. The method of any one of claims 24 to 26, wherein the amount of lusutrombopag is from about 20-100 nM.

28. The method of any one of claims 24 to 27, wherein the medium comprises stem cell factor, optionally wherein the amount of stem cell factor is from about 2.5-25 ng / ml.

29. The method of any one of claims 24 to 28, wherein the medium comprises one or more of insulin, transferrin, selenium, ethanolamine, penicillin, streptomycin, and L-glutamine.

30. The method of any one of claims 24 to 29, wherein the medium comprises a human hematopoietic stem cell self-renewal agonist.

31. The method of any one of claims 24 to 30, wherein the medium comprises a solubilizing agent, optionally wherein the solubilizing agent is an amphiphilic polymer.

32. The method of any one of claims 24 to 31, wherein the medium comprises from about 20- 40 nM or 25-35 nM lusutrombopag, from about 5-15 ng / ml or 8-12 ng / ml stem cell factor, and one or more of insulin, transferrin, sodium selenite, ethanolamine, penicillin, streptomycin, and L-glutamine.

33. The method of any one of claims 24 to 32, wherein the basal medium is selected from minimal essential medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), and Iscove's Modified Dulbecco's Medium (IMDM).

34. The method of any one of claims 24 to 33, wherein the medium is substantially free of albumin, animal serum, polyvinyl alcohol, butyzamide, and a phosphatidylinositol 3-kinase (PI3K) activator.

35. A method for ex vivo production of lymphoid cells, the method comprising culturing mammalian hematopoietic stem and progenitor cells (HSPCs) in a chemically defined basal medium comprising an amount of lusutrombopag and an amount of stem cell factor.

36. The method of claim 35, wherein the lymphoid cells are selected from one or more of T cells, natural killer cells, B cells, and dendritic cells.

37. A method for ex vivo production of myeloid cells, the method comprising culturing mammalian hematopoietic stem and progenitor cells (HSPCs) in a chemically defined basal medium comprising an amount of lusutrombopag and an amount of stem cell factor.

38. The method of claim 37, wherein the myeloid cells are selected from one or more of erythrocytes, platelets, neutrophils, macrophages, and dendritic cells.

39. A method for expanding a population of mammalian hematopoietic stem and progenitor cells (HSPCs) ex vivo, the method comprising culturing the HSPCs in a chemically defined basal medium comprising an amount of lusutrombopag and an amount of stem cell factor.

40. The method of claim 39, wherein the culturing is performed for a period of from 7-28 days, preferably at least 14-28 days.

41. The method of any one of claims 11 to 40, wherein the mammalian HSPCs are human HSPCs.

42. The HSPC culture medium of claim 6, wherein the solubilizing agent is polyvinyl caprolactam-poly vinyl acetate-polyethylene.

43. The HSPC culture medium of claim 6, wherein the solubilizing agent is polyvinyl alcohol.

44. The method of claim 8, 17 or 31, wherein the solubilizing agent is polyvinyl caprolactampolyvinyl acetate-polyethylene.

45. The method of claim 8, 17 or 31, wherein the solubilizing agent is polyvinyl alcohol.

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