therapy

Abstract

The present invention relates to a method for expanding hematopoietic stem cells and progenitor cells (HSPCs) for use in therapy.

Description

[Technical Field]

[0001] This invention relates to a method for expanding hematopoietic stem cells and progenitor cells. Also disclosed herein are pluripotent cells expanded using this method for use in therapy. [Background technology]

[0002] Hematopoietic stem cell transplantation and progenitor cell transplantation (HSCT) are the most successful and widely used stem cell therapies to date. HSCT is used to treat conditions in which the normal immune system is impaired, such as hematological disorders or chemoradiotherapy. The use of HSCT has also been clinically proven in gene therapy and is expected to be further expanded by new genome editing technologies. Nevertheless, transplantation still presents challenges, as tissue must be matched to the recipient and demand often exceeds supply.

[0003] Initially, hematopoietic stem cells and progenitor cells (HSPCs) for transplantation were derived solely from bone marrow (BM). More recently, it has been discovered that umbilical cord blood (UCB) also contains HSPCs capable of engrafting in the bone marrow and producing blood cells throughout the recipient's lifetime. Using UCB as a source of HSPCs for use in HSCT has several advantages over more conventional BM, as UCB is tested and banked prior to use and therefore more readily available, and UCB also contains more immature stem cells and shows less associated graft-versus-host disease due to tissue type incompatibility. However, transplantation using cells derived from UCB is limited by the number of cells present in a single UCB unit. This quantitative limitation cannot be overcome by transplanting multiple UCB units into a single subject, as engraftment of HSPCs from only one UCB unit is predominant. Therefore, to date, these transplants have been restricted to use in children.

[0004] Before transplantation, the entire umbilical cord blood unit is first separated, red blood cells are discarded, and then the cell population is further enriched with nucleated cells by sorting for either CD34+ or CD133+ cells. The markers CD133 and CD34 are found among numerous progenitor cells / stem cells, including those of the hematopoietic system. Furthermore, it has been shown that these are the CD133+ and CD34+ compartments of hematopoietic cells where cells that regrow over time reside. Therefore, increasing the number of these specific cell types can significantly improve engraftment and make it possible to apply UCB HSCT to treating older children and adults. Unfortunately, using conventional culture methods, hematopoietic stem cells (HSCs) characterized by the expression of markers CD133, CD34, CD90, and CD49f are rapidly depleted during proliferation and subsequently differentiate into cell types with limited potency. Therefore, there is great interest in developing culture conditions that allow for the expansion of these HSCs without compromising the characteristics of stem cells.

[0005] Several strategies exist to increase the total number of cells in a UCB unit by attempting to mimic the niche or environment in which these cells normally reside. In the 1970s, it was established that conditions containing serum and specific cytokines, mainly stem cell factor (SCF), thrombopoietin (TPO), interleukin-3 (IL3), interleukin-6 (IL6), and granulocyte colony-stimulating factor (G-CSF), could be used in vitro for HSC expansion. By the early 1990s, the first clinical trial was conducted using UCB cells expanded for 10 days in serum-free medium containing SCF, G-CSF, and MGDF (megakaryocyte growth and development factor). This expansion method resulted in a 56-fold expansion of total nucleated cells (TNCs) and a 4-fold expansion of CD34+ cells. Patients were injected with one manipulated fraction and one unmanipulated fraction, either together or at 10-day intervals. This trial demonstrated the feasibility and overall safety of expanding UCB units ex vivo. Of the 37 patients treated, all showed engraftment, but only 12 were still alive after 30 months. Further studies using different combinations of cytokines have led to more defined protocols in in vitro expansion, some of which have shown promise in preclinical models. These include SCF, TPO, fms-like tyrosine kinase 3-ligand (FLT3LG), IL3 and IL6, and more recently, Wnt1, bone morphogenetic protein 7 (BMP7), angiopoietin-like protein 5 ANGPTL5, and insulin growth factor-binding protein 2 (IGFBP2).

[0006] Early observations during in vitro expansion of hematopoietic cells indicated that accelerated cell proliferation was associated with the simultaneous differentiation of these cells into more determined precursors or more ultimately differentiated cells. This led to the hypothesis that fate determination is most likely controlled at the epigenetic level, with specific gene sets being transcribed or silenced at different stages. Therefore, controlling or altering the epigenome would affect the overall phenotype of cells and their behavior. Studies using histone modifiers, including histone deacetylase inhibitors, have yielded promising results. The first such studies used 5-aza-2'-deoxycytidine and trichostatin A. Using this regime, UCB CD34+ / CD90+ cells expanded four times more than cells expanded with cytokines alone, and furthermore, retained the ability to re-breed NOD / SCID mice. By extending these studies to include alternative histone modifying enzymes, it became clear that other HDAC inhibitors also possessed similar properties, with valproic acid and scriptide being particularly effective among them. This has also been reported in "Enriched and expanded human cord blood stem cells for treatment of hematological disorders" by Chaurasia, P. & Hoffman, R. (2014), "Expansion of human umbilical cord blood SCID-repopulating cells using chromatin-modifying agents" by Araki, H. et al. (2006), and WO2014 / 189781.

[0007] Several target molecules were obtained through chemical library screening to identify molecules that preferentially enable the expansion of CD34+ cells and prevent their differentiation. Of particular note is the aryl hydrocarbon receptor antagonist StemRegenin1, which is currently being used in clinical trials to expand cells. Another set of notable compounds are the pyrimidoindole derivatives UM729 and UM171, which were found to preferentially expand HSCs with long-term engraftment ability, so-called long-term HSCs (LT-HSCs), determined by the presence of markers CD34, CD90(Thy1), and CD49f, as well as the absence of CD38 and CD45RA. Other molecules found to preferentially expand CD34+ cells include resveratrol, GSK-3 inhibitors, p18 protein inhibitors, and others.

[0008] In particular, the receptor tyrosine kinase, RET, is noteworthy and has been shown to be expressed in mouse hematopoietic stem cells (HSCs). When activated by Grial-derived neurotrophic factor (GDNF) family ligands and co-receptors, RET plays a crucial role in enhancing their in vivo survival and elongation, and mediates Bcl-2 expression. As reported by Grey et al. in “Activation of the receptor tyrosine kinase RET improves long-term hematopoietic stem cell outgrowth and potency” (2020), RET has been shown to mediate improved sustained cell growth, stress tolerance, and overall cell survival throughout in vitro expansion.

[0009] Amifostine, a prodrug of WR1065, was developed by the U.S. military as a radioprotective compound and approved for clinical use in 1995 under the name ethiol. Although the exact mechanism by which the drug exerts its effects is still under investigation, it is metabolized in vivo to WR1065, a ROS scavenger that prevents DNA damage through p53 activation. In vitro studies of the effects of this compound on hematopoietic cells have shown that pretreatment of bone marrow-derived CD34+ cells with WR1065 increased the formation of both CFU-GEMM and BFU-E colonies by approximately 38-fold.

[0010] WO9625045 discloses thiols containing amiphostin for hematopoietic stem cell growth.

[0011] WO2020 / 084310 and WO2020 / 084310 disclose the use of a combination of aminothiol and HDAC inhibitors combined to form an expanded population of HSPCs, which can then be used in therapy.

[0012] EPCR (CD201) has recently emerged as the strongest indicator of LT-HSC engraftment ability. EPCR expression has been confirmed in mouse bone marrow. Subsequently, CD201-positive cells from mouse BM and fetal liver were shown to exhibit regrowth potential upon transplantation.

[0013] Later, in 2017, human umbilical cord blood CD34 + For the first time, a small subset of cells expressed the endothelial protein C receptor (EPCR / CD201 / PROCR) upon exposure to the HSC self-renewal agonist UM171. In contrast to EPCR-negative cells, EPCR-positive UM171-treated cells exhibited robust multi-series recollection and sequential rearrangement capabilities in immunodeficient mice.

[0014] In 2020, expanded HSCs phenotypically marked by the expression of stem cell markers CD34, CD90, and EPCR (CD201) were shown to be highly rich in LT-HSCs.

[0015] In 2020, the HSC group CD34 + EPCR + (CD38 / CD45RA) - (simply EPCR) + These HSCs (as HSCs) have been shown to possess high regrowth and self-renewal capabilities, reaching a stem cell frequency of approximately 1 in 3 cells, which is the highest described to date. Therefore, it is desirable to create protocols that produce a higher proportion and number of expanded HSCs expressing EPCR. [Overview of the project]

[0016] It is possible to achieve HSPC expansion by culturing cells in the presence of an HDAC inhibitor, such as scriptide, in combination with an aminothiol compound such as WR1065. The present invention is based at least in part on data presented herein showing improvements to three exemplary protocols of the present invention (PTC13325.1, PTC13325.2, and PTC13325.3) compared to controls, as well as the protocols disclosed in WO2020 / 084310 and WO2020 / 084310 (PTC13303). The presence of an additional culture step, before culturing again in the presence of an HDAC inhibitor and then an aminothiol compound, is thought to result in cell expansion and an increase in the total number of nucleated cells.

[0017] Furthermore, it was surprisingly found that the protocol of the present invention also produces enlarged cells in which a high percentage of HSCs express the marker EPCR. EPCR is a potent indicator of LT-HSC engraftment ability, and therefore, a higher percentage of expression of this marker in the enlarged population is particularly important for engraftment, and is a desirable feature leading to the conclusion that the protocol of the present invention produces enlarged cells with superior engraftment ability.

[0018] Even more surprisingly, the protocol of the present invention has been found to exhibit a higher percentage of early lymphocytes and myeloid progenitor cells in an expanded population of cells (especially when compared to PTC13303). Since the recovery of platelets and neutrophils are important short-term indicators of treatment success, this is a desirable feature in any therapeutic product. Delayed recovery of platelets and neutrophils is dangerous for patients and results in longer hospital stays.

[0019] Thus, the expanded cells produced according to the method of the present invention can generate a therapeutic product that exhibits both excellent engraftment ability in patients and increased platelet and neutrophil recovery.

[0020] Thus, a first aspect of the present invention is a method for expanding hematopoietic stem and progenitor cells (HSPC), the method comprising i) obtaining an isolated population of HSPC; ii) culturing the isolated population of HSPC for at least 48 hours to form a first cultured population; iii) adding a histone deacetylase inhibitor (HDAC inhibitor) and further culturing the cells to form a second cultured population; iv) adding to the second cultured population an aminothiol compound having the formula RNH(C n H 2n )NH(C n H 2n )SX, wherein R is hydrogen, aryl, acyl, or an alkyl group containing 1 to 7 carbon atoms, each n has a value of 2 to 6, and X is H or PO3H2, or a pharmaceutically acceptable salt thereof, and further culturing the cells to form an expanded population of cells.

[0021] A second aspect is a composition comprising an expanded population of cells for use in therapy, wherein the cells i) Obtain an isolated population of HSPCs, ii) Culturing an isolated population of HSPCs for at least 48 hours to form a first cultured population, iii) Add a histone deacetylase inhibitor (HDAC inhibitor) and further culture the cells to form a second cultured population. iv) an aminothiol compound, wherein the aminothiol compound is of the formula RNH(C n H 2n )NH(C n H 2n The present invention relates to a composition expanded by a method comprising adding an aminothiol compound, or a pharmaceutically acceptable salt thereof, having SX, in which R is hydrogen, aryl, acyl, or an alkyl group containing 1 to 7 carbon atoms, each n having a value of 2 to 6, and X being H or PO3H2, to a second cultured population, and further culturing the cells to form an expanded population of cells.

[0022] The third aspect is a method of treatment, i) A step to obtain an isolated population of HSPCs, ii) A step of culturing an isolated population of HSPCs for at least 48 hours to form a first cultured population, iii) Adding a histone deacetylase inhibitor (HDAC inhibitor) and further culturing the cells to form a second cultured population, iv) an aminothiol compound, wherein the aminothiol compound is of the formula RNH(C n H 2n )NH(C n H 2n A method comprising the steps of adding an aminothiol compound, or a pharmaceutically acceptable salt thereof, having SX, wherein R is hydrogen, aryl, acyl, or an alkyl group containing 1 to 7 carbon atoms, each n having a value of 2 to 6, and X is H or PO3H2, to a second cultured population and further culturing the cells to form an expanded population of cells.

[0023] A fourth aspect is the use of a composition comprising an enlarged population of cells in the manufacture of a pharmaceutical product for use in therapy, wherein the cells i) Obtain an isolated population of HSPCs, ii) Culturing an isolated population of HSPCs for at least 48 hours to form a first cultured population, iii) Add a histone deacetylase inhibitor (HDAC inhibitor) and further culture the cells to form a second cultured population. iv) an aminothiol compound, wherein the aminothiol compound has the formula RNH(C n H 2n )NH(C n H 2n The use is expanded by a method comprising adding an aminothiol compound, or a pharmaceutically acceptable salt thereof, having SX, in the formula, where R is hydrogen, aryl, acyl, or an alkyl group containing 1 to 7 carbon atoms, each n having a value of 2 to 6, and X being H or PO3H2, to a second cultured population, and further culturing the cells to form an expanded population of cells.

[0024] A fifth aspect is a kit for expanding HSPC as defined above, wherein the kit comprises a sterile element for expanding HSPC, an HDAC inhibitor, and an aminothiol compound of the formula RNH(C n H 2n )NH(C n H 2n A kit comprising an aminothiol compound, or a pharmaceutically acceptable salt thereof, having SX, where R is hydrogen, aryl, acyl, or an alkyl group containing 1 to 7 carbon atoms, each n having a value of 2 to 6, and X is H or PO3H2.

[0025] The sixth aspect is an enlarged population of cells obtained by the method described above. [Brief explanation of the drawing]

[0026] [Figure 1]This paper presents a gating strategy for identifying long-term human CB hemoglobin sclerosing cells (HSCs) using flow cytometry. [Figure 2A] Protocols: This shows the magnification change in the total number of TNCs compared to pre-magnified cells from two independent UCB donors (Donor 1 and Donor 2) after magnification using Ctrl, PTC13303, and PTC13325.1. Combined data are shown in the plot on the left. [Figure 2B] The estimated percentage of Lin- / CD34+ cells from donor 1 and donor 2 after treatment with the protocols, namely Ctrl (control), PTC13303, and PTC13323.1, is shown. [Figure 2C] The calculation percentage of long-term HSC (LT-HSC) cells in (i) the TNC population or (ii) the CD34+ cell population from donor 1 and donor 2 (defined by the markers CD34+ / CD38- / CD45RA- / CD90+ / CD49f+). [Figure 2D] The total number of LT-HSCs (defined by the markers CD34+ / CD38- / CD45RA- / CD90+ / CD49f+) is shown, calculated using the number of TNCs and percentage of LT-HSCs within the TNC population for Donor 1 and Donor 2. [Figure 2E] We compare the in vitro protocols PTC13303, PTC13325.1, and PTC13325.2 using UCB from two additional donors (Donors 3 and 4). [Figure 2F] This shows the percentage of different populations of hematopoietic progenitor cells from donor 3 after processing with protocols Ctrl, PTC13303, PTC13323.1, and PTC13325.2. [Figure 3] (i) The total number of colonies scored for expanded cells from Donor 1 and Donor 2, and (ii) the number of CFU-GEMM colonies correlated with the number of the most primitive HSCs. These CFU-GEMM colonies are capable of multi-lineage hematopoiesis and reflect their potency in forming mixed-form colonies. [Figure 4A](i) Percentage of human cells determined by human CD45 expression in mouse bone marrow aspirates (BMA) at 10 and 16 weeks post-transplantation of enlarged cells (each dot represents the percentage of human CD45 cells in the BMA of an individual animal), and (ii) Human hematopoietic cell lineages within engrafted human CD45-positive cells, analyzed in mouse BMA using human lineage-specific antibodies. [Figure 4B] (i) the percentage of human cell engraftment in mouse bone marrow after animal sacrifice at 18 weeks post-transplantation, as determined by human-specific CD45 antibody, and (ii) the percentage of human LT-HSC cells (CD34+ / CD38- / CD45RA- / CD90+ / CD49f+) within the population of engrafted human CD45 cells. [Figure 5]A: UMAP plot of sample density by protocol, B: UMAP of Leiden clustering assignment, C: Dot plot of cluster markers using log-normalized expression by color and percentage of cells expressing genes in clusters by size. High expression of CD34, SPINK, and JUN indicates clusters 1, 4, and 6, consisting of potential LT-HSC / HSPCs, while clusters 5 and 9 consist of circulating HSPCs. D: Violin and box plots of U statistics for HSC signatures (CD34+, PROCR+, CD38-) show a higher score of abundance in PTC13325.2 compared to control expanded medium and PTC13303. The box plot of abundance variation analysis as neighborhood log2(magnification change) distribution per cluster, colored by E: log2(magnification change) and sized by spatial FDR (sFDR), shows the median (thick bar) and the first and third quartiles (boxes) with a 1.5* interquartile range (whiskers), colored by E: log2(magnification change), and shows statistically significant positive abundance of LT-HSC / HSPC in cultures treated with PTC13325.2 compared to control expansion medium. ns = not significant, sFDR > 0.05 (GMP: granulocyte-monocyte progenitor cells, HSPC: hematopoietic stem cells and progenitor cells, MKP: megakaryocyte progenitor cells, MEP: megakaryocyte / erythrocyte progenitor cells, MEMP: megakaryocyte / erythrocyte / mast cell precursors, DC: dendritic cells). [Figure 6A] We will compare protocols Ctrl, PTC13303, and PTC13325.2 ex vivo using UCBs from up to six independent donors (n=4-6). [Figure 6B] We compare the magnification changes in the number of primitive, engraftable HSCs ex vivo for protocols Ctrl, PTC13303, and PTC13325.2 (n=4~6). [Figure 6C]We will compare the expression of the phenotypic markers, (i) CFD49f and (ii) EPCR, of LT-HSCs in CD34+CD38-CD45RA-CD90+ and CD34+CD38-CD45RA-CD90- cell populations in ex vivo enlarged cells using protocols Ctrl, PTC13303, and PTC13325.2. [Figure 7A] We will compare protocol Ctrl, RET agonist, and PTC13325.3 ex vivo using UCB from up to six independent donors (n=4-6). [Figure 7B] We compare the magnification changes in the number of primitive, engraftable HSCs ex vivo using protocol Ctrl, RET agonist, and PTC13325.3 (n=4~6). [Figure 7C] This shows the expression of (i) CD49f and (ii) EPCR, phenotypic markers for LT-HSCs, in CD34+CD38-CD45RA-CD90+ and CD34+CD38-CD45RA-CD90- cell populations in ex vivo enlarged cells according to protocol PTC13325.3. [Figure 7D] This shows the evaluation of the number of total nucleated cells (TNTs) produced relative to the number of cells in the LT-HSC compartment for four independent donors, using protocols Ctrl, PTC13303, PTC13325.2, and PTC13325.3. [Figure 7E] The left image shows the total number of scored colonies for cells expanded ex vivo using protocols Ctrl, PTC13303, RET agonist, PTC13325.2, and PTC13325.3 (n=3), and the right image shows the total number of scored CFU-GEMM colonies for cells expanded ex vivo. [Modes for carrying out the invention]

[0027] As used herein, the terms hematopoietic stem cells and progenitor cells (HSPCs) refer to cells found in bone marrow, umbilical cord blood, and peripheral blood that can differentiate and / or proliferate to form blood cells, and examples of blood cells include, but are not limited to, monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, dendritic cells, megakaryocytes, platelets, T cells, B cells, and natural killer cells.

[0028] As used herein, the terms “hematopoietic stem cell” or “HSC” refer to pluripotent or pluripotent cells that have the ability to differentiate into all lineages of blood cells and to regenerate themselves while maintaining their pluripotency.

[0029] As used herein, the term “long-term hematopoietic stem cells (LT-HSCs)” refers to self-renewing hematopoietic stem cells (HSCs). That is, LT-HSCs can sustain the hematopoietic system throughout the life of an animal. LT-HSCs preferably include Lin-, CD34+, CD133+, CD38-, CD45RA-, CD90+, CD49f+, and / or CD201+ (EPCR). In the terms “CD34+”, “CD133+”, “CD90+”, and “CD49f+”, the (+) notation indicates that the specified differentiation antigen (CD) is expressed by the cell and present on the cell surface. In the terms “CD38-”, and “CD45RA-”, the (-) notation indicates that the specified CD is not expressed by the cell or is poorly expressed. However, human embryonic stem cells and any cells resulting from the destruction of human embryos are not within the scope of this invention.

[0030] As used herein, the term “isolated population” refers to a sample of cells obtained from a source, where the cells may be commercially obtained or obtained from the subject. Sources of isolated populations are not limited to, but include, umbilical cord blood, bone marrow, and peripheral blood. “Isolated populations” may be obtained from fresh or frozen sources, with fresh sources not frozen before use. If the sample is frozen, the cells are thawed before use in this method.

[0031] As used herein, the term “cultured population” refers to an isolated population of cells grown ex vivo in an artificial medium. The type of artificial medium to be used will be obvious to those skilled in the art, and an example of a suitable medium is StemSpan ACF medium (Stem Cell Technologies), which may be referred to herein as the base medium or basal medium. The artificial medium may also be supplemented with other factors or cytokines to improve cell growth, and examples of supplements, though not limited to, include stem cell factor (SCF), fms-related tyrosine kinase 3-ligand (FLT3LG), and thrombopoietin (TPO). It may also be supplemented with IL-6. The isolated population can be cultured / grown over several days to form a cultured population. In some embodiments, the culture time is 2 to 20 days, more preferably 3 to 20 days, and most preferably 4 to 15 days. For example, culture / grown can be carried out over 20, 15, 10, 9, 8, 7, 6, 5, or 4 days. The total culture time includes the time required to perform steps (ii), (iii), and (iv).

[0032] As used herein, the term c-Jun N-terminal kinase (JNK) inhibitor refers to a compound that inhibits the activity of JNK. JNK belongs to the mitogen-activated protein kinase family and responds to stress stimuli such as cytokines, ultraviolet irradiation, heat shock, and osmotic shock. They play a role in T cell differentiation and cellular apoptosis pathways. As used herein, the term JNK inhibitor refers to a compound that can inhibit the activity of any JNK.

[0033] Examples of JNK inhibitors, though not limited to them, include SP600125, AS601245, JNK-IN-8, JNK-IN-7, JNK Inhibitor VIII, IQ-3, DB07268, IQ-1S, and AS602801. In some embodiments, the JNK inhibitor is SP600125.

[0034] As used herein, the term aryl hydrocarbon receptor antagonist (AhR antagonist) refers to a compound that interferes with or inhibits the activity of an aryl hydrocarbon receptor. AhRs are members of a family of basic helix-loop-helix transcription factors that regulate gene expression as sensors for heterologous biochemicals such as aryl hydrocarbons, and as regulators of enzymes such as cytochrome P450.

[0035] Examples of AhR antagonists, though not limited to them, include SR1, PD98059, GNF351, BAY2416964, CH-223191, PDM-11, and BAY-218. In some embodiments, the AhR inhibitor is SR1.

[0036] As used herein, the term histone deacetylase inhibitor (HDAC inhibitor) refers to a compound that inhibits the activity of the enzyme histone deacetylase. Histone deacetylases are classified into four classes: Class I, Class II, Class III, and Class IV. Based on their sequence homology and domain organization, Class II inhibitors can be further subdivided into Class IIa and Class IIb. As used herein, the term histone deacetylase inhibitor refers to a compound that can inhibit the activity of any of the classes of histone deacetylase.

[0037] Examples of HDAC inhibitors, though not limited to them, include scriptide, vorinostat, takezinarin, RG2833, RGFP966, trichostatin A, LMK235, tubastatin A, xinostat, LBH589, PXD101, ITF2357, PCI-24781, FK228 MS-275, MGCD0103, sodium phenylbutyrate, valproic acid, AN-9, Baceca, and Savicol.

[0038] As used herein, the term receptor tyrosine kinase (RTK) agonist refers to a compound that enhances the activity of enzyme receptor tyrosine kinases. RTKs play crucial roles in cellular processes, including growth, motility, differentiation, and metabolism, by catalyzing phosphoryl transfer to tyrosine residues in protein substrates using ATP as a phosphate donor. Therefore, dysregulation of RTK signaling leads to the classification of human diseases, including cancer. There are 20 classifications of RTKs.

[0039] Examples of RTK classifications, though not restricted, include the RET receptor family. Examples of RET agonists, though not restricted, include GDNF, GFRα1, BT-13, Q525, and BT44.

[0040] One aspect of the present invention is the formula RNH(CnH 2n )NH(C n H 2n The use of an aminothiol compound having SX, wherein R is hydrogen, aryl, acyl, or an alkyl group containing 1 to 7 carbon atoms, each n having a value of 2 to 6, and X is H or PO3H2, or a pharmaceutically acceptable salt thereof.

[0041] As used herein, “aryl” means monocyclic, bicyclic, or tricyclic monovalent or divalent (where appropriate) aromatic radicals such as phenyl, biphenyl, naphthyl, and anthracenyl, which are optionally, preferably, C1-C6 alkyl, hydroxy, C1-C3 hydroxyalkyl, C1-C3 alkoxy, C1-C3 haloalkoxy, amino, C1-C3 monoalkylamino, C1-C3 bisalkylamino, C1-C3 acylamino, C1-C3 aminoalkyl, mono(C1-C3 alkyl)aminoC1-C3 alkyl, and bis(C It can be substituted with up to five substituents selected from the group consisting of 1-C3 alkyl)amino C1-C3 alkyl, C1-C3-acylamino, C1-C3 alkylsulfonylamino, halo, nitro, cyano, trifluoromethyl, carboxy, C1-C3 alkoxycarbonyl, aminocarbonyl, monoC1-C3 alkylaminocarbonyl, bisC1-C3 alkylaminocarbonyl, -SO3H, C1-C3 alkylsulfonyl, aminosulfonyl, monoC1-C3 alkylaminosulfonyl, and C1-C3 alkylaminosulfonyl.

[0042] As used herein, “alkyl” means a C1-C7 alkyl group that can be linear or branched. Preferably, it is a C1-C6 alkyl moiety. More preferably, it is a C1-C4 alkyl moiety. Examples include methyl, ethyl, n-propyl, and t-butyl. It may be a divalent, for example, propylene.

[0043] As used herein, acyl is an alkyl group as defined above, containing a carbonyl group (C=O).

[0044] Each of the alkyl and acyl groups may optionally be substituted with an aryl, cycloalkyl (preferably C3-C10), or heteroaryl group. They may also be substituted with halogens (e.g., F, Cl), NH2, NO2, or hydroxyl groups.

[0045] As used herein, the term “umbilical cord blood” has its conventional use in the art, namely, generally the blood remaining in the umbilical cord and placenta after childbirth. Human umbilical cord blood is within the scope of the present invention and can be obtained with prior written consent and ethical approval.

[0046] As used herein, the term “peripheral blood” has its conventional use in the art, i.e., generally refers to blood circulating throughout the circulatory system. Human peripheral blood is within the scope of the present invention and can be obtained with prior written consent and ethical approval.

[0047] As used herein, the term “bone marrow” has its conventional use in the art, namely, the gelatinous tissue present in bone cavities. The tissue includes red bone marrow, a subset of bone marrow having populations of hematopoietic stem cells, progenitor cells, and precursor cells. Human bone marrow is within the scope of the present invention and can be obtained with prior written consent and ethical approval.

[0048] As used herein, the term “enlarged cells” refers to cells that have been cultured ex vivo under appropriate conditions and are undergoing cell division to increase their number.

[0049] As used herein, the term “cell expansion” refers to the amplification of the number of cells by ex vivo culture of cells under appropriate conditions, where the number of cells present at the end of the culture is greater than the number of cells present at the start of the culture.

[0050] Within a cell, which may be an isolated population of cells, a cultured population of cells, or part of an expanded cell, there exist cell subtypes. Examples of cell subtypes, though not restricted, include hematopoietic stem cells, hematopoietic progenitor cells, and cells defined by their phenotypic markers. Non-restrictive examples of phenotypic markers are Lin, or CD38, or CD34, or CD133, or CD45RA, or CD90, or CD49f, or CD201, and cells can also be defined by combinations of these phenotypic markers. As used herein, the term “rich” is used to refer to a set of cells that contain a high proportion of a particular subset / subtype of cells, and a set of cells may contain 2%, or 5%, or 10%, or 15%, or 20%, or 25%, or 30%, or 35%, or 40%, or 45%, or 50%, or 55%, or 60%, or 65%, or 70%, or 75%, or 80%, or 85%, or 90% of a particular subset / subtype of cells. Within the present invention, the term “rich” can be used to refer to a population of cells that have undergone expansion and in which a particular subtype of cells has increased in number proportionally to the other cells in the population. This abundant population of cells contains a significant proportion of a particular cell subtype, which may be 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the total population.

[0051] As used herein, the term “serum-free tissue culture system” refers to culturing cells in a culture medium that is not supplemented with animal-derived serum.

[0052] As used herein, the term “feeder-free tissue culture system” refers to a method of culturing cells without utilizing a layer of connective tissue cells to support growing cells and provide metabolites.

[0053] As used herein, the term “total cell expansion” refers to an increase in the total number of nucleated cells.

[0054] As used herein, the term “total culture time” refers to the time during which steps ii), iii), and iv) are performed. Here, step ii) comprises culturing an isolated population of HSPCs for at least 48 hours to form a first cultured population; step iii) comprises adding a histone deacetylase inhibitor (HDAC inhibitor) to form a second cultured population; and step iv) comprises an aminothiol compound having the formula RNH(C n H 2n )NH(C n H 2n The process involves adding an aminothiol compound, or a pharmaceutically acceptable salt thereof, to a cultured population of HSPCs, having SX, where R is hydrogen, aryl, acyl, or an alkyl group containing 1 to 7 carbon atoms, each n having a value of 2 to 6, and X is H or PO3H2, to form enlarged cells. The cells are grown on a suitable medium, such as basal medium, for the total culture time, and the end of the total culture time is indicated by the cells being harvested, pooled, or analyzed.

[0055] As used herein, the term “pre-culture step” refers to step ii), which includes culturing an isolated population of HSPCs for at least 48 hours to form a first cultured population, and is separate from the isolation step of step i).

[0056] As used herein, the term “subject” refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, dogs, cats, rodents, etc., that is a recipient of therapy using the present invention. In particular, human subjects are envisioned. “Patient” is used herein to refer to a human subject.

[0057] The embodiments of the present invention are defined above. The embodiments described below are applicable to all embodiments of the present invention.

[0058] The enlarged population of cells produced by the methods described herein may be rich in HSCs, and these enlarged cells have been shown to possess long-term engraftment ability and the ability to regrow mammalian bone marrow.

[0059] The kit of the present invention is described herein. In preferred embodiments, the kit also contains apparatus and / or materials for obtaining isolated populations of HSPCs. Those skilled in the art will know of suitable elements, apparatus, and / or materials. Examples include magnetic bead isolation, MACS bead isolation column, CliniMACS (Miltenyi), or FACS sorting.

[0060] In one embodiment of the present invention, the therapy or treatment is the therapy or treatment of a hematological disorder, an immunological disorder, a metabolic disorder, or a neurodegenerative disorder.

[0061] In some embodiments, the therapy or treatment includes regrowth of mammalian bone marrow.

[0062] In preferred embodiments, the therapy or treatment is for acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, non-Hodgkin lymphoma, severe aplastic anemia, severe combined immunodeficiency, or sickle cell disease.

[0063] In one embodiment, the therapy or treatment is gene therapy.

[0064] In one embodiment, the treatment method further includes the step of administering a target to enlarged cells.

[0065] In one embodiment, step i) further includes selecting cells that are CD133+ and / or CD34+. In some embodiments, the isolated population includes cells that are CD133+ and / or CD34+.

[0066] In some embodiments of the present invention, step i) comprises culturing the cells for about 12 to about 36 hours, preferably 24 hours. This allows them to recover from harvesting, transport, cooling, freezing, thawing, or any other such operations that may precede a pre-culture step (step ii). This culture may be referred to in the art as “recovery” or “priming.” Preferably, the cells are recovered in StemSpan ACF medium (Stem Cell Technologies) containing 100 ng / mL SCF, 100 ng / mL FLT3LG, and 20 ng / mL TPO (all from Miltenyi Biotec) in a 96-well round-bottom suspension plate in a humidified incubator at 37°C containing 5% CO2.

[0067] When isolated populations of HSPCs are obtained from a frozen source, it may be preferable to select cells that are CD34+. When isolated populations of HSPCs are obtained from a fresh source, it may be preferable to select cells that are CD133+. Suitable methods for selecting cells by cell surface markers, for example, by using magnetically activated cell sorting (MACS) or fluorescence-activated cell sorting (FACS), are known in the art. Preferably, the isolated population includes cells that are CD38-, or CD34+, or CD133+, or CD45RA-, or CD90+, or CD49f+, or any combination thereof.

[0068] In some embodiments of the present invention, the culture process in step ii) is separate from the isolation (i.e., recovery or priming) process in step i).

[0069] In some embodiments of the present invention, step ii) includes culturing an isolated population of HSPCs for a period of time sufficient to increase the total number of viable nucleated cells. In some embodiments, the total number of CD34+ cells will decrease.

[0070] In some embodiments of the present invention, step ii) includes culturing the cells for about 48 hours to about 96 hours, preferably 72 hours.

[0071] In one embodiment, the cells in the first cultured population are less pluripotent than the isolated population of HSPCs, and the number of cells in the first cultured population is increased.

[0072] In one embodiment, the cells in the first cultured population proliferate more than the cells in the second cultured population.

[0073] In one embodiment, step iii) includes adding an HDAC inhibitor when the cells from the first cultured population have become less pluripotent than the isolated population of HSPCs.

[0074] In some embodiments of the present invention, step iii) includes culturing the cells for about 24 hours to about 72 hours, preferably 48 to 72 hours.

[0075] In some embodiments, the cells in the first cultured population in step ii) proliferate more than the cells in the second cultured population in step iii).

[0076] In some embodiments of the present invention, step iv) includes culturing the cells for about 16 to about 24 hours, preferably 24 hours.

[0077] In some embodiments of the present invention, step ii) includes culturing cells in the presence of a basal medium.

[0078] In some embodiments of the present invention, step (ii) comprises culturing cells in the presence of compounds known to expand CD34+CD38-HSPC. Suitable compounds are listed in the following documents, incorporated herein by reference: Sakurai Masatoshi et al: “Chemically defined cytokine-free expansion of human haematopoietic stem cells” (Nature 2023), and Saiyin et al: “Clinical Outcomes of Umbilical Cord Blood Transplantation Using Ex Vivo Expansion: A Systematic Review and Meta-Analysis of Controlled Studies” (Transplantation and Cellular Therapy 2022). In some embodiments, step (ii) comprises culturing cells in the presence of nicotinamide, pyrimide-indole derivatives such as UM171 and UM729, p38MAPK inhibitors, Notch ligands, Wnt agonists, JNK inhibitors, cytokines, and / or AhR antagonists.

[0079] In one embodiment of the present invention, step ii) includes culturing cells in the presence of a JNK inhibitor.

[0080] In some embodiments of the present invention, step ii) further comprises culturing cells in the presence of an aryl hydrocarbon receptor antagonist.

[0081] In some embodiments of the present invention, step ii) further comprises culturing cells in the presence of cytokines, preferably interleukins, more preferably interleukin-6 (IL-6).

[0082] In one embodiment of the present invention, the JNK inhibitor is selected from SP600125, AS601245, JNK-IN-8, JNK-IN-7, JNK Inhibitor VIII, IQ-3, DB07268, IQ-1S, and AS602801. In a preferred embodiment, the JNK inhibitor is SP600125.

[0083] In one embodiment of the present invention, the AhR antagonist is selected from SR1, PD98059, GNF351, BAY2416964, CH-223191, PDM-11, and BAY-218. In a preferred embodiment, the AhR antagonist is SR1.

[0084] In another embodiment of the present invention, step ii) further comprises culturing cells in the presence of at least one, preferably at least two, RET agonists.

[0085] In one embodiment of the present invention, at least one, preferably at least two, RET agonists are selected from GDNF, GFRα1, BT-13, Q525, and BT44. In a preferred embodiment, at least one, preferably at least two, RET agonists are selected from GDNF and GFRα1. In some embodiments of the present invention, two RET agonists are used in the culture of step ii).

[0086] In one embodiment of the present invention, the HDAC inhibitor is selected from a broad-spectrum inhibitor or a selective class I, class IIa, class IIb, class III, or class IV inhibitor. Preferably, the HDAC inhibitor is selected from a broad-spectrum inhibitor or a selective class I, class Iia, class III, or class IV inhibitor. More preferably, the HDAC inhibitor is a broad-spectrum inhibitor, a class I, or a class IIa inhibitor.

[0087] In preferred embodiments, the HDAC inhibitor is selected from scriptides, RG2833, RGFP966, LMK235, tubastatin A, xynostat, and sodium phenylbutyrate. In some embodiments of the present invention, the HDAC inhibitor is a scriptide or xynostat. In preferred embodiments, the HDAC inhibitor is a scriptide having the following structure. [ka]

[0088] In a preferred embodiment, R is hydrogen.

[0089] In some embodiments, the aminothiol compound of the present invention is amifostin [ka] Or WR1065 [ka] That is the case.

[0090] In some embodiments of the present invention, the cells are washed between steps ii) and iii), and preferably, the cell culture medium in stage iii) or later is substantially free of JNK inhibitors and / or aryl hydrocarbon antagonists.

[0091] In one embodiment of the present invention, the JNK inhibitor is used at a concentration of 0.01 μM to 50 μM, preferably 0.1 μM to 10 μM, and more preferably at a concentration of 0.2 μM.

[0092] In one embodiment of the present invention, the antagonistaryl hydrocarbon acceptor is used at a concentration of 0.01 μM to 50 μM, preferably 0.1 μM to 10 μM, and more preferably at a concentration of 1 μM.

[0093] In one embodiment of the present invention, IL-6 is used at a concentration of 0.01 μg / ml to 50 μg / ml, preferably 0.05 μg / ml to 10 μg / ml, and more preferably the antagonistaryl hydrocarbon acceptor is used at a concentration of 0.1 μg / ml.

[0094] In one embodiment of the present invention, at least one, preferably at least two, RET agonists are used at a concentration of 100 ng / ml. If two or more RET agonists are present, they are used in approximately equal amounts (1:1) at a concentration of 100 ng / ml.

[0095] In one embodiment of the present invention, the HDAC inhibitor is used at a concentration of 0.01 μM to 50 μM, preferably 0.1 μM to 10 μM, and more preferably at a concentration of 0.3 μM.

[0096] In preferred embodiments of the present invention, the aminothiol compound, for example WR1065, is used at a concentration of 50 μM to 500 μM, preferably 50 μM to 150 μM.

[0097] In some embodiments of the present invention, steps ii), iii), and iv) are carried out over a total time sufficient for the isolated population of HSPCs to form enlarged cells.

[0098] In some embodiments of the present invention, steps ii), iii), and iv) are carried out over a period of 2 to 10 days, preferably 3 to 8 days. In preferred embodiments, steps ii), iii), and iv) are carried out over a period of 3 to 8 days. In more preferred embodiments, steps ii), iii), and iv) are carried out over a period of about 5 days.

[0099] In one embodiment of the present invention, steps iii) and iv) are initiated up to 72 hours before the end of the total culture time (i.e., the end of step iv).

[0100] In some embodiments of the present invention, step iv) is started 16 to 24 hours before the end of the total culture time.

[0101] Preferably, step iii) is performed 48 to 72 hours before the end of the total culture time, and step iv) is performed 24 hours before the end of the total culture time.

[0102] Preferably, cells are cultured in a serum-free tissue culture system. In some embodiments of the present invention, cells are cultured in a feeder-free tissue culture system. The culture system does not contain serum and / or feeders, but various nutrients may be added to provide cells with appropriate growth and expansion conditions. Examples of suitable media include, but are not limited to, StemSpan ACF medium (Stem Cell Technologies), StemPro34 serum-free medium (Invitrogen), Stemline II (Thermo Fisher), HPC expansion medium DXF (PromoCell), QBSF-60 (Quality Biological), and StemMACS HSC expansion medium XF (Miltenyi Biotec). In a preferred embodiment of the present invention, cells are cultured in StemSpan ACF medium (Stem Cell Technologies). The suitable medium may also contain various additives and components, which may be chemical or biological components. These components may be incorporated into the suitable medium individually or in combination, and those skilled in the art will be able to select suitable components as needed. These components may also be incorporated during culture as needed. Examples of both biological and chemical components, though not limited to them, include amino acids, vitamins, cytokines, growth factors, hormones, antibiotics, fatty acids, sugars, sodium, calcium, potassium, magnesium, phosphorus, agar, agarose, methylcellulose, collagen, insulin, transferrin, lactoferrin, cholesterol, ethanolamine, sodium pyruvate, 2-mercaptoethanol, polyethylene glycol, and sodium selenite.

[0103] Various cytokines can be incorporated into the culture medium and / or during culture. Examples of suitable cytokines are, but are not limited, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-13 (IL-13), and Examples include tarleukin-14 (IL-14), interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), interferon-α (INF-α), interferon-β (INF-β), interferon-γ (INF-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), Wnt1, bone morphogenetic protein 7 (BMP7), angiopoietin-like protein 5 (ANGPTL5), insulin growth factor-binding protein 2 (IGFBP2), erythropoietin (EPO), thrombopoietin (TPO), and Fms-like tyrosine kinase 3-ligand (FLT3LG). In one embodiment of the present invention, the culture medium is supplemented with SCF, TPO, and FLT3LG.

[0104] Various growth factors can be incorporated into the culture medium and / or during culture. Examples of suitable growth factors, though not limited to them, include insulin-like growth factor (IGF), epidermal growth factor (EGF), human epidermal growth factor (hEGF), platelet-derived growth factor (PDGF), fibroblast growth factor 1 (FGF1), nerve growth factor (NGF), macrophage inflammatory protein 1-α (MIP-1α), and leukemia inhibitory factor (LIF).

[0105] In one embodiment, the isolated population is cultured at a temperature of 32°C to 39°C, preferably 36°C to 38°C. In another embodiment, the cells are cultured in a humidified incubator containing about 1% to about 50% CO2, preferably about 1% to about 25% CO2, more preferably about 1% to about 10% CO2. The present invention can be carried out in culture vessels suitable for animal cell culture. In one embodiment, the present invention is carried out in a Nanex hematopoietic stem / progenitor cell (HSPC) expansion plate, or a TC-treated Corning 24-well plate, or a suspension Greiner Bio 24-well plate. In a preferred embodiment, the present invention is carried out in a conventional cell culture plate, or in a suitable closed system such as a cell culture bag (e.g., VueLife®) or a stirred bioreactor.

[0106] In one embodiment of the present invention, the enlarged cells are rich in hematopoietic stem cells (HSCs) and long-term hematopoietic stem cells (LT-HSCs). In some embodiments, the enlarged cells are rich in Lin-, or CD38-, or CD34+, or CD133+, or CD45RA-, or CD90+, CD201+, or CD49f+, or CD201, or any combination thereof. Preferably, the cells are rich in CD34+, CD133+. More preferably, the enlarged cells are rich in CD38-, CD34+, CD133+. Most preferably, the enlarged cells are rich in Lin-, CD38-, CD34+, CD133+, CD45RA-, CD90+, CD49f+, and preferably CD201.

[0107] In one embodiment of the present invention, the enlarged cells are rich in Lin-, CD38-, CD34+, CD133+, CD45RA-, CD90+, CD201+, and CD49f+, or any combination thereof.

[0108] In preferred embodiments of the present invention, the total cell expansion is approximately 2 to 50 times, or approximately 5 to 50 times, or approximately 10 to 20 times. The total cell expansion is determined by measuring the total number of nucleated cells at the start of the culture time and comparing it to the total number of nucleated cells present at the end of the culture time.

[0109] In preferred embodiments, the expansion of Lin-, CD38-, CD34+, CD133+, CD45RA-, CD90+, CD201+, and / or CD49f+ cells is 200 to 2,000 times, more preferably 400 to 1,000 times, and most preferably 800 times. The expansion of Lin-, CD38-, CD34+, CD133+, CD45RA-, CD90+, CD201+, and / or CD49f+ cells is determined by measuring the number of Lin-, CD38-, CD34+, CD133+, CD45RA-, CD90+, CD201+, and / or CD49f+ cells present at the start of culture and comparing it to the number of Lin-, CD38-, CD34+, CD133+, CD45RA, CD90+, CD201+, and / or CD49f+ cells present at the end of culture. In one embodiment, the enlargement of CD34+, CD45RA-, and CD90+ cells is 50 to 800 times, more preferably 400 to 600 times, and most preferably 500 times. The enlargement of CD38-, CD34+, CD45RA-, and CD90+ cells is determined by measuring the number of CD38-, CD34+, CD45RA-, and CD90+ cells present at the start of the culture time and comparing it to the number of CD38-, CD34+, CD45RA-, and CD90+ cells present at the end of the culture time.

[0110] Preferred methods for determining cell expansion are known in the art and include, for example, multicolor flow cytometry combined with total cell counting, the use of absolute counting beads combined with flow cytometry, and cell counting based on imaging analysis of cell aliquots using manual or automated hemocytometers (Viacell, Countess, Nucleocounter, Nexcelome).

[0111] Disclosed herein are an expanded population of cells, preferably HSCs, which are rich in Lin-, CD38-, CD34+, CD45RA-, CD90+, CD201+, and / or CD49f+.

[0112] In one embodiment of the present invention, an isolated population of cells is obtained from umbilical cord blood, bone marrow, or peripheral blood.

[0113] In a preferred embodiment, an isolated population of cells is obtained from umbilical cord blood.

[0114] In some embodiments, the cells are obtained from mammals (e.g., mice, rats, dogs, or humans). Embodiments in which the cells are obtained from humans are preferred.

[0115] As described above, methods for producing enlarged cells for therapeutic use, methods of treatment, and use in the manufacture of pharmaceuticals may include any of the additional features provided herein.

[0116] Cells enlarged by the methods presented herein can be used as cell transplants. Cells enlarged by the methods presented herein can be used to regrow mammalian bone marrow. Thus, one embodiment of the present invention is an enlarged population of cells for use in the treatment of hematological, immunological, metabolic, or neurodegenerative disorders. Here, the subject is administered an enlarged population of cells enlarged according to the methods described above. In a particular embodiment, the enlarged population of cells is for use in the treatment of hematological disorders.

[0117] Enlarged populations of cells can be used as grafts for hematopoietic stem cell therapy, as an alternative to conventional bone marrow, umbilical cord blood, or peripheral blood transplants. Transplantation of enlarged populations of cells can be performed in the same manner as conventional bone marrow, umbilical cord blood, or peripheral blood transplants. The graft may contain the enlarged population of cells along with one of the following components: buffer, antibiotic, or pharmaceutical compound.

[0118] Examples of disorders that can be treated using an expanded population of cells include acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, non-Hodgkin lymphoma, severe aplastic anemia, severe combined immunodeficiency, sickle cell disease, chronic granulomatosis, severe combined immunodeficiency syndrome, adenosine deaminase (ADA) deficiency, agammaglobulinemia, immunodeficiency syndromes such as Wiscott-Aldrich syndrome, Chediak-Higashi syndrome, and acquired immunodeficiency syndrome (AIDS), congenital anemia such as C3 deficiency and thalassemia, hemolytic anemia due to enzyme deficiency and sickle cell anemia, lysosomal storage disorders such as Gaucher disease and mucopolysaccharidosis, adrenoleukodystrophy, various types of cancer and tumors, particularly hematological malignancies such as acute or chronic leukemia, Fanconi syndrome, aplastic anemia, and granulocyte anemia. Gramulocytopenia, lymphopenia, thrombocytopenia, idiopathic thrombocytopenic purpura, thrombotic thrombocytopenic purpura, Kasabach-Merritt syndrome, malignant lymphoma, Hodgkin's disease, multiple myeloma, chronic liver disease, renal failure, massive blood transfusions in blood banks or during surgery, hepatitis B, hepatitis C, severe infections, systemic lupus erythematosus, rheumatoid arthritis, xerodermosteosis, systemic sclerosis, polymyositis, dermatomyositis, mixed connective tissue disease, polyarteritis nodosa, Hashimoto's disease, Graves' disease, myasthenia gravis, insulin-dependent diabetes mellitus, autoimmune hemolytic anemia, snake bite, hemolytic uremic syndrome, disseminated erythema, hemorrhage, Bernard-Souliers syndrome, Glanzmann thrombocytopenia, uremia, myelodysplastic syndrome, polycythemia vera These include rubra vera, erythremia, essential thrombocythemia, myeloproliferative disorders, traumatic spinal cord injury, nerve injury, neuritis, skeletal muscle injury, scarring, diabetes mellitus, cerebral infarction, myocardial infarction, and obstructive arteriosclerosis.

[0119] The enlarged population of cells may be administered via the following routes: subcutaneous, intraparietal, intramuscular, intravenous, intratumoral, intraocular, intraretinal, intravitreous, or intracranial.

[0120] The enlarged population of cells may be combined with pharmaceutically acceptable excipients, diluents, or carriers to improve and enhance administration, stability, homogeneity, bioavailability, or any combination thereof. In certain embodiments, the extracellular vesicles or cells of this disclosure are administered suspended in a sterile solution. In certain embodiments, the solution contains 0.9% NaCl. In certain embodiments, the solution further comprises a buffer, e.g., acetate, citrate, histidine, succinate, phosphate, bicarbonate, or hydroxymethylaminomethane (Tris); a surfactant, e.g., polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), or poloxamer 188; a polyol / disaccharide / polysaccharide, e.g., glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, or dextran 40; an amino acid, e.g., glycine or arginine; an antioxidant, e.g., ascorbic acid or methionine; and a chelating agent, e.g., one or more of EGTA or EGTA.

[0121] The enlarged population of cells produced by this method can be used in gene therapy. To introduce the therapeutic gene into a patient, the gene of interest should be transfected into isolated HSC populations. The therapeutic gene can be introduced using a viral or nonviral method. Suitable viral vectors include retroviruses, adenoviruses, adeno-associated viruses, and herpes simplex viruses. Cells containing the gene of interest can be enlarged according to this method before being introduced into a patient.

[0122] The following examples illustrate the present invention.

[0123] [Examples]

[0124] Protocol for cell expansion Introduction Umbilical cord blood (UCB) is a valuable source of hematopoietic stem cells (HSCs) for patients requiring allogeneic transplantation, particularly ethnic minority patients for whom identifying HLA-matched donors is difficult. Despite its ability to reduce the strictness of HLA matching and provide superior overall survival and relapse-free survival, UCB transplantation is constrained by the limited number of HSCs in each UCB unit, the variability in expansion between UCB units, and delayed hematopoietic recovery compared to GCSF-mobilized peripheral blood or BM transplantation.

[0125] There are different approaches to expanding UCB CD34+ cells without using regenerative capabilities.

[0126] Currently, there are two companies offering expanded UCB: ExCellthera and Gamida Cell (the latter being the most advanced, in Phase 3 clinical trials).

[0127] material and method cell We obtained cryopreserved human umbilical cord blood (UCB) cells rich in CD34+ cells (after sorting with magnetic beads). All experiments were performed starting from thawed frozen cell stocks.

[0128] material 1. Base medium: StemSpan (Stem Cell Technologies) 2. Growth factors [Table 1] 3. Chemical compounds [Table 2]

[0129] Protocol for cell expansion Day 0: CD34-positive UCB cells were thawed and pooled in StemSpan ACF medium (Stem Cell Technologies) containing 100 ng / mL SCF, 100 ng / mL FLT-3L, and 20 ng / mL TPO (all from Miltenyi Biotec). After counting, the cells were plated in the above medium at 20,000 cells / well in 96-well round-bottom suspension plates. The cells were recovered overnight under these conditions in a humidified incubator at 37°C with 5% CO2.

[0130] Day 1: Recovered CD34-positive cells were harvested from the counted plates and seeded at a rate of 20,000 cells / well in 0.5 mL of basal medium containing the following components into 24-well Nanex plates (Compass Biomedical) or standard tissue culture-treated plates. [Table 3]

[0131] PCT13303 is disclosed in WO2020 / 084310 and WO2020 / 084310, and is incorporated herein for comparative purposes. PTC13303 lacks step (ii) of the present invention, namely the pre-culture step before adding HDAC and aminothiol.

[0132] Day 4: Cells were collected separately from each well, centrifuged, and placed in fresh culture medium according to the corresponding protocol. [Table 4]

[0133] Day 6: WR-1065 was directly added to wells containing cell cultures for protocols PTC13303 (comparison), PTC13325.1, PTC13325.2, and PTC13325.3 up to a final concentration of 100 μM for up to 24 hours. Cells from the control group were left untreated.

[0134] Day 7: Cells were collected from individual wells, centrifuged, and resuspended in culture medium / buffer as needed, following the procedure below. 1. Flow cytometry 2. Colony assay 3.Animal transplantation 4. RNA sequencing

[0135] Flow cytometry of enlarged cells Cells were collected, washed, and stained in 3% FBS in PBS using a panel of pre-conjugated antibodies as shown in Table 1. Cells were incubated with antibodies on ice for 30 minutes, then washed twice with 3% FBS in PBS, resuspended in 250 μL of buffer, and subsequently analyzed using a BD Canto II flow cytometer (Figure 1). [Table 5]

[0136] Human hematopoietic colony formation assay in semi-solid culture medium CFU assays were performed using the MethoCult Classic kit (Stem Cell Technologies) and the reagents shown in Table 2, following the manufacturer's instructions. Briefly, aliquots of cells (either a known number or volume) were mixed with 3 mL of MethoCult medium, plated using a blunt needle, and injected into one well of a 6-well suspension plate. The plates were incubated for 2 weeks in a humidified incubator at 37°C with 5% CO2 without changing the medium. After 2 weeks, colonies were photographed and counted under a dissecting microscope. The proportion of different types of colonies was then scored, and the proportion / sample was calculated. [Table 6]

[0137] animal transplant NSG mice were gamma-irradiated 24 hours prior to transplantation. For each mouse, the total number of cells expanded in vitro from each well was intravenously transplanted. The expanded cell count was considered equivalent to the initial number of 20,000 pre-expanded cells per well.

[0138] Mouse bone marrow aspirations were performed at weeks 10 and 16 for analysis of human cell engraftment (CD45 content by flow cytometry). Mice were sacrificed 18 weeks after transplantation, bone marrow was collected, and the human cell content was analyzed by flow cytometry using the markers CD34, CD45RA, CD90, and CD49f to determine the percentage of human leukocytes (CD45+) and human LT-HSCs (CD34+ / CD45RA- / CD90+ / CD49f+) in mouse bone marrow.

[0139] Single-cell RNA sequencing Expanded human CD34 cells were sorted for the CD34+ / CD45RA- marker by flow cytometry of LT-HSC cells. This abundant population was processed for single-cell RNA sequencing using standard procedures.

[0140] Donor deconvolution FASTQ files were sorted to the human genome hg38 release 108 using STAR v.2.7.10a and the following parameters: --soloType CB_UMI_Simple --soloUMIlen 12 --outSAMtype BAM SortedByCoordinate --runRNGseed 1 --outSAMattributes NH HI AS nM CB UB GX GN. The sorted BAM files were indexed using samtools v.1.16.1 with the samtools index function.

[0141] I downloaded a custom VCF file as shown in the souporcell GitHub repository (https: / / github.com / wheaton5 / souporcell), which contains SNPs from a 1K genome project filtered by MAF > 0.02.

[0142] I used bcftools v.1.12 to index VCF files, utilizing the bcftools index and bcftools tabs functionality.

[0143] In all protocols, cells were genotyped using cellsnp-lite v1.2.2 with a custom VCF as the reference, barcodes filtered by CellRanger as the input barcodes, and parameters --minMAF 0.1--minCOUNT 20.

[0144] Next, using the same VCF file and N=3 donors as reference, we deconvolved the donors using cellsnp-lite input with vireo v0.2.3.

[0145] We imported the R v.4.2.1 protocol VCF file and matched genotyped donors across the protocol by calculating the Spearman correlation of allele depth (AD) for each donor and each variant of the protocol. Three distinct clusters emerged, which we used to assign matching donor identifications across the protocol.

[0146] Filtering, dimensionality reduction, and integration For quantification, the FASTQ files were sorted and quantified using CellRanger v3 with default parameters. The resulting filtered count matrix was loaded into R using the DropletUtils R / Bioconductor package v1.18.1.

[0147] Donor identification from the deconvolution step was added to each dataset, and cells marked "unassigned," which were doublets and / or cells that failed genotyping, were discarded.

[0148] All counting matrices were pooled together while maintaining sample identification. Cells were retained according to the following criteria for quality control. 1. Total number of readings within the three median absolute deviations (MADs) of the median for each protocol (filtering both low and high readings). 2. Percentage of mitochondrial transcripts within three MADs, median (high filtering) for each control group. 3. Percentage of MALAT1 expression within the three MADs.

[0149] All filtering was performed using the isOutlier function from the scater R / Bioconductor package v1.26.1. This filtering resulted in a total of 16,481 cells.

[0150] Next, the numbers were normalized using the computeSumFactors pooling method from the scran R / Bioconductor package v1.26.1, which includes pre-clustering step clustering within the protocol and donor combination coefficients.

[0151] By fitting nonlinear tendencies to mean-variance relations (blocked by protocol) and retaining the top 2000 genes, highly variable genes were identified and ordered by their residuals from the fit, as implemented in the modelGeneVar from scran.

[0152] For linear dimensionality reduction, log-normalized highly variable genes were used as input to the runPCA function from scran, resulting in 50 principal components. Using the rounded square root of the number of cells as n_neighbors and setting min_dist=0.7, UMAP non-linear dimensionality reduction was performed on the first 20 dimensions of the PCA embedding using the umap function from the uwot R package v0.1.14.

[0153] For the integration, the first 20 main components of PCA were used as input to the fastMNN function from the batcher R / Bioconductor package v1.18.1, using donor and protocol factors combined as a batch. During integration, UMAP reduction was performed again using the same parameters. UMAP was used only for visualization and to quickly assess integration efficiency by comparing plots before and after integration.

[0154] Multi-resolution clustering and cell type annotation First, we set the number of shared neighbors to k=10 and constructed a shared nearest neighbor (SNN) graph in the unified space (first 20 dimensions) by weighting the edges between any two nodes of those shared neighbors using the Jaccard coefficients. This was done using the makeSNNgraph function from the bluster R / Bioconductor package v1.8.0. Next, we applied a community detection algorithm based on the Leiden graph to the SNN using the cluster_leiden function from the igraph R package v1.3.5, with 30 iterations and three different values ​​for the resolution parameter (0.2, 0.5, 0.8, 1).

[0155] Three methods were used to evaluate the most likely clustering resolution. 1. Build a clustering tree using the cluster tree R package v0.5.0 and select resolutions that do not result in crossovers between them. 2. Calculate the pairwise modularity ratio between clusters using the pairwiseModularity function from bluster. 3. As implemented in the approximateSilhouette function from bluster, the distance from each cell to each cluster center point is calculated, and the resolution that maximizes the overall silhouette width is selected to calculate the approximate silhouette width. The combination of these criteria resulted in a clustering resolution of 0.46.

[0156] Identification of cluster markers Using Wilcoxon's rank-sum test and the wilcoxauc function from the presto R package (v.1.0.0), we determined "cluster markers" as genes specifically overexpressed in one cluster compared to the rest of the data. We ordered the genes within each cluster by the difference between the percentage of cells expressing them and the percentage of the rest of the cells in the cluster, and plotted dot plots using the join of the top three genes in each cluster.

[0157] Cell type signature analysis The U-statistic was calculated using the UCell R / Bioconductor package v2.6.2 with CD34+, PROCR+, and CD38- as inputs, with no other changes to the default parameters.

[0158] Differential abundance analysis Using the R / Bioconductor package MiloR (v.1.10.0), we evaluated protocol-related differences in local abundance using multiple donors. Neighborhoods were constructed on the fastMNN-corrected space using k=30, considering only enlarged cells (i.e., excluding cells from uncultured samples). Then, using a design that included both protocol and sex as covariates, the testNhoods function was used to test for differential abundance, with the function automatically performing spatial FDR correction.

[0159] Data visualization All figures of the single-cell analysis results were plotted using the ggplot2 R package (v.3.4.4).

[0160] Example 1: In vitro expansion of human UCB CD34-rich cells using PTC13303, PTC13325.1, and PTC13325.2 increases the number of long-term HSCs. Cells from four independent donors were expanded using protocol Ctrl-control, PTC13303, PTC13325.1, and PTC13325.2. Cells were collected 7 days after expansion. Cell counts resulting from expansion were quantified by flow cytometry compared to bead counts used as an internal control. Cell populations were evaluated by flow cytometry using the antibody panel shown in Table 1.

[0161] As shown in Figure 2A, compared to the control treatment or treatment with PTC13303, treatment with PTC13325.1 resulted in a greater increase in the total number of nucleated cells (TNCs) compared to the input number of cells. Specifically, treatment with PTC13303 reduced the number of TNCs compared to the control treatment.

[0162] The percentage of CD34+ cells increased after treatment with both PTC13303 and PTC13325.1 compared to the control treatment (Figure 2B). However, as shown in the graphs for the two independent donors, the magnitude of this effect may be donor-dependent. Similarly, treatment with both PTC13303 and PTC13325.1 increased the percentage of long-term HSCs (LT-HSCs defined as CD34+ / CD38- / CD45RA- / CD90+ / CD49f+, Table 1, Figure 1), estimated as the percentage of LT-HSCs within the TNC population (Figure 2C(i)) or within the CD34-positive cell population (Figure 2C(ii)). The magnitude of this effect may also be donor-dependent.

[0163] However, when calculating the total number of long-term HSCs using the number of TNCs and the percentage of LT-HSCs within the TNC population, a very clear donor-independent effect is observed. As shown in Figure 2D, treatment with PTC13325.1 dramatically increases the number of LT-HSCs compared to both the control treatment and the PTC13303 treatment.

[0164] PTC13325.1 and PTC13325.2 were compared in vitro using UCBs from two additional donors. Cells were processed using donors 1 and 2, as in previous experiments. By the end of expansion, cells were evaluated by flow cytometry, and their number and phenotype were assessed using two different marker panels: CD34+ / CD38- / CD45RA- / CD90+ / CD49f+ and CD34+ / CD38- / CD45RA- / CD90+ / CD201(EPCR)+. The results are shown in Figure 2E.

[0165] Figure 2E(i) shows the magnification change in the total number of TNCs in two independent UCB donors (Donor 3 and Donor 4) after expansion using protocols Ctrl, PTC13303 (comparison), PTC13325.1, and PTC13325.2, compared to pre-expanded cells. Figure 2E(ii) shows the estimated percentage of Lin- / CD34+ and Lin- / CD34+ / CD45RA- cells in Donor 3 and Donor 4 after treatment with protocols Ctrl, PTC13303, PTC13323.1, and PTC13325.2. Figure 2E(iii) shows the total number of LT-HSCs (defined by either the marker CD34+ / CD38- / CD45RA- / CD90+ / CD49f+ or the marker CD34+ / CD38- / CD45RA- / CD90+ / CD201+ (EPCR)) calculated using the number of TNCs and percentage of LT-HSCs within the TNC population for Donor 3 and Donor 4. Figure 2E(iv) shows the percentage of CD201+ or CD49f+ cells in the population of CD34+ / CD38- / CD45RA- / CD90+ cells (Donor 4) after treatment with Protocol:Ctrl, PTC13303, PTC13323.1 and PTC13325.2.

[0166] Example 2: Hematopoietic colony formation assay in semi-solid medium of expanded cells in vitro A standard in vitro test that provides an estimate of the efficacy of HSCs for engraftment is the hematopoietic colony formation assay in semi-solid medium. This assay assesses the number of colony-forming units (CFUs) and their phenotype (erythrocyte, bone marrow, lymphoid, or mixed (GEMM) colonies) corresponding to the number and type of hematopoietic progenitor cells in the sample. GEMM colonies represent the most primitive type of undetermined hematopoietic progenitor cell, capable of producing both erythrocytes, bone marrow, and lymphoid blood cells.

[0167] Using Ctrl, PTC13303, and PTC13325.1, 1000 cells collected 7 days after expansion of CD34-rich UCB cells from two independent donors were mixed with MethoCult semi-solid medium and double-placed for human hematopoietic colony formation for a period of two weeks. The number and type of hematopoietic colonies (colony-forming units, CFUs) were scored under a microscope. As shown in Figure 3, cells treated with PTC13325.1 generally produced a substantially larger number of hematopoietic colonies (Figure 3(i)), specifically, mixed GEMM phenotype colonies (Figure 3(ii)). Interestingly, cells expanded with PTC13303 had a higher potency for producing GEMM colonies compared to cells expanded with Ctrl (Figure 3(ii)), but the total number of hematopoietic colonies was smaller (Figure 3(i)). This suggests that PTC13325.1 expands both the progenitor cell compartment and the primitive stem cell compartment, while PTC13303 (comparison) prioritizes only the expansion of primitive stem cells at the expense of progenitor cell expansion. This is important for engraftment and the reconstruction of the immune system.

[0168] Example 3: Engraftment of enlarged human CD34-positive cells in NSG mice One of the most reliable and widely accepted criteria for evaluating the efficacy of hematopoietic stem cells and progenitor cells is their ability to reconstruct the hematopoietic system in transplanted animal recipients. NSG mice have a not fully developed functional immune system and are used as model recipients for hematopoietic xenografts at sublethal irradiation. To test the reconstitution potential of expanded cells in vitro, human UCB CD34-positive cells pooled from four donor units and expanded by Ctrl and PTC13325.2 were transplanted into NSG mice. The percentage of hematopoietic reconstitution by human cells was estimated using a human-specific CD45 antibody (Figure 4).

[0169] Human / mouse chimeras were initially evaluated using bone marrow aspiration (BMA) from live animals at 10 and 16 weeks post-transplant (Figure 4A). The percentage of human cells was significantly higher in the BM of mice transplanted with cells augmented by PTC13325.2 compared to the control protocol (Figure 4A(i)), indicating increased development of both lymphoid and myeloid cells (Figure 4A(ii)). Eighteen weeks after transplantation, animals were sacrificed and the presence of human cells was evaluated in the BM by flow cytometry using a panel of markers shown in Table 1. The percentage of engrafted human cells was significantly higher in mice transplanted with cells augmented by PTC13325.2, reaching nearly 100% in some animals (Figure 4B(i)). Furthermore, the percentage of human LT-HSCs, defined by the CD34+ / CD38- / CD45RA- / CD90+ / CD49f+ (CD49f+HSC) markers, was increased in the same animals compared to mice transplanted with cells expanded by the control protocol (Figure 4B(ii)).

[0170] Example 4: Single-cell transcriptomics analysis Analysis was performed by single-cell RNA sequencing using cells either freshly isolated from umbilical cord blood (uncultured) or expanded for 7 days, under one of the following conditions: control, protocol PTC13325.2, or protocol PTC13303. Three UCB donors were pooled under each condition, with the control, as well as protocols PTC13325.2 and PTC13303, sharing the same three donors. A 10,000-point genomic SNP catalog was used as a reference for common variants to assign donor identification to each cell, and three donors per sample were identified to perform Bayesian variant-based donor deconvolution. After assigning each cell that passed minimal QC filtering to each donor, an additional round of QC-based filtering was performed (see Methods), resulting in 3721 cells (uncultured), 7039 cells (control), 4975 cells (protocol PTC13303), and 4558 cells (protocol PTC13325.2). The cells were integrated into a shared reduced-dimensional space to remove sex and protocol-associated differences and projected into two dimensions using UMAP (homogeneous manifold approximation and projection) (Figure 5A). Multi-resolution Leiden clustering was performed, and 12 clusters were identified at a resolution of 0.5 (Figure 5B), which were annotated according to the expression of typical markers such as CD34, SPINK2 (HSPC), MKI67, TOP2A (circulating cells), CLU, ITGA2B (megakaryocyte progenitor cells), KLF1 (erythrocyte progenitor cells), GATA2, CPA3 (early megakaryocyte / erythrocyte / mast cell progenitor cells), CEBPD, MPO (granulocyte-monocyte progenitor cells, GMP), IRF8, and SERPINF1 (monocyte progenitor cells) (Figure 5C).

[0171] To better measure the increase in stem cell content caused by culturing in different media, the signature score of each single cell in each cluster was derived and compared across protocols on a cluster-by-cluster basis (Figure 5D). Both PTC13303 and PTC13325.2 showed an increase in the amount of cells with higher HSC signatures compared to the control expansion medium in clusters containing HSCPs and circulating HSPCs (clusters 4, 5, 6, and 9), with PTC13325.2 showing a greater increase than PTC13303.

[0172] The selection of cluster numbers in in vitro expansion of stem cell populations (or mixtures of progenitor cells) is actually rather arbitrary, as cells exist on a continuum rather than as a defined set of subpopulations. To better characterize this continuum, data were divided into partially overlapping neighborhoods to provide finer analysis while maintaining even transcriptional space sampling (see Methods). Since there were three matched replicates between the control and protocol PTC13325.2 conditions, we used miloR to estimate neighborhood-level abundance variations (Figure 5E) and were able to observe how small but statistically significant sets of neighborhoods were positively overrepresented in protocol PTC13325.2 containing cells corresponding to the expanded HSPC / MPP clusters (Cluster 1, Figure 5E). Conversely, the control baseline protocol appeared to be more biased towards other progenitor cell compartments, such as monocyte progenitor cells (Cluster 13).

[0173] In summary, all these findings are consistent with observations from phenotypic determination and in vivo experiments, pointing to preferential expansion of long-term HSC compartments driven by protocol PTC13325.2 compared to controls.

[0174] Example 5: Discovery of a protocol for expanding hematopoietic progenitor cells and stem cells using the CombiCult screening platform and ex vivo expanded UCB cultures. Ex vivo culture of UCB cells using cytokine cocktails is known to promote cell division. These proliferation steps are associated with a low engraftment rate, rapid loss of pluripotency in HSCs, and accumulation of advanced progenitor and differentiated cells. The end products of such ex vivo cultures, despite having higher total cell numbers, do not offer long-term benefits for clinical use. Following one week of ex vivo culture in a cytokine cocktail supplemented with the chemical combinations of protocols PTC13303 and PTC13325.2, a reproducible abundance of CD34+ was observed (Figure 6A(i)), and most importantly, a more primitive CD34+CD38- compartment was observed (Figure 6A(ii)). Both protocols were capable of significantly enriching populations of engraftable CD34+CD38-CD45RA-CD90+ cells (Figure 6A(iii)), which expressed markers associated with the phenotype of long-term regrowth cells (LT-HSCs, CD49f+EPCR+) (Figure 6A(iv), Figure 6C-FACS plot). Interestingly, upregulation of both the CD49f and EPCR markers was observed in both CD90+ and CD90- cell populations compared to pre-expanded cells on day 1 and cells expanded with cytokines alone (control), suggesting that the culture medium components in protocols PTC13303 and PTC13325.2 positively regulated the expression of those markers (Figure 6C).

[0175] The magnitude of the observed effect was donor-dependent, but compared to protocol PTC13303, increased production of phenotypically engraftable ex vivo-treated cells was observed in cells enlarged by protocol PTC13325.2 per unit of starting material (Figure 6B).

[0176] This example demonstrates that ex vivo-enlarged UCB cultures are rich in HSCs.

[0177] Example 6: Expansion of primitive HSCs Mechanically, both protocols PTC13303 and PTC13325.2 utilize the use of HDAC inhibitors (scriptides) combined with additional small molecules, which have functions relatively studied in the context of stem cells. StemRegenin1 (SR1) is an AHR inhibitor that positively affects HSC expansion, while the JNK inhibitor (SP600125) and WR play various roles in stress response, cell cycle, and differentiation. Following a comparison of both protocols in ex vivo culture, the similarities in the expansion of primitive cell compartments may be attributable to the use of HDAC inhibitors (shared by both protocols, Figure 6A), but the greater number of primitive cells obtained with protocol PTC13325.2 suggests that the addition of an expansion step during the first three days of culture worked to our advantage (Figure 6B).

[0178] The first proliferation step of protocol PTC13325.2 was assumed to also be achievable by using other ex vivo amplifiers for hematopoietic cell count. In fact, when cells were first treated in vitro with RET and then with HDAC inhibitors and WR (protocol PTC13325.3, Figures 7A, B), a similar significant expansion in the primitive HSC count was observed. In a similar manner, both CD90+ and CD90- cell populations showed increased expression of both CD49f and EPCR (Figure 7C).

[0179] When plotting the number of total nucleated cells (TNTs) produced against the number of cells in the LT-HSC compartment, all three protocols produced tight clusters, independently of the donor response to the treatment. As a result, protocols PTC13325.2 and PTC13325.3 had similar effects that favored both total cell number and highly engraftable cell compartments (Figure 7D).

[0180] This final observation was supported by the overall and most significant increase in CFU-GEMM colony count produced by cells expanded ex vivo by protocols PTC13325.2 and PTC13325.3 compared to protocol PTC13303 (Figure 7E).

[0181] Therefore, the combination of the proliferation step and HDAC inhibitors is important for the expansion of primitive HSCs.

[0182] Consideration The example demonstrates a method for expanding UCB CD34 cells that increases the number of CD34 cells in a short period (7 days), increases the amount of cells with phenotypic characteristics of long-term regrowing cells, and produces excellent engraftment results upon transplantation in a well-characterized animal model (pre-conditioned NSG mice).

[0183] In vitro data show that cells in PTC13325.1 and PTC13325.2 media expanded in greater quantities compared to cells grown in control basal medium containing cytokines alone (TNT population). When placed in culture, HSCs typically differentiate and lose expression of stem cell markers. In contrast, cells grown in the expansion media of protocols PTC13303 (comparative), PTC13325.1, and PTC13325.2 had a higher percentage of CD34+ cells compared to control basal medium alone. The most primitive ("true," or undetermined) HSCs were phenotypic-characterized for the number of marker CD34+ / CD38- / CD45RA- / CD90+ / CD49f+ / CD201(EPCR)+ expression. Such cells have been shown to exhibit long-term repopulation potential in animal xenograft models. The percentage of this population was shown to be high after cell expansion with PTC13303, PTC13325.1, and PTC13325.2, and the total number of cells with this phenotype was significantly higher after expansion with protocols PTC13325.1 and PTC13325.2 compared to PTC13303 or control basal medium alone. This is an important finding as it indicates that cells expanded according to the protocols of the present invention have better engraftment ability than cells expanded according to methods of the prior art.

[0184] One of the surrogate in vitro tests for evaluating the functional efficacy of HSCs is the colony formation assay. Cells with stem cell or progenitor cell potential can divide and produce colonies with distinct morphological phenotypes when placed in a semi-solid medium containing several cytokines, which helps assess the quantity and type of progenitor cells in a sample. The most primitive cells give rise to colonies with mixed GEMM morphology, indicating that these cells are higher in the hierarchy of hematopoietic cell lineages compared to more deterministic progenitor cells. Our results for colony formation showed a dramatic increase in both the total number of colonies and, specifically, the number of GEMM colonies after cell expansion at PTC13325.1. These data correlate with flow analysis and strongly indicate that the LT-HSCs and hematopoietic progenitor cells produced by expansion at PTC13325.1 are functional cells. Notably, cells enlarged by PTC13303 produced a greater number of GEMM colonies compared to the control; however, PTC13325.1 was superior in both the number of functional progenitor cells and the number of true HSCs.

[0185] To test whether cells enlarged with PTC13325.1 were actually functional cells capable of hematopoietic rearrangement, cells enlarged with PTC13325.2 were pooled from four donors and transplanted into NSG mice. Engraftment was tracked over time by bone marrow aspiration (at weeks 10 and 16) and analysis of endpoint bone marrow rearrangement at week 18. The percentage of human cells (by CD45 marker) was high at all time points in animals treated with PTC13325.2, reaching nearly 100% in individual animals.

[0186] PTC13325.2 differed from PTC13325.1 in terms of the length of cell exposure to compounds SR1, SP600125, and IL-6 (see Materials and Methods), however, cell exposure to scriptide and WR-1065 remained the same in both time and length. In PTC13325.1, the exposure times to SR1, SP600125, and IL-6 were shorter because the cells were washed before the addition of HDAC / aminothiol.

[0187] To confirm that PTC13325.2 contributed to an increase in the number of long-term HSCs, single-cell transcriptomics analysis was performed to compare basal expansion medium, PTC13303, and PTC13325.2. By applying unbiased clustering of the integrated data and comparing the proportion of double-positive CD34 / EPCR cells in all clusters, we were able to see that PTC13325.2 consistently increased the number of LT-HSCs compared to basal expansion medium, as observed in phenotypic analysis and in vivo transplantation assays.

[0188] When protocols PTC13325.1 and PTC13325.2 were compared in vitro using UCBs from two additional donors (Figure 2E), an increase in total nucleated cell (TNC) count was observed when cells were treated with both protocols. This increase was significant compared to the control protocol (cytokines alone). The magnitude of the proliferation effect was donor-dependent, but the trend was always the same.

[0189] In ex vivo culture, hematopoietic stem cells (HSCs, CD34+ cells) can proliferate but rapidly lose their "stem cell characteristics," as defined by the expression of markers such as CD34+ alone or in combination with CD38- (CD34+ / CD38- are thought to be more primitive and less differentiated HSCs). It is important to define which cell types proliferated in expansion culture and whether the "stem cell" characteristics of the cells were preserved or diminished. To this end, we analyzed the percentage of CD34+ and CD34+ / CD38- cells within the TNC population for cells expanded by protocols PTC13303, PTC13325.1, and PTC13325.2, as well as for controls (cytokines only). We showed that cells treated with the control protocol rapidly lost their stem cell characteristics (CD34+, and more notably, CD34+ / CD38- phenotype; Lin- is a lineage-negative population except for the most advanced progenitor cells), but their stem cell nature was preserved after treatment with all three PTC protocols disclosed herein. We conclude that PTC13325.1 and PTC13325.2 are capable of significantly increasing cell proliferation while preserving the stem cell phenotype. This is important for engraftment and therapeutic potential in conditions of impaired immune systems.

[0190] In the field of hematopoietic stem cells (HSCs), ongoing research exists to define a set of phenotypic markers that reliably correlate with the long-term engraftment ability of cells at transplantation. Long-term engraftment is thought to be mediated by the most primitive population of HSCs that have not yet been determined to belong to any hematopoietic cell lineage (LT-HSCs). Historically, the set of markers for this LT-HSC population was thought to be Lin- / CD34+ / CD38- / CD45RA- / CD90+ / CD49f+, however, a novel marker, CD201 (EPCR), has recently emerged, which is thought to be more predictive of engraftment ability. Since previous in vitro experiments (including those disclosed in WO2020 / 084310 and WO2020 / 084310) evaluated engraftment ability using CD49f as a surrogate marker, we decided to use CD201 as a marker and compare our protocol (Figure 2E, Figure 5, Figure 6).

[0191] Single-cell RNA sequencing data were analyzed from CD34+ / CD45RA-HSCs generated using protocols PTC13303, PTC13325.2, and controls, and from freshly isolated CD34+ / CD45RA-HSCs without culture from UCB (Figure 5). Cell populations were characterized by unbiased clustering (Figure 5B), and cluster marker expression was measured (Figure 5C). Cell identification was also confirmed by measurement of HSC and progenitor cell type signatures (Figure 5D). Focusing on cell clusters containing LT-HSCs, the following was demonstrated using this method: 1. The percentage of LT-HSCs increases after treatment with the PTC protocol in the examples compared to the control. 2. The percentage increase is more pronounced after treatment with PTC13325.2 than after treatment with PTC13303.

[0192] Multiparametric flow cytometry is a proven method for immunophenotyping of various subpopulations of HSCs and progenitor cells. When protein markers were measured using this technique, an increase in the percentage of CD201+ and CD49f+ cells was observed in the CD34+CD38-CD45RA-CD90+ cell population after processing with the PTC13325.1 and PTC13325.2 protocols compared to control medium and the PTC13303(comparative) protocol (Figure 2E(iv)).

[0193] The number of LT-HSCs in the enlarged culture was calculated based on the total number of nucleated cells (TNCs) and the percentage of LT-HSCs in the TNC population. Compared to the original protocol PTC13303, in addition to a higher percentage of LT-HSCs in the present invention protocols PTC13325.1 and PTC13325.2, the total number of LT-HSCs was also shown to be much higher when cells were enlarged by PTC13325.1 and PTC13325.2 (Figure 2E(iii)). The magnitude of these results is donor-dependent, but the trend remains the same. This leads to the conclusion that the present invention protocols produce enlarged cell preparations with superior engraftment ability.

[0194] Individual enlarged cell populations were further phenotypic-characterized after treatment with control medium or protocols PTC13303, PTC13325.1, and PTC13325.2, and the results are shown in Figure 2F. We used a well-established set of markers CD34, CD38, and CD45RA, and the expression of each of them indicates the determinant of HSCs to more mature hematopoietic progenitor cells. Acquisition of CD38 and CD45RA expression indicates stepwise progression through the differentiation process, but CD38+ / CD45RA+ cells differentiate more than CD38+ / CD45RA- or CD38- / CD45RA+ cells. Loss of CD34 expression indicates a more advanced differentiation step. Therefore, we analyzed only progenitor cells that retained the CD34 marker.

[0195] We show that cells treated with the control protocol most rapidly accumulate a more differentiated phenotype expressing the CD38+ / CD45RA+ marker. Treatment with all three protocols, PTC13303(comparative), PTC13325.1, and PTC13325.2, reduces the proportion of differentiated cells; however, protocol PTC13303(comparative) supports an undifferentiated state of cells (CD34+ / CD38- / CD45RA-), while the present protocols PTC13325.1 and PTC13325.2 preferentially promote the accumulation of marker-expressing progenitor cells [CD38- / CD45RA+]. These phenotypic changes support rapid and stable hematopoietic rearrangement of recipients by cells expanded with protocols PTC13325.1 and PTC13325.2. This is also supported by extensive detection of precursor signatures in single-cell datasets (Figures 5D, 5E).

[0196] The protocol of the present invention differs from the comparative example in that a pre-culture step is included before the addition of the HDAC inhibitor and aminothiol. In the protocol of the present invention, this pre-culture step is performed in the presence of SR1 and / or SP600125, however, neither of these compounds is considered essential. The act of having an additional culture step at least 48 hours before the addition of HDAC / aminothiol is considered necessary to achieve the results shown herein.

Claims

1. A method for expanding hematopoietic stem cells and progenitor cells (HSPCs), wherein the method is i) Obtain an isolated population of HSPCs, ii) The isolated population of HSPCs is cultured for at least 48 hours to form a first cultured population, iii) Adding a histone deacetylase inhibitor (HDAC inhibitor) and further culturing the cells to form a second cultured population, iv) an aminothiol compound, wherein the aminothiol compound is of the formula RNH(C ｎ H ２ｎ )NH(C ｎ H ２ｎ ) has SX, where R is hydrogen, aryl, acyl, or an alkyl group containing 1 to 7 carbon atoms, each n has a value of 2 to 6, and X is H or PO ３ H ２ A method comprising adding an aminothiol compound, or a pharmaceutically acceptable salt thereof, to the second cultured population, and further culturing the cells to form an enlarged population of cells.

2. A composition comprising an enlarged population of cells for use in therapy, wherein the cells are i) Obtain an isolated population of HSPCs, ii) The isolated population of HSPCs is cultured for at least 48 hours to form a first cultured population, iii) Adding a histone deacetylase inhibitor (HDAC inhibitor) and further culturing the cells to form a second cultured population, iv) an aminothiol compound, wherein the aminothiol compound is of the formula RNH(C ｎ H ２ｎ NH(C ｎ H ２ｎ ), where R is hydrogen, aryl, acyl, or an alkyl group containing 1 to 7 carbon atoms, each n has a value of 2 to 6, and X is H or PO ３ H ２ An aminothiol compound or a pharmaceutically acceptable salt thereof is added to the second cultured population, and the cells are further cultured to form an expanded population of cells. A composition expanded by a method comprising:

3. It is a method of treatment, i) A step to obtain an isolated population of HSPCs, ii) A step of culturing the isolated population of HSPCs for at least 48 hours to form a first cultured population, iii) Adding a histone deacetylase inhibitor (HDAC inhibitor) and further culturing the cells to form a second cultured population, iv) an aminothiol compound, wherein the aminothiol compound is of the formula RNH(C) ｎ H ２ｎ )NH(C ｎ H ２ｎ ) has SX, where R is hydrogen, aryl, acyl, or an alkyl group containing 1 to 7 carbon atoms, each n has a value of 2 to 6, and X is H or PO ３ H ２ A method comprising the steps of adding an aminothiol compound, or a pharmaceutically acceptable salt thereof, to the second cultured population, and further culturing the cells to form an enlarged population of cells.

4. The use of a composition comprising an enlarged population of cells in the manufacture of a pharmaceutical product for use in therapy, wherein the cells are i) Obtain an isolated population of HSPCs, ii) The isolated population of HSPCs is cultured for at least 48 hours to form a first cultured population, iii) Adding a histone deacetylase inhibitor (HDAC inhibitor) and further culturing the cells to form a second cultured population, iv) an aminothiol compound, wherein the aminothiol compound is of the formula RNH(C) ｎ H ２ｎ )NH(C ｎ H ２ｎ ) has SX, where R is hydrogen, aryl, acyl, or an alkyl group containing 1 to 7 carbon atoms, each n has a value of 2 to 6, and X is H or PO ３ H ２ An expanded use, comprising adding an aminothiol compound, or a pharmaceutically acceptable salt thereof, to the second cultured population, and further culturing the cells to form an expanded population of cells.

5. The composition according to claim 2, the method according to claim 3, or the use according to claim 4, wherein the therapy or treatment is the therapy or treatment of a hematological disorder, an immunodeficiency, a metabolic disorder, or a neurodegenerative disorder.

6. The composition according to claim 2, the method according to claim 3, the use according to claim 4, or the composition, method, or use according to claim 5, wherein the therapy or treatment comprises regenerating mammalian bone marrow.

7. The composition according to claim 2, the method according to claim 3, the use according to claim 4, or the composition, method, or use according to claim 5 or 6, wherein the therapy or treatment is acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, non-Hodgkin lymphoma, severe aplastic anemia, severe combined immunodeficiency, or sickle cell disease.

8. The composition according to claim 2, the method according to claim 3, or the use according to claim 4, wherein the therapy or treatment is gene therapy.

9. The method according to claim 3, further comprising the step of administering the enlarged cells to the subject.

10. A method, composition, or use according to any one of the prior claims, comprising step i) selecting cells that are CD133+.

11. A method, composition, or use according to any one of the prior claims, comprising step i) selecting cells that are CD34+.

12. A method, composition, or use according to any one of the prior claims, wherein step ii) comprises culturing the cells for about 48 hours to about 96 hours, preferably about 72 hours.

13. A method, composition, or use according to any one of the prior claims, wherein step iii) comprises culturing the cells for about 24 hours to about 72 hours, preferably about 48 hours to 72 hours.

14. The method, composition, or use according to any one of the prior claims, wherein the cells in the first cultured population proliferate more than the cells in the second cultured population.

15. A method, composition, or use according to any one of the prior claims, wherein step iv) comprises culturing the cells for about 16 to about 24 hours, preferably about 24 hours.

16. A method, composition, or use according to any one of the prior claims, wherein step ii) comprises culturing the cells in the presence of a basal medium.

17. The method, composition, or use according to any one of the prior claims, further comprising step ii) culturing the cells in the presence of interleukin-6 (IL-6).

18. A method, composition, or use according to any one of the prior claims, comprising step ii) culturing the cells in the presence of nicotinamide, pyrimido-indole derivatives such as UM171 and UM729, a p38 MAPK inhibitor, a Notch ligand, a Wnt agonist, a JNK inhibitor, a cytokine, at least one RET agonist, and / or an aryl hydrocarbon receptor antagonist.

19. A method, composition, or use according to any one of the prior claims, comprising step ii) culturing the cells in the presence of a JNK inhibitor and / or an aryl hydrocarbon receptor antagonist.

20. The method, composition, or use according to any one of the prior claims, wherein the cells are washed between step ii) and step iii), and preferably the cell culture medium from stage iii) onward is substantially free of the JNK inhibitor and / or the aryl hydrocarbon antagonist.

21. The method, composition, or use according to any one of the prior claims, wherein the JNK inhibitor is SP600125.

22. The method, composition, or use according to any one of the prior claims, wherein the antagonist of the aryl hydrocarbon acceptor is SR1.

23. A method, composition, or use according to any one of the prior claims, wherein step ii) comprises culturing the cells in the presence of at least one, preferably at least two, RET agonists.

24. The method, composition, or use according to any one of the prior claims, wherein the at least one, preferably at least two, RET agonists are selected from GDNF, GFRα1, BT-13, Q525, and BT44, and preferably the RET agonists are selected from GDNF and GFRα1.

25. The method, composition, or use according to any one of the prior claims, wherein the HDAC inhibitor is a scriptide or xinostat.

26. The method, composition, or use according to any one of the prior claims, wherein the HDAC inhibitor is a scriptide.

27. The aminothiol compound is amifostin: 【Chemistry 1】 The method, composition, or use according to any one of the prior claims.

28. The aminothiol compound is WR1065: 【Chemistry 2】 The method, composition, or use according to any one of claims 1 to 26.

29. The isolated population obtained from umbilical cord blood, according to the method, composition, or use of any one of the prior claims.

30. The method, composition, or use according to any one of claims 1 to 28, wherein the isolated population is obtained from peripheral blood.

31. The method, composition, or use according to any one of claims 1 to 28, wherein the isolated population is obtained from bone marrow.

32. The method, composition, or use according to any one of the prior claims, wherein steps ii), iii), and iv) are performed for a total time sufficient to allow the isolated population of HSPCs to form expanded cells.

33. The method, composition, or use according to any one of the prior claims, wherein steps ii), iii), and iv) are performed over a total period of time of about 3 to 8 days.

34. The method, composition, or use according to any one of the prior claims, wherein the cells are cultured in a serum-free or feeder-free tissue culture system.

35. The cell is obtained from a mammal, preferably a human, according to the method, composition, or use described in any one of the prior claims.

36. The method, composition, or use according to any one of the prior claims, wherein the enlarged cells are rich in hematopoietic stem cells (HSCs) and / or long-term hematopoietic stem cells (LT-HSCs).

37. The method, composition, or use according to any one of the prior claims, wherein the enlarged cells are rich in Lin-, CD38-, CD34+, CD45RA-, CD90+, CD201+, and CD49f+.

38. The method, composition, or use according to any one of the prior claims, wherein the total cell expansion is approximately 5 to approximately 20 times, preferably approximately 10 to approximately 20 times.

39. The method, composition, or use according to any one of the prior claims, wherein the magnification of the Lin-, CD38-, CD34+, CD45RA-, CD90+, and CD49f+ cells is approximately 500 times.

40. The method, composition, or use according to any one of the prior claims, wherein the JNK inhibitor is used at a concentration of 0.01 to 50 μM, preferably 0.2 μM.

41. The method, composition, or use according to any one of the prior claims, wherein the antagonistaryl hydrocarbon acceptor is used at a concentration of 0.01 to 50 μM, preferably 1 μM.

42. The method, composition, or use according to any one of the prior claims, wherein the IL-6 is used at a concentration of 0.01 to 50 μg / ml, preferably 0.1 μg / ml.

43. The method, composition, or use according to any one of the prior claims, wherein at least one, preferably two, RET agonists are used at a concentration of 100 ng / ml.

44. The method, composition, or use according to any one of the prior claims, wherein the HDAC inhibitor is used at a concentration of 0.01 to 50 μM, preferably 0.3 μM.

45. The method, composition, or use according to any one of the prior claims, wherein the aminothiol compound is used at a concentration of 50 to 500 μM, preferably 100 μM.

46. A kit for expanding HSPC according to claim 1, wherein the kit comprises a sterilizing element for expanding HSPC, an HDAC inhibitor, and an aminothiol compound of the formula RNH(C) ｎ H ２ｎ )NH(C ｎ H ２ｎ ) has SX, where R is hydrogen, aryl, acyl, or an alkyl group containing 1 to 7 carbon atoms, each n has a value of 2 to 6, and X is H or PO ３ H ２ A kit comprising an aminothiol compound, or a pharmaceutically acceptable salt thereof.

47. The kit according to claim 46, having any of the additional features described in claims 10 to 45.

48. An enlarged population of cells obtained by the method according to claim 1 or any one of claims 10 to 45.

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