Methods for detecting hematopoietic stem and progenitor cells (HSPCS)

Abstract

The present disclosure, in various aspects and embodiments, provides methods for detecting specific hematopoietic lineages derived from HSPCs. Methods may involve culturing HSPCs in a culture medium for a sufficient time to obtain an output population of cells that includes a lymphoid lineage cell(s), and one or both of a myeloid lineage cell(s) and an erythroid lineage cell(s). Methods of simultaneously obtaining lymphoid, myeloid and erythroid lineage cells from HSPCs obtained from different sources are also described. Methods of the present disclosure may reliably detect multilineage differentiation potential of HSPCs in vitro.

Description

METHODS FOR DETECTING HEMATOPOIETIC STEM AND PROGENITOR CELLS (HSPCs)CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of United States Provisional Patent Application No. 63 / 706,400, filed October 11 , 2024, the entire contents of which is hereby incorporated by reference in its entirety.TECHNICAL FIELD

[0002] This disclosure relates to HSPC-related applications, and more specifically to detection of HSPCs, and still more specifically to detection of specific cell lineages derived from HSPCs.BACKGROUND

[0003] The hematopoietic system of mammals is one of the most highly regenerative systems and is composed of billions of erythrocytes, platelets, myeloid cells, innate and adaptive immune cells. The hematopoietic system is maintained by a small fraction of cells, hematopoietic stem and progenitor cells (HSPCs), that consist of and / or can produce multilineage and unilineage progenitors that subsequently differentiate into mature blood cells. Even a single hematopoietic stem cell (HSC) can produce long-term and multipotent reconstitution of the entire hematopoietic system. Therefore, it is important to assess the multilineage capabilities of HSPCs to understand their functional capabilities further.

[0004] Functional assays such as transplantation assays and in vitro colony formation are the “gold standard” methods currently used to assess the function of HSPCs or hematopoietic stem cells (HSC). The colony formation assay, better known as the colony-forming unit (CFU) assay, is one of the most widely used assays for HSPCs and involves measuring the proliferation and differentiation capabilities of individual cells within a sample. Colonies of differentiated cells produced by each input progenitor cell are manually observed to assess the lineage potential of HSPCs. Manual counting and accurate identification of different colony types are challenging aspects of the CFU assay and it is further limited by its inability to identify cells with lymphoid potential. Another functional assay identifies long-term culture-initiating cells (LTC-IC), and relies on co-culture of HSPCs with stromal systems for typically 5-6 weeks followed by assessment of lineage potential by CFU-assay. Transplantation assays such as in vivo xenograft assays involve the transplantation of HSCs into immunodeficient mice and tracking the output over the course of 4-6 months, and may further involve secondary transplants into mice from the primary recipient.

[0005] Therefore, functional assays for assessing multilineage potential of HSPCs suffer from various limitations, including complexity, duration, inability to detect all major hematopoietic lineages, reliance on numerous reagents and specialized equipment, and these methods are extremely time-consuming and may not be entirely physiologically relevant to the human system.

[0006] The present disclosure provides methods, assays, and media for reliably and robustly detecting multilineage differentiation potential of one or more HSPCs. Advantageously, the disclosed methods, media and kits provide a stroma-free system that may be performed in a liquid phase in one assay to simultaneously differentiate HSPCs into erythroid, myeloid, and lymphoid (EML) lineages. This disclosure presents a significant advance for functionally assessing the differentiation potential of HSPCs, and contributes greatly to research, medicine, and cell and gene therapy applications.SUMMARY

[0007] In one aspect of this disclosure, methods are provided for detecting multilineage differentiation potential of one or more hematopoietic stem and progenitor cells (HSPCs). The methods may comprise (i) providing one or more HSPCs; and (ii) culturing the one or more HSPCs in a culture medium for a sufficient time to obtain an output population of cells comprising lymphoid lineage cell(s), and one or both of myeloid lineage cell(s) and erythroid lineage cell(s).

[0008] One or more HSPCs of this disclosure may be derived from an input population of cells, wherein the input population is cord blood-derived, bone marrow-derived, peripheral blood-derived, mobilized peripheral blood-derived or pluripotent stem cell-derived (PSC). One or more HSPCs of this disclosure may be of human or non-human origin. One or more HSPCs of this disclosure may be depleted of red blood cells (RBCs). One or more HSPCs of this disclosure may be CD34+.

[0009] Culturing one or more HSPCs of this disclosure may be performed in liquid phase. Accordingly, in such embodiment, culturing is not performed in a semi-solid or solid phase. Culturing one or more HSPCs of this disclosure may be in the absence of feeder or stromal cells.

[0010] A culture medium of this disclosure may comprise one or more cytokines. A culture medium of this disclosure may comprise one or any combination of IL-2, IL-15, and erythropoietin. In one embodiment, the one or more cytokines comprise one or any combination of IL-2, IL-7, IL-15 and erythropoietin. One or more cytokines may beexogenously added. A culture medium of this disclosure may serum-free, or a culture medium of this disclosure may comprise serum (e.g. ranging from between about 1% and 30%).

[0011] Methods of any aspect of this disclosure may further comprise obtaining a population of cells comprising a lymphoid lineage cell or cells, and one or both of a myeloid lineage cell or cells and an erythroid lineage cell or cells after culturing one or more HSPCs in a culture medium for a sufficient time. A sufficient culturing time to obtain an output population of cells comprising a lymphoid lineage cell or cells, and one or both of a myeloid lineage cell or cells and an erythroid lineage cell or cells may elapse 6 weeks or less, such as about 1 week ± 1 day, 2 weeks ± 2 days, 3 weeks ± 3 days, 4 weeks ± 4 days, 5 weeks ± 5 days or 6 weeks ± 6 days. An output population of cells comprising a lymphoid lineage cell or cells, and one or both of a myeloid lineage cell or cells and an erythroid lineage cell or cells may be obtained in the culture medium after culturing the one or more HSPCs for about 2 weeks, about 3 weeks, or about 4 weeks.

[0012] An erythroid lineage cell of this disclosure may comprise an erythroblast and / or a megakaryocyte. A myeloid lineage cell of this disclosure may comprise a myeloblast cell, a basophil, a neutrophil, an eosinophil, a monocyte, a dendritic cell and / or a mast cell. A lymphoid lineage cell of this disclosure may comprise a natural killer (NK) cell, an early T cell progenitor, a lymphoid progenitor, a T lymphocyte and / or a B lymphocyte.

[0013] An output population of cells obtained by the methods of this disclosure may comprise: a) one of or any combination of a natural killer (NK) cell, an early T cell progenitor, a lymphoid progenitor, a T lymphocyte, and a B lymphocyte; b) one or both of an erythroblast and a megakaryocyte; and / or c) one of or any combination of a myeloblast cell, a mast cell, a promyelocyte, a myelocyte, a basophil, a neutrophil, an eosinophil, a monocyte, and a dendritic cell.

[0014] Methods of this disclosure may further comprise comparing an output population of cells when provided one or more HSPCs are isolated from a first donor / individual and at least a second donor / individual to identify differences in multilineage differentiation potential.

[0015] Methods of this disclosure may further comprise exposing one or more HSPCs to a test condition comprising one or more compound prior to or while culturing the one or more HSPCs in a culture medium to assess an effect of the compound (relative to a reference compound) on the multilineage differentiation potential of the one or more HSPCs. One or more compound may comprise a small molecule, a chemical compound, a protein, a peptide, a nucleic acid or a nanoparticle.

[0016] Methods of this disclosure may further comprise quantifying a probability of obtaining a lymphoid lineage cell or cells, and one or both of a myeloid lineage cell or cells and an erythroid lineage cell or cells among an output population of cells after culturing, such as via an in vitro limiting dilution analysis. Methods of such aspects may comprise: (i) providing one or more HSPCs; (ii) preparing at least one cell dose comprising an input number of the one or more HSPCs; (iii) culturing the one or more HSPCs in a culture medium for a sufficient time to obtain an output population of cells comprising a lymphoid lineage cell, and one or both of a myeloid lineage cell and an erythroid lineage cell; and (iv) measuring a probability of obtaining a lymphoid lineage cell, and one or both of a myeloid lineage cell and an erythroid lineage cell of the output population of cells after the culturing. A probability of obtaining a lymphoid lineage cell or cells, and one or both of a myeloid lineage cell or cells and an erythroid lineage cell or cells may be obtained or calculated by applying a Poisson statistical analysis (e.g. Extreme Limiting Dilution Analysis (ELDA) algorithm). Methods, media and kits of this disclosure may yield an about 5-20% probability of obtaining an erythroid lineage cell or cells, and / or about 25-55% probability of obtaining a myeloid lineage cell or cells, and / or about 15- 40% probability of obtaining a lymphoid lineage cell or cells using an in vitro limiting dilution assay as described herein.

[0017] In a related or different aspect of this disclosure, systems or kits are provided for detecting multilineage differentiation potential of one or more hematopoietic stem and progenitor cells (HSPCs) comprising a culture medium for culturing the one or more HSPCs and at least one supplement comprising one or more cytokines. A culture medium comprised in the kits of this disclosure may comprise one or more cytokines. A culture medium comprised in the kits of this disclosure may comprise one or more cytokines, wherein the one or more cytokines comprise one of or any combination of IL-2, IL-3, IL-6 IL-7, IL-15, G-CSF, GM-CSF or erythropoietin.

[0018] In a related or different aspect of this disclosure, media are provided for culturing one or more HSPCs, the media comprising a basal medium and one or more cytokines. One or more cytokines may comprise IL-2, IL-3, IL-6, IL-7, IL-15, G-CSF, GM-CSF or erythropoietin. Media of this disclosure may be serum-containing (e.g. ranging between about 1% and 30%), or albumin-containing (but serum-free).

[0019] A medium of this disclosure may support multilineage differentiation potential of one or more HSPCs to obtain an output population of cells comprising a lymphoid lineage cell or cells, and one or both of a myeloid lineage cell and an erythroid lineage cell.

[0020] In related or different aspects of this disclosure, various methods, assays or applications are provided in relation to measuring / detecting / assessing / modulatingmultilineage differentiation potential of one or more HSPCs when such one or more HSPCs are exposed to, or contacted with a treatment condition, and as may be compared against a parallel or previously determined reference condition. In such aspects, multilineage differentiation potential of one or more HSPCs of a donor / individual / patient may be compared against multilineage differentiation potential of one or more HSPCs of at least one different donor / individual / patient / source. In such aspects, multilineage differentiation potential of one or more HSPCs from the same donor / individual / patient / source may be compared where the one or more HSPCs have undergone a manipulation, and thus pre- and post-manipulation multilineage differentiation potential may be compared. In such aspects, multilineage differentiation potential of one or more HSPCs comprising a first unique molecular feature may be compared against multilineage differentiation potential of one or more HSPCs either comprising at least a second or an established molecular feature. In such aspects, multilineage differentiation potential of one or more HSPCs derived from a first PSC line and at least a second PSC line may be compared to identify cell line-dependent differences in multilineage differentiation potential. In such aspects, multilineage differentiation potential of one or more HSPCs derived from a PSC line cultured in a first culture condition may be compared against multilineage differentiation potential of one or more HSPCs derived from the PSC line cultured in a second culture condition to identify differences in multilineage potential arising due to culture conditions. In such aspects, multilineage differentiation potential of one or more HSPCs obtained before, after or before and after a treatment or a procedure may be compared to quantify an effect of the treatment or the procedure on the multilineage differentiation potential. In such aspects, multilineage differentiation potential of one or more HSPCs exposed to a test condition may be compared against a parallel or previously determined reference or control condition. In such aspects, multilineage differentiation potential of one or more gene-edited HSPCs or HSPCs derived from gene- edited PSCs may be compared against a parallel or previously determined multilineage differentiation potential of alternate gene-edited HSPCs or alternate HSPCs derived from gene-edited PSCs.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] For a better understanding of the various embodiments described herein, and to show more clearly how these various embodiments may be carried into effect, reference will be made, by way of example, to the accompanying drawings which show at least one example embodiment, and which are now described. The drawings are not intended to limit the scope of the teachings described herein.

[0022] Figures 1 A(i-xi) and 1 B(i-vi) show a comprehensive and simplified exemplary flow cytometry sequential gating strategy, respectively, for identifying erythroid, myeloid and lymphoid lineage cells. In both workflows, cells were initially refined using a light scatter gate followed by the exclusion of doublets and dead cells. In Figure 1A, cells were subsequently gated on the basis of expression of phenotype markers for CD45, CD34, CD41 , CD33, CD19, Glycophorin A (GPA), FCeRla, CD56, CD15, CD11C, CD14 and CD11b, while in Figure 1 B cells were subsequently gated on the basis of expression of phenotype markers for CD33, CD15, CD19, CD56, Glycophorin A (GPA) and CD41. Unstained and FMO (Fluorescence minus one) controls were used for each sample. FSC: Forward scatter, SSC: Side scatter, MK: Megakaryocytes.

[0023] Figures 2A(i-iv) and 2B(i-iv) show the total log 10 counts for CD34+(i), Erythroid (ii), Myeloid (iii), and lymphoid (iv) lineage cells by flow cytometry at Week 3 in either serumcontaining (Figure 2A) or serum-free (Figure 2B) media conditions. In Figure 2A), erythroid, myeloid, lymphoid (EML) assay performance was assessed in EML medium comprising 12.5% serum. Data represents the mean ± SD of 3 experiments. In Figure 2B), EML assay output was assessed in serum-free media formulations relative to IMDM + 12.5% FBS as a control. Data represents the mean ± SD of 3 technical replicates from 1 experiment.

[0024] Figures 3A(i-vii) and 3B(i-iv) show the impact of cytokine supplementation on EML assay output. Bar graphs show the total log 10 counts of monocytes (i), myeloid cells (ii), dendritic cells (iii), B cells (iv), erythroid cells (v), NK cells (vi) and neutrophils (vii) after 3 weeks in EML medium comprising or excluding IL-2 (Figure 3A). Bar graphs show the total log 10 counts of B cells (i), Erythroid (ii), NK cells (iii) and neutrophils (iv) after 3 weeks in EML medium comprising a lower (IL-2-low) or higher (IL-2-high) IL-2 concentration, or IL-15 (Figure 3B). Data represents the mean ± SD of 2 technical replicates from 1 experiment.

[0025] Figure 4(i-viii) shows EML assay output for cord blood-derived HSPCs in EML media containing or lacking exogenously added SCF. Bar graphs show the total log 10 counts of CD34+cells (i), erythroid cells (ii), megakaryocytes (iii), NK cells (iv), B cells (v), myeloid cells (vi), neutrophils (vii) and monocytes (viii) obtained after 3 weeks of culture. Data represents the mean ± SD of 3 technical replicates from 1 experiment.

[0026] Figure 5(i-viii) shows EML assay output when assaying fresh or pre-cultured (for 7 days) HSPC for multilineage potential. Bar graphs show the total log 10 counts of CD34+cells (i), neutrophils (ii), myeloid cells (iii), B cells (iv), megakaryocytes (MK) (v), NK cells (vi), erythroid cells (vii) and monocytes (viii) obtained after 3 weeks. Data represents the mean ± SD of 2 technical replicates from 1 experiment.

[0027] Figures 6A(i-iii) and 6B(i-iii) show EML assay output of HSPCs obtained from 11 different cord blood samples (Figure 6A) and 6 different bone marrow donors (Figure 6B) after 3 weeks. The log 10 % of viable erythroid (i), myeloid (ii), and lymphoid (iii) lineage cells were calculated by flow cytometry for each of the donor samples. Grey line represents the mean of all samples. Data represents 2-3 technical replicates per sample and corresponding mean.

[0028] Figure 7(i-vi) shows the impact of small molecule supplementation in EML medium on output of cord-blood derived CD34+HSPCs. EML medium comprised SCF, TPO and Flt3L (denoted as STF) and was further supplemented with one or more chemical compounds (e.g. small molecule): (i) UM729; (ii) a cocktail comprising a histone deacetylase inhibitor (HDACi), a histone demethylase inhibitor (HDMi) and a histone methyltransferase inhibitor (HMTi); (iii) an inhibitor of Enhancer of Zeste Homolog 2 (EZH2) (denoted as EZH2i); and (iv) an 5- hydroxytryptamine 1 receptor (5-HT1 F-R) agonist. Fresh HSPC controls were not precultured with small molecules prior to an EML assay. Data represents the mean ± SD of 5 donors from 5 experiments for the fresh cells, and for the conditions including STF, STF+UM729, STF+HDACi+HDMi+HMTi, of 3 donors from 3 experiments for the STF+EZH2i condition, and of 1 donor from 1 experiment for the STF+5-HT 1 F-R agonist condition.

[0029] Figures 8A and 8B show an EML assay of HSPCs in an EML medium in the absence (Figure 8A) or presence of a notch ligand (~2.7 pmol / ml)(Figure 8B) to detect B cells, lymphoid progenitors (“LP”) / T lymphocytes, CD34+, erythroid, MK, myeloid, and NK cells. Bar graphs show the log 10 % of viable cells obtained after 3 weeks. Data represents the mean ± SD of 1 experiment.DETAILED DESCRIPTION

[0030] This disclosure relates to methods and assays for detecting and / or assessing and / or modulating multilineage differentiation potential of one or more HSPCs. This disclosure also relates to media used in the foregoing methods and assays.

[0031] Where used in this disclosure, the term “HSPC” refers to hematopoietic stem and progenitor cells some of which are capable of long-term and multipotent reconstitution of the entire hematopoietic system. The term may be used interchangeably with the term ‘hematopoietic stem cell’ or “HSC”. HSPC may be characterized by the presence / absence of one or more markers that distinguish over other non-HSPC cell types. A well-known characteristic of primary or PSC-derived HSPC is the expression of the transmembrane phosphoglycoprotein CD34; thus, HSPC may be referred to as CD34+cells. Examples of other markers of HSPCs include CD38, CD90, CD45RA, CD45RO, CD10, CD109, CD166, HLA- DR, CD201 ,CD49f, integrin-alpha3, EPCR (Endothelial protein C receptor), RET (RET protooncogene, GPRC5C (G-protein coupled receptor family group 5 member C), CD117, CD133,CD59, CD45, GPI-80, CD43, CD44, CD9, CD48, CD84 and CD244. Human HSPCs may be further defined and distinguished by expression of CD45 and CD34. HSPCs may lack expression, or have only low expression, of markers such as Glycophorin A, CD3, CD4, CD8, CD14, CD15, CD19, CD20 and CD56; which may characterize more mature hematopoietic cells.

[0032] Where used in this disclosure, the term ‘hierarchy’ refers to the hematopoietic hierarchy. HSCs which are defined as cells that have self-renewal capacity and the potential to give rise to all hematopoietic cell types may reside at the top of the hierarchy. HSCs may give rise to the Multi-Potent Progenitor (MPP) population that has diminished self-renewal capacity compared to HSCs. MPPs may further advance towards oligopotent progenitors and segregate into populations with increasingly limited differentiation and self-renewal potentials. Some of these populations may include common myeloid progenitors (CMPs), multi-lymphoid progenitors (MLPs), megakaryocyte-erythroid progenitors (MEPs), and granulocyte-monocyte progenitors (GMPs), and Pre-B / NK cells. These progenitors may differentiate into mature effector cells such as erythrocytes, megakaryocytes, platelets, granulocytes, monocytes, B- cells, NK cells, T-cells and dendritic cells.

[0033] Where used in this disclosure, the term “multilineage potential” refers to the capacity of HSPCs to give rise to multiple cell lineages, which may ultimately give rise to all the cells of the hematopoietic hierarchy cells. Particularly, multilineage potential as used herein may refer to differentiating one or more HSPCs to obtain an output population of cells comprising a lymphoid lineage cell or cells, and one or both of a myeloid lineage cell or cells and an erythroid lineage cell or cells.

[0034] Where used in this disclosure, the term “EML” refers to erythroid, myeloid and lymphoid lineages of cells. The term “EML assay” may refer to an assay, test, environment, or experiment to determine the multilineage differentiation potential, such as of one or more HSPCs, to obtain an output population of cells comprising a lymphoid lineage cell or cells, and one or both of a myeloid lineage cell or cells and an erythroid lineage cell or cells.

[0035] Where used in this disclosure, the term “input population” refers to a starting population comprising one or more HSPCs. An input population may comprise non-purified, purified, (pre-) cultured, or non-cultured cells. The term is not particularly limiting and the skilled person will know that single cell HSPCs may also be considered an input HSPC population.

[0036] Where used in this disclosure, the term “output population” refers to a population of cells obtained following an EML assay of this disclosure. An output population of cells may comprise a lymphoid lineage cell or cells, and one or both of a myeloid lineage cell or cellsand an erythroid lineage cell or cells, as may be obtained in a culture medium of this disclosure.

[0037] Where used in this disclosure, the term “pluripotent stem cell” or “PSC” refers to a cell having the potential for self-renewal and differentiation into specialized cell types of the three germ layers (ectoderm, endoderm and mesoderm). PSCs may be embryonic stem cells (ESCs) and / or induced pluripotent stem cells (iPSCs). PSCs may be isolated, derived or induced from any source species, but in this disclosure, stem cells are preferably mammalian.

[0038] Where used in this disclosure, the term “marker” refers to a biomolecule or biocompound that can be used to distinguish one cell type from another. A marker may be a protein, lipid, glycosylation mark, or the like, and may be expressed extracellularly on the cell surface or inside a cell as an intracellular molecule or compound.

[0039] Where used in this disclosure, the term “one or more compound” refers to an individual or a combination of compounds which may be naturally occurring, synthetic, organic or inorganic. Exemplary compounds include, but are not limited to, small molecules (e.g. small molecule inhibitors / antagonists or activators / agonists) and biologies (e.g. antibodies, hormones, proteins / peptides, nucleic acids, cytokines (e.g. SCF, FLT-3L, TPO and IL-6), and extracellular matrix factors). Exposing one or more HSPCs to one or more compound prior to or while culturing the one or more HSPCs in a culture medium may or may not modulate multilineage differentiation potential of the one or more HSPCs.Methods

[0040] In one aspect of this disclosure, methods are provided for detecting multilineage differentiation potential of one or more HSPCs. In one aspect of this disclosure, methods are provided for assessing multilineage differentiation potential of one or more HSPCs, such as in advance of research, medical (e.g. clinical, therapeutic), or banking applications. In one aspect of this disclosure, methods are provided for modulating multilineage differentiation potential of one or more HSPCs, such as in advance of research, medical (e.g. clinical, therapeutic), or banking applications.

[0041] Methods of this disclosure may comprise providing one or more HSPCs, as may be comprised in an input population of cells. One or more HSPCs may be of human or non-human origin (e.g. primate, rodent, fish, canine, equine, porcine, etc). Such one or more HSPCs are not limited provided that it / they may, in methods and assays of this disclosure, be assessed for multilineage differential potential to determine capacity / potential for generating or differentiating to one or more cell lineages of the hematopoietic hierarchy.

[0042] One or more HSPCs may be derived from any source or sample, including but not limited to, cord blood, bone marrow, (mobilized) peripheral blood, or any other tissue or organ, such as lymph, heart, spleen, intestine, muscle, kidney, liver, lung, gingiva or brain. One or more HSPCs may also be derived / differentiated from PSCs using commercially available or publicly disclosed media and methods. Preferably, one or more HSPCs may be derived from an input population of cells that may be cord blood-, bone marrow-, (mobilized) peripheral blood-, or PSC-derived.

[0043] One or more HSPCs may or may not be purified from a sample, and a sample may correspond to one from a healthy donor, a donor having a disease, infection, or other illness / condition. If purified, isolated, or enriched, one or more HSPCs may be positively selected (on the basis of a desired phenotypic marker, such as CD34+and / or CD45) or negatively selected, such as by immunomagnetic separation. In addition or in the alternative, one or more HSPCs may be depleted of undesirable cells, such as red blood cells (RBCs) via magnetic cell separation, chemical lysis, sedimentation, centrifugation, erythrocyte aggregation agents, and microbubbles.

[0044] In a specific embodiment, one or more HSPCs may express the transmembrane phosphoglycoprotein CD34; thus, one or more HSPCs may be CD34+. A CD34+HSPC may further express one or a combination of other markers (e.g. CD45, etc).

[0045] One or more HSPCs, as may be comprised in an input population of cells, may be precultured, such as to expand the one or more HSPCs and / or expose the one or more HSPCs to a (putative) therapeutic / expansion agent. Pre-culturing or -expanding one or more HSPCs, as may be comprised in an input population of cells, may be for a sufficient time in an appropriate culture medium (e.g. a StemDiff™ or StemSpan™ branded medium, STEMCELL Technologies). Alternatively, one or more HSPCs may be used fresh without pre-culturing or one or more cryopreserved HSPCs may be used. One or more HSPCs, as may be comprised in an input population of cells, may be gene-edited, by any gene editing technique known in the art, such as to correct a genetic defect or to introduce a new / alternative marker or function.

[0046] One or more HSPCs, as may be comprised in an input population of cells, may comprise a plurality of HSPCs or may comprise a single HSPC. A single HSPC may be obtained / isolated from a population of cells by sorting, enriching or purifying based on a presence / absence of one or more phenotypic marker. If an input population of cells comprises a plurality of HSPCs, such population may comprise 2 through 300000 or more HSPCs. In one embodiment, an input population of cells comprises at least one cell dose comprising an input number of one or more HSPCs, including but not limited to at least 1 , 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more HSPCs. A single or plural HSPC may generate one, each or any combination of erythroid, myeloid and lymphoid lineage cells.

[0047] Methods of this disclosure may be performed in the absence of support from or interaction with supporting cells, such as Mesenchymal Stromal Cells (MSCs), macrophages, endothelial cells, osteoblasts, adipocytes or sympathetic nerve cells, as may be found in the bone marrow microenvironment. More specifically, methods of this disclosure may be performed in the absence of support from or interaction with stromal / feeder cells (e.g. MS-5 cells, OP9-DL1 / DL4 cells, or other engineered cells).

[0048] Methods of this disclosure may comprise culturing one or more HSPCs, as may be comprised in an input population of cells, in a culture medium for a sufficient time to obtain an output population of cells comprising a lymphoid lineage cell (or a plurality of lymphoid lineage cells), and one or both of a myeloid lineage cell (or a plurality of myeloid lineage cells) and an erythroid lineage cell (or a plurality of erythroid lineage cells). In a specific embodiment, culturing one or more HSPCs occurs in a liquid phase. In a specific embodiment, culturing one or more HSPCs does not occur in a solid or semi-solid phase.

[0049] Culture media of this disclosure comprise a basal medium, and may be further supplemented with one or more factors that facilitate / promote / direct differentiation of one or more cell lineages downstream of a cultured HSPC(s). Basal media typically include one or more of: amino acids, vitamin(s), organic and / or inorganic salt(s), buffer(s), antioxidant(s), protein(s), energy (e.g., carbon) source(s), and the like for supporting the growth of cells. If basal media do not include one or more of the foregoing components, and if essential, they may be added by supplementation. Numerous basal media are known and commercially available, including Dulbecco's Modified Eagle's Medium (DMEM), F12, Roswell Park Memorial Institute Medium (RPMI) 1640, Iscove's Modified Dulbecco's Medium (IMDM), Advanced DMEM, Advanced DMEM / F-12, Minimum Essential Medium (MEM), a-MEM, StemSpan™-branded basal media, MyeloCult™-branded media, and others marketed specifically for the culture of HSPCs. In a specific embodiment, a basal medium is MyeloCult™ H5100, StemSpan™ SFEM, StemSpan™ SFEM II media, a-MEM with nucleosides, or IMDM.

[0050] Culture media of this disclosure may be further supplemented, such as to facilitate / promote / direct differentiation of one or more HSPCs, as may be comprised in an input population, to obtain an output population of cells comprising a lymphoid lineage cell (or cells), and one or both of a myeloid lineage cell (or cells) and an erythroid lineage cell (or cells). Numerous appropriate factors are known, including one or more of cytokines, growth factors, small molecules, serum / albumin / serum replacement, and the like. Exemplary one ormore cytokines may be selected from chemokines, hematopoietic cytokines, interferons, interleukins, tumor necrosis factor (TNF) or colony-stimulating factors (CSF) such as granulocyte-colony stimulating factor (G-CSF). Specifically, one or more cytokines comprised in a culture medium of this disclosure may be one or any combination of IL-2, IL-3, IL-6, IL-7 IL-15, erythropoietin (EPO), stem cell factor (SCF), Thrombopoietin (TPO), Fms-like tyrosine kinase 3 (Flt3) ligand, GM-CSF or G-CSF. More specifically, one or more cytokines comprised in a culture medium of this disclosure may be one or any combination of IL-2, IL-3, IL-6, IL-7, IL-15, G-CSF, GM-CSF or EPO. In one embodiment, one or more cytokines comprised in a culture medium of this disclosure are one or more of IL-3, G-CSF, GMCSF and / or erythropoietin. In one embodiment, one or more cytokines comprised in a culture medium of this disclosure are one or more of IL-2, G-CSF and / or erythropoietin. In one embodiment, one or more cytokines comprised in a culture medium of this disclosure are one or more of IL-2, IL-6 and / or erythropoietin. In one embodiment, one or more cytokines comprised in a culture medium of this disclosure are one or more of IL-2 and / or IL-15, and / or erythropoietin. In one embodiment, one or more cytokines comprised in a culture medium of this disclosure are one or more of or any combination of IL-2, IL-7, IL-15, and erythropoietin. In one embodiment, one or more cytokines comprised in a culture medium of this disclosure are one or more of or any combination of IL-2, IL-7, and erythropoietin. One or more cytokines comprised in a culture medium of this disclosure may be exogenously added.

[0051] If IL-2 is present in a culture medium, an effective IL-2 concentration does not result in any or in significant levels of cell toxicity / death of desired cells. A concentration of IL-2 may range between about 0.1 ng / ml and 200 ng / ml, between about 1 ng / ml and 100 ng / ml, between about 2 ng / ml and 80 ng / ml, between about 5 ng / ml and 50 ng / ml, between about 5 ng / ml and 15 ng / ml, between about 35 ng / ml and 45 ng / ml or any value therebetween. In embodiments, a concentration of IL-2 is about 40 ng / ml ± 5 ng / ml, or about 10 ng / ml ± 5 ng / ml, or any value therebetween.

[0052] If IL-3 is present in a culture medium, an effective IL-3 concentration does not result in any or in significant levels of cell toxicity / death of desired cells. A concentration of IL-3 may range between about 0.1 ng / ml and 200 ng / ml, between about 1 ng / ml and 100 ng / ml, between about 2 ng / ml and 80 ng / ml, or between about 5 ng / ml and 50 ng / ml. In embodiments, a concentration of IL-3 ranges between about 5 ng / ml and 15 ng / ml. In one embodiment, a concentration of IL-3 is about 10 ng / ml ± 5 ng / ml.

[0053] If IL-6 is present in a culture medium, an effective IL-6 concentration does not result in any or in significant levels of cell toxicity / death of desired cells. A concentration of IL-6 may range between about 0.1 ng / ml and 200 ng / ml, between about 1 ng / ml and 100 ng / ml, betweenabout 2 ng / ml and 80 ng / ml, or between about 5 ng / ml and 50 ng / ml. In one embodiment, a concentration of IL-6 ranges between about 5 ng / ml and 15 ng / ml. In one embodiment, a concentration of IL-6 is about 10 ng / ml ± 5 ng / ml.

[0054] If IL-7 is present in a culture medium, an effective IL-7 concentration does not result in any or in significant levels of cell toxicity / death of desired cells. A concentration of IL-7 may range between about 0.1 ng / ml and 200 ng / ml, between about 1 ng / ml and 100 ng / ml, between about 2 ng / ml and 80 ng / ml, or between about 5 ng / ml and 50 ng / ml. In one embodiment, a concentration of IL-7 ranges between about 10 ng / ml and 40 ng / ml. In one embodiment, a concentration of IL-7 ranges between about 20 ng / ml and 30 ng / ml.

[0055] If IL-15 is present in a culture medium, an effective IL-15 concentration does not result in any or in significant levels of cell toxicity / death of desired cells. A concentration of IL-15 may range between about 0.1 ng / ml and 200 ng / ml, between about 1 ng / ml and 100 ng / ml, between about 2 ng / ml and 80 ng / ml, or between about 5 ng / ml and 50 ng / ml. In one embodiment, a concentration of IL-15 ranges between about 10 ng / ml and 30 ng / ml. In one embodiment, a concentration of IL-15 is about 20 ng / ml ± 5 ng / ml.

[0056] If G-CSF is present in a culture medium, an effective G-CSF concentration does not result in any or in significant levels of cell toxicity / death of desired cells. A concentration of G- CSF may range between about 0.1 ng / ml and 200 ng / ml, between about 1 ng / ml and 100 ng / ml, between about 2 ng / ml and 80 ng / ml, or between about 5 ng / ml and 50 ng / ml. In embodiments, a concentration of G-CSF ranges between about 5 ng / ml and 40 ng / ml, or between about 5 ng / ml and 15 ng / ml.

[0057] If EPO is present in a culture medium, an effective EPO concentration does not result in any or in significant levels of cell toxicity / death of desired cells. A concentration of EPO may range between about 0.1 units / ml and 200 units / ml, between about 1 units / ml and 100 units / ml, between about 2 units / ml and 80 units / ml, or between about 5 units / ml and 50 units / ml. In one embodiment, a concentration of EPO ranges between about 1 units / ml and 10 units / ml. In one embodiment, a concentration of EPO is about 3 units / ml ± 2 units / ml.

[0058] Culture media of this disclosure may be serum-containing or serum-free. If serumcontaining, a culture medium may comprise FCS, FBS, or the like. If serum-free, a culture medium may comprise an albumin (e.g. BSA, HSA, or the like), whether recombinant or not, or a different serum replacement (BIT 9500 Serum Substitute, STEMCELL Technologies, or the like). If recombinant, an albumin may be expressed and isolated from an appropriate cell line, such as a bacterial, fungal, plant, or animal cell line.

[0059] If culture media comprise an albumin (and not serum), the albumin concentration may be expressed as a percentage of the total culture medium volume or as grams per liter (g / L) or milligrams per milliliter (mg / mL). In one embodiment, a culture medium may comprise an albumin at a concentration of about 0.1 mg / ml to about 200 mg / ml. Albumin may be comprised in a culture medium at a concentration ranging between about 0.01% and 25%, such as at a concentration of about 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. In one embodiment, a culture medium may comprise an albumin at a concentration ranging between about 0.1% and 5%. In one embodiment, a culture medium may comprise an albumin at a concentration of about 0.1% ± 0.05%.

[0060] If culture media comprise serum or a serum-like component, the serum concentration therein may be expressed as a percentage of the total culture medium volume. Serum may be comprised in a culture medium at a concentration ranging between about 1% and 30%, such as at a concentration of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30%. In embodiments, a culture medium comprises serum at a range between about 10% to 30%, between about 15% to 30%, or between about 5% to 15%. Serum-like components may comprise chemically defined additives, formulations with reduced serum content, engineered serum and the like known in the art.

[0061] A culture medium may be devoid of one or more components routinely used in hematopoietic applications. In one embodiment, a culture medium does not comprise SCF.

[0062] If an output of specific lymphoid progenitors (e.g. CD5+CD7+) or T lymphocytes is desired, a culture medium may comprise a notch ligand. A notch ligand may be a full-length protein, a fusion protein, or a peptide. In one embodiment, a notch ligand may comprise a delta-like protein, such as DLL1 or DLL4, or a functional equivalent thereof.

[0063] Methods of this disclosure may further comprise obtaining an output population of cells comprising a lymphoid lineage cell (or cells), and one or both of a myeloid lineage cell (or cells) and an erythroid lineage cell (or cells) after culturing one or more HSPCs, as may be comprised in an input population of cells, in a culture medium for a sufficient time.

[0064] A time sufficient for culturing (or differentiating) one or more HSPCs, as may be comprised in an input population of cells, in a culture medium of this disclosure to obtain an output population of cells may comprise about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or 6 weeks. Preferably, culturing according to the methods of this disclosure elapses between about 3 weeks to 6 weeks.

[0065] Following culture for a sufficient period of time, methods of this disclosure may comprise obtaining an output population of cells comprising a lymphoid lineage cell (or cells), and one or both of a myeloid lineage cell (or cells) and an erythroid lineage cell (or cells). In one embodiment, each of a lymphoid lineage cell (or cells), a myeloid lineage cell (or cells), and an erythroid lineage cell (or cells) are obtained from one or more HSPCs following culture for a sufficient period of time in a culture medium of this disclosure. In one embodiment, each of a lymphoid lineage cell (or cells), a myeloid lineage cell (or cells), and an erythroid lineage cell (or cells) are obtained from a single HSPCs following culture for a sufficient period of time in a culture medium of this disclosure. In one embodiment, each of a lymphoid lineage cell (or cells), a myeloid lineage cell (or cells), and an erythroid lineage cell (or cells) are obtained from a plurality of HSPCs following culture for a sufficient period of time in a culture medium of this disclosure.

[0066] Following culture for a sufficient period of time, methods of this disclosure may comprise obtaining an output population of cells comprising a lymphoid lineage cell (or cells), and one or both of a myeloid lineage cell (or cells) and an erythroid lineage cell (or cells) within 2 weeks, 3 weeks, or 4 weeks of culturing one or more HSPCs in a culture medium of this disclosure. At least one representative cell type of each of lymphoid, myeloid and erythroid lineages may be obtained within 2 weeks, 3 weeks or 4 weeks of culturing one or more HSPCs in a culture medium of this disclosure. Methods of this disclosure may comprise simultaneously obtaining an output population of cells comprising a lymphoid lineage cell (or cells), and one or both of a myeloid lineage cell (or cells) and an erythroid lineage cell (or cells) from a same culture vessel within 2 weeks, 3 weeks, 4 weeks, 5 weeks or 6 weeks of culturing one or more HSPCs in a culture medium of this disclosure. Methods of this disclosure may comprise obtaining > 5% of an output population of cells comprising a lymphoid lineage cell (or cells), and one or both of a myeloid lineage cell (or cells) and an erythroid lineage cell (or cells) after culturing the one or more HSPCs in the culture medium for a sufficient time.

[0067] In the methods disclosed, an erythroid lineage cell may arise / output from an appropriate progenitor cell, such as a myeloid progenitor cell and / or a megakaryocyte / erythroid progenitors (MEPs). An erythroid lineage cell of this disclosure may comprise erythroid lineage cells and megakaryocytes arising from an MEP. An Erythroid lineage cell may comprise an erythroid cell precursor, an erythrocyte or an erythroblast. Megakaryocytes obtained by the methods of this disclosure may further differentiate into platelets.

[0068] In the methods disclosed, a myeloid lineage may arise from an appropriate progenitor cell, such as a myeloid progenitor cell and / or a common myeloid progenitor (CMP) which mayfurther give rise to granulocyte / macrophage progenitors (GMPs). Myeloid lineage cells may comprise myeloblast cells, granulocytes (including basophils, neutrophils, and eosinophils), monocytes, macrophages, dendritic cells and mast cells.

[0069] In the methods disclosed, a lymphoid lineage may arise from an appropriate progenitor cell, such as a common lymphoid progenitor (CLP) which may differentiate into lineage- restricted progenitors such as Pro-B (B-cell progenitors), CD5+CD7+Early T cell progenitors / lymphoid progenitors, Pro-T (T cell progenitors), Pro-NK (NK cell progenitors) and Pro-DC (DC progenitors) cells. Lymphoid lineage cells may comprise B-lymphocytes, early T cell progenitors, lymphoid progenitors, T-lymphocytes, natural killer (NK)-cells or dendritic cells. T-lymphocytes may further comprise helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, NK-T cells, gamma delta T cells or innate-like T cells. B lymphocytes may comprise plasma B cells or Memory B cells.

[0070] Methods of this disclosure may comprise yielding or obtaining an output population of cells comprising a lymphoid lineage cell or cells, and one or both of a myeloid lineage cell or cells and an erythroid lineage cell or cells. In one embodiment, an output population comprises: (i) a lymphoid lineage cell or cells and a myeloid lineage cell or cells; (ii) a lymphoid lineage cell or cells and an erythroid lineage cell or cells; or (iii) a lymphoid lineage cell or cells, a myeloid lineage cell or cells, and an erythroid lineage cell or cells. A lymphoid lineage cell (or cells) may comprise one or any combination of a natural killer (NK) cell, an early T cell progenitor, a lymphoid progenitor, a T lymphocyte, and a B lymphocyte. An early T cell progenitor or a lymphoid progenitor may comprise a cell capable of differentiating into a mature T cell and may comprise one or more markers such as CD7, CD5 and / or CD3. An early T cell progenitor or a lymphoid progenitor may lack expression of one or more markers comprising CD4 and / or CD8. A myeloid lineage cell (or cells) may comprise one or any combination of a myeloblast cell, a mast cell, a promyelocyte, a myelocyte, a basophil, a neutrophil, an eosinophil, a monocyte, and a dendritic cell. An erythroid lineage cell (or cells) may comprise one or any combination of erythroblast and a megakaryocyte.

[0071] Methods of this disclosure may comprise yielding or obtaining an output population of cells comprising B cells and lymphoid progenitors / T lymphocytes. Such methods may comprise culturing one or more HSPCs in a culture medium as described above, but additionally comprising a Notch ligand and / or lacking an SCF. In one embodiment, a notch ligand comprise DLL1 or DLL4, or a functional equivalent thereof. In one embodiment, a notch ligand may be immobilized on a solid phase, such as a wall of a container (or the like) or on a bead / particle (or the like).

[0072] Culturing one or more HSPCs in a culture medium to obtain an output population of cells comprising a lymphoid lineage cell or cells, and one or both of a myeloid lineage cell or cells and an erythroid lineage cell or cells may be performed in any appropriate cell culture vessel or dish known in the art, such as one or more wells of a 384-, 96-, 48-, 24-, 12-, or a 6- well plate.

[0073] An output population of cells comprising a lymphoid lineage cell or cells, and one or both of a myeloid lineage cell or cells and an erythroid lineage cell or cells may be analysed for its composition such as by flow cytometry, microscopy (e.g confocal / fluorescent), qPCR / RT-PCR, RNA-seq, deep learning or machine learning. Analyzing an output population of cells by flow cytometry may comprise combining an output population of cells with one or more antibodies against one or more markers of a lymphoid, myeloid and / or an erythroid lineage cell. By way of non-limiting example, markers of a lymphoid lineage cell may comprise one or any combination of CD45, CD19, CD56, CD7 or CD5; markers of a myeloid lineage cell may comprise one or any combination of CD45, CD33, FCeRla, CD11 b, CD14, CD15 or CD11c and markers of an erythroid lineage cell may comprise one or any combination of CD235a (GlyA, Glycophorin A or GPA), CD45 or CD41 .

[0074] Methods of this disclosure may further comprise analysing an output population of cells using a flow cytometry method comprising 1 , 2 or 3 lineage-specific markers to identify a representative cell or cells of the lymphoid, and myeloid and / or erythroid lineages. Such a flow cytometry method may comprise: identifying a lymphoid lineage cell by CD19 and / or CD56; and / or identifying a myeloid lineage cell by CD33 and / or CD15; and / or identifying an erythroid lineage by GPA and / or CD41 .

[0075] Methods of this disclosure may further comprise culturing one or more HSPCs in a static or a suspension cell culture system. Regardless of whether one or more HSPCs are cultured in a static or a suspension culture environment, it may be preferable that such environment is in the absence of stromal cells, feeder cells, or the like.Media and Kits

[0076] In one aspect of this disclosure, (liquid) culture media and / or kits are provided for detecting multilineage differentiation potential of one or more HSPCs. In one aspect of this disclosure are provided media and / or kits for assessing multilineage differentiation potential of one or more HSPCs, such as in advance of research, medical (e.g. clinical, therapeutic), or banking applications. In one aspect of this disclosure are provided media and / or kits for modulating multilineage differentiation potential of one or more HSPCs, such as in advance of research, medical (e.g. clinical, therapeutic), or banking applications.

[0077] Media for culturing one or more HSPCs to obtain an output population of cells comprising a lymphoid lineage cell, and one or both of a myeloid lineage cell and an erythroid lineage cell may be as described herein. Briefly, culture media of this aspect may comprise a basal medium, serum at a concentration ranging from between about 1% and 30% and one or more cytokines as described above and at the concentrations as described above, which cytokines may comprise one or any combination of IL-2, IL-3, IL-6, IL-7, IL-15, G-CSF, GM- CSF, SCF, Flt3-L or EPO.

[0078] Media for culturing one or more HSPCs to obtain an output population of cells comprising a lymphoid lineage cell, and one or both of a myeloid lineage cell and an erythroid lineage cell may be as described above. Briefly, culture media of this aspect may comprise a medium comprising a basal medium and one or more of: a serum albumin (e.g. BSA); and one or more cytokines (e.g. IL-2, IL-7, IL-15, EPO, etc.). In one embodiment, the medium is serum-free.

[0079] Kits of this disclosure may comprise a basal culture medium for culturing one or more HSPCs and one or more supplements to be added thereto. An exemplary supplement may comprise one or more cytokines, the one or more cytokines comprising one or any combination of IL-2, IL-3, IL-6, IL-7, IL-15, G-CSF, GM-CSF or EPO. Another exemplary supplement may comprise an albumin and / or serum. An exemplary supplement may comprise a Notch ligand and / or a small molecule. If one of a cytokine supplement and / or an albumin / serum supplement is not included in a kit, then it may either be comprised in the basal medium or separately provided by a user. Thus, kits of this disclosure may further comprise instructions for use.

[0080] A kit may comprise a basal medium, one or more supplement to obtain an output population of cells comprising a lymphoid lineage cell, and one or both of a myeloid lineage cell and an erythroid lineage cell. In one embodiment, a kit may comprise a basal medium and a supplement to obtain an output population of cells comprising a lymphoid lineage cell. In one embodiment, a kit may comprise a basal medium and a supplement to obtain an output population of cells comprising a myeloid lineage cell. In one embodiment, a kit may comprise a basal medium and a supplement to obtain an output population of cells comprising an erythroid lineage cell.

[0081] Each of the foregoing may be provided as separate kits, or may be combined together in any combination to provide a kit for the complete workflow for detecting multilineage differentiation potential of one or more HSPCs.

[0082] In some embodiments, kits of the disclosure may not include basal media, and may only include supplements described herein. Thus, any of the described supplements may be sold independently as a standalone supplement.

[0083] A concentration of the components comprised in a supplement may be higher than when diluted in or combined with a basal medium, as described herein.Specific assays and applications

[0084] Methods, media and kits of this disclosure may further relate to detecting / identifying multilineage potential of novel HSPC populations, which may comprise unique molecular features, within a HSPC hierarchy. Accordingly, such aspects may comprise: (i) providing one or more HSPCs comprising unique molecular features; and (ii) culturing the one or more HSPCs comprising unique molecular features in a culture medium to obtain an output population of cells comprising a lymphoid lineage cell or cells, and one or both of a myeloid lineage cell or cells and an erythroid lineage cell or cells. Unique molecular features may comprise one or more cell surface markers, transcription factors / regulators, or a signaling pathway component. Such aspect may facilitate discovery and research of multilineage potential of novel HSPC populations within the HPSC hierarchy that may not have been identified using legacy assays for detecting lineage potential of HSPCs. Such aspect may further comprise comparing an output population of cells obtained from one or more HSPCs comprising unique molecular features to an output population of cells obtained from one or more HSPCs comprising either established molecular features (e.g CMP, GMP, and MEP) or alternate unique molecular features to identify differences in multilineage potential.

[0085] Methods, media and kits of this disclosure may further relate to identifying donordependent differences in multilineage differentiation potential. Accordingly, such aspects may comprise: (i) providing one or more HSPCs of a first donor / individual; (ii) culturing the one or more HSPCs in a culture medium for a sufficient time to obtain an output population of cells comprising a lymphoid lineage cell or cells, and one or both of a myeloid lineage cell or cells and an erythroid lineage cell or cells, and (iii) comparing the output population of cells of the first donor / individual to an output population of at least a second donor / individual to identify donor- or individual-dependent differences in multilineage differentiation potential. An output population of at least a second donor / individual may have been previously determined or obtained in parallel (but in a separate container) to the output population of cells of the first donor / individual. HSPCs from different donors / individual may present variable / incongruent multilineage differentiation potential, and thus output populations therefrom may comprise different proportions of lymphoid lineage cells and one or both of myeloid and erythroid lineage cells.

[0086] Methods, media and kits of this disclosure may further relate to identifying multilineage differentiation potential of one or more HSPCs prior to or after developing / manufacturing i) a cell or gene therapy product, or ii) a tissue transplant. Accordingly, such aspects may comprise: (i) providing a first source of one or more HSPCs; (ii) culturing such one or more HSPCs in a culture medium for a sufficient time to obtain an output population of cells comprising a lymphoid lineage cell or cells, and one or both of a myeloid lineage cell or cells and an erythroid lineage cell or cells; and (iii) quantifying the output population of cells of the first source of one or more HSPCs for the lymphoid lineage cell or cells, and one or both of the myeloid lineage cell or cells and the erythroid lineage cell or cells. Such aspect may further comprise comparing an output population of cells obtained from the first source of one or more HSPCs to an output population of cells obtained from at least a second source of one or more HSPCs, to identify a candidate source of one or more HSPCs for developing / manufacturing the i) cell or gene therapy product, or ii) a tissue transplant. An output population of cells obtained from at least a second source of one or more HSPCs may have been previously determined or obtained in parallel (but in a separate container) to the output population of cells obtained from the first source of one or more HSPCs. HSPCs from different sources may present variable / incongruent multilineage differentiation potential, and thus output populations therefrom may comprise different proportions of lymphoid lineage cells and one or both of myeloid and erythroid lineage cells.

[0087] Methods, media and kits of this disclosure may further relate to assessing / screening efficiency of PSC differentiation to HSPCs. Accordingly, such aspects may comprise: (i) providing one or more HSPCs derived from a first PSC cell line; and (ii) culturing the one or more HSPCs derived from the first PSC cell line in a culture medium to obtain an output population of cells comprising a lymphoid lineage cell or cells, and one or both of a myeloid lineage cell or cells and an erythroid lineage cell or cells; and (iii) comparing the output population of cells of the first PSC cell line to an output population of at least a second PSC cell line to identify cell line-dependent differences in multilineage differentiation potential. An output population of at least a second PSC line may have been previously determined or obtained in parallel (but in a separate container) to the output population of cells of the first PSC cell line. HSPCs derived from different PSC lines may present variable / incongruent multilineage differentiation potential, which may be due to origin, and thus output populations therefrom may comprise different proportions of lymphoid lineage cells and one or both of myeloid and erythroid lineage cells. Such aspect may further comprise comparing an output population of cells obtained from a PSC line cultured in a first culture condition to an output population of cells obtained from the PSC line cultured in a second culture condition to identify differences in multilineage potential arising due to the culture conditions used. A first and / or asecond culture condition may comprise differences including, but not limited to, cell culture medium used, culture volume or scale (e.g. small-scale or large-scale), and culture dimension (e.g. 2D or 3D). Such aspect may comprise a gene-edited PSC line and may comprise comparing multilineage differentiation potential before, after or before and after genetically modifying a PSC line to identify an impact of genetic modification of a PSC line on multilineage potential of one or more PSC-derived HSPCs.

[0088] Methods, media and kits of this disclosure may further relate to assessing / screening multilineage potential of a source of one or more HSPCs obtained before, after, or before and after a treatment or a procedure. Accordingly, such aspects may comprise: (i) providing a source of one or more HSPCs obtained before, after or before and after a treatment or a procedure; (ii) culturing the one or more HSPCs in a culture medium for a sufficient time to obtain an output population of cells comprising a lymphoid lineage cell or cells, and one or both of a myeloid lineage cell or cells and an erythroid lineage cell or cells; and (iii) comparing the output population obtained from the source of one or more HSPCs to quantify an effect of the treatment or the procedure on the multilineage differentiation potential of the one or more HSPCs. A treatment or a procedure may comprise expanding, genetically modifying, purifying, cryopreserving, transporting or storing the one or more HSPCs. A treatment or a procedure may comprise transplantation of one or more HSPCs into an individual, a radio- or chemotherapy treatment of an individual, or storage of HSPCs in a blood bank. A treatment or a procedure may be performed in vitro, in vivo or ex vivo. Such aspect may assess a multilineage potential of HSPCs prior to transplantation of one or more HSPCs into suitable hosts or prior to clinical use of one or more HSPCs for treating autoimmune diseases, cancers, blood disorders, immune disorders, genetic diseases or other indications. Exemplary cancers include leukemia, lymphoma, solid tumors and multiple myeloma. Exemplary non-cancerous indications include thalassemia, sickle cell disease, anemia, severe combined immunodeficiency (SCID), Wiskott-Aldrich Syndrome and metabolic disorders.

[0089] Methods, media and kits of this disclosure may further relate to screening responsiveness of one or more HSPCs to a test condition (e.g. one or more compound) to determine the effect of the compound on multilineage differentiation potential of the one or more HSPCs. Accordingly, such aspect may comprise: (i) providing one or more HSPCs; (ii) exposing the one or more HSPCs to a test condition (e.g. one or more compound); and (iii) culturing the one or more HSPCs in a culture medium for a sufficient time to obtain an output population of cells comprising a lymphoid lineage cell or cells, and one or both of a myeloid lineage cell or cells and an erythroid lineage cell or cells. Such aspect may further comprise comparing an output population of cells comprising a lymphoid lineage cell or cells, and oneor both of a myeloid lineage cell or cells and an erythroid lineage cell or cells obtained with one or more compound to a reference or control compound. An output population of cells from a reference or control compound may have been previously determined or obtained in parallel (but in a separate container / well) to the output population of cells exposed to the test condition. Such one or more compound is not limited, and in a specific embodiment may comprise a small molecule, a chemical compound, a protein, a peptide, a nucleic acid or a nanoparticle. Exemplary compounds may comprise cytokines, cytokine analogs, growth factors, pharmacological agents, signaling pathway modulators / inhibitors, metabolic redox regulators, adhesion receptors / ligands, extracellular matrix (ECM) components, chemotherapeutic agents, myelosuppressive drugs, receptor agonists / antagonists, steroids, alkylating agents, nucleoside analogs, proteasome inhibitors, immunosuppressants (e.g. calcineurin inhibitors, corticosteroids), antibodies and antibody-drug conjugates . Such compounds may be well known in the art including, but not limited to, histone deacetylase (HDAC) Inhibitors, histone acetyltransferases modulators, DNA methyltransferase (DNMT) inhibitors, lysine-specific demethylase 1 inhibitors, bromodomain and extra-terminal (BET) inhibitors, glycogen synthase kinase-3 (GSK-3) inhibitors, p38 and JNK kinase inhibitors, mTOR inhibitors (e.g., Rapamycin), phosphoinositide 3-kinase (PI3K) inhibitors, aryl hydrocarbon receptor antagonists, copper chelators, NAD+ / SIRT1 Modulators, prostaglandin pathway modulators, redox-active compounds and metallic / lipid / polymeric nanoparticles. In one embodiment, exemplary small molecules may comprise one or any combination of a pyrimido-indole derivative, a histone deacetylase inhibitor (HDACi), a histone demethylase inhibitor (HDMi), a histone methyltransferase inhibitor (HMTi), an inhibitor of Enhancer of Zeste Homolog 2 (EZH2i) and an 5-hydroxytryptamine 1 receptor (5-HT1 F-R) agonist. One or more compound may comprise combinations of one or more compounds described above.

[0090] Methods, media and kits of this disclosure may further relate to assessing multilineage differentiation potential of gene-edited HPSCs. Accordingly, such aspect may comprise: (i) providing one or more gene-edited HSPCs; and (ii) culturing the one or more gene-edited HSPCs in a culture medium for a sufficient time to obtain an output population of cells comprising a lymphoid lineage cell or cells, and one or both of a myeloid lineage cell or cells and an erythroid lineage cell or cells. Such aspect may further comprise comparing an output population of cells obtained from a first gene-edited HSPC to an output population of a second gene-edited HSPC. An output population of cells from a second gene-edited HSPC may have been previously determined or obtained in parallel (but in a separate container / well) to the output population of cells from a first gene-edited HSPC. Such aspect may involve comparing multilineage differentiation potential before, after or before and after genetically modifying one or more HSPCs.

[0091] Methods, media and kits of this disclosure may further relate to assessing toxicity of a compound on one or more HSPCs and associated effects on multilineage differentiation potential. Accordingly, such aspect may comprise: (i) providing one or more HSPCs; (ii) exposing the one or more HSPCs to a compound; and (ii) culturing the one or more HSPCs in a culture medium for a sufficient time to obtain an output population of cells comprising a lymphoid lineage cell or cells, and one or both of a myeloid lineage cell or cells and an erythroid lineage cell or cells. Such aspect may further comprise comparing an output population of cells comprising a lymphoid lineage cell or cells, and one or both of a myeloid lineage cell or cells and an erythroid lineage cell or cells obtained with a compound (whose toxicity is being assessed) to a reference or control compound. An output population of cells from a reference or control compound may have been previously determined or obtained in parallel (but in a separate container / well) to the output population of cells exposed to the test compound. Such one or more compound is not limited, and may comprise environmental chemicals, toxins, heavy metals, chemotherapeutic agents, antibiotics, immunosuppressants, specific proteins, peptides, or nucleic acids. In one embodiment, a compound may comprise a small molecule.

[0092] Methods, media and kits of this disclosure may further relate to performing an in vitro limiting dilution analysis to quantify a probability of obtaining a lymphoid lineage cell or cells, and one or both of a myeloid lineage cell or cells and an erythroid lineage cell or cells among an output population of cells after culturing one or more HSPCs in a culture medium as described above. Accordingly, such aspect may comprise: (i) providing one or more HSPCs;(ii) preparing at least one cell dose comprising an input number of the one or more HSPCs;(iii) culturing the one or more HSPCs in a culture medium for a sufficient time to obtain an output population of cells comprising a lymphoid lineage cell or cells, and one or both of a myeloid lineage cell or cells and an erythroid lineage cell or cells; and (iv) quantifying a probability of obtaining a lymphoid lineage cell or cells, and one or both of a myeloid lineage cell or cells and an erythroid lineage cell or cells among the output population of cells after the culturing. A cell dose comprising an input number of the one or more HSPCs may comprise at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more HSPCs per well of a culture vessel. Two or more replicates may be prepared for each cell dose comprising an input number of the one or more HSPCs. A probability of obtaining a lymphoid lineage cell or cells, and one or both of a myeloid lineage cell or cells and an erythroid lineage cell or cells may be quantified or calculated using any statistical method known in the art. Preferably, a probability may be calculated using any software or computer-generated methods known in the art, and preferably applying a Poisson statistical analysis (e.g. Extreme Limiting Dilution Analysis (ELDA) algorithm). Methods, media and kits of this disclosure may yield an about 5-20% probability of obtaining an erythroid lineage cell or cells, and / or about 25-55% probability ofobtaining a myeloid lineage cell or cells, and / or about 15-40% probability of obtaining a lymphoid lineage cell or cells using an in vitro limiting dilution assay as described herein. In some embodiments, an output population of cells may comprise a lymphoid lineage cell or cells, a myeloid lineage cell or cells, an erythroid lineage cell or cells or combinations thereof. Determining a probability of obtaining each lineage (lymphoid, erythroid or myeloid) using the methods of this disclosure could be advantageous for effectively predicting starting cell numbers and resultant cell yields for large-scale or clinical HSPC-related applications. An in vitro limiting dilution analysis as described herein may be used in conjunction with any of the methods, media, kits, assays and applications of the disclosure.

[0093] In any of the described applications and assays, the nature of the one or more HSPCs, source of the one or more HSPCs, culture media, sufficient time of culture, culture conditions, output populations, and in vitro limiting dilution methodology may be as described herein, and is thus incorporated by reference.

[0094] Any of the described methods, applications and assays may be in vitro or ex vivo.

[0095] The following non-limiting examples are illustrative of the present disclosure.ExamplesExample 1: Preparation of HSPCs

[0096] CD34+HSPCs were isolated from either cord blood (CB), adult mobilized peripheral blood (mPB), or bone marrow (BM) by immunomagnetic separation, such as with the EasySep™ Human CD34 Positive Selection Kit II (STEMCELL Technologies). When differentiating PSC (e.g. SCTi003-A or STiPS-F016) to CD34+cells, PSCs were maintained in a TeSRTM-branded medium before differentiation using the STEMdiff™ Hematopoietic-EB Medium and Supplements (STEMCELL Technologies) according to manufacturer’s instructions.Example 2: EML assay workflow

[0097] Unless otherwise specified herein, EML assays were performed in either 24-well or 96- well formats as follows. A cryovial containing CD34+cells purified as described in Example 1 was thawed in a 37°C water bath, and 1 ml of medium (e.g. IMDM with 10% FBS) was added dropwise to the cryovial. The suspension was added dropwise into 10 mL of medium in a 15 mL Falcon Tube, centrifuged at 1200 rpm for 10 minutes, and after removing the supernatant the cell pellet was resuspended in 500 pL of an appropriate basal medium of this disclosure. Viability and cell counts were determined using a NucleoCounter® with staining dyes acridine orange (AO) and / or DAPI, and cell density was adjusted with a culture medium (hereinafterreferred to as EML medium, comprising a basal medium (e.g. IMDM, alpha-MEM with nulceosides), FBS, 2-mercaptoethanol, L-glutamine, erythropoietin and IL-2) in order to seed ~5000 cells in 1000 pL / well of a 24-w plate, or ~ 1000 cells in 200 pL / well of a 96-w plate.

[0098] Cells were cultured for up to 4-6 weeks, with half medium changes weekly. If harvesting cells at any point for further analysis (as described in Example 3), 90 pL (96-w) or 480 pL (24-w) of cells were transferred into a plate or a tube respectively. For 24-w plate samples, harvested cells were centrifuged at 1200 rpm for 5 min. To maintain cultures, at the end of Week 1 roughly half the medium was exchanged with fresh EML medium (~100 pL for 96-w and -~500 pL for 24-w). At the end of Weeks 2, 3, 4, 5 or 6, cells were mixed and replenished with additional fresh EML medium (~100 pL for 96-w and ~500 pL for 24-w) and incubated at 37°C for further culturing.

[0099] To output specific lymphoid progenitors or T lymphocytes (e.g. CD5+CD7+), a notch ligand (e.g DLL4-coated bead) was included in an EML medium comprising a basal medium (e.g. IMDM), FBS, 2-mercaptoethanol, L-glutamine, erythropoietin and IL-2 and lacking an SCF) in the described EML workflow.Example 3: EML assay analysis & flow cytometry assessments

[0100] Cell counts and viability measurements were determined essentially as described in Example 2. Flow cytometry assessments were performed at the end of each week of culture, as described below. Briefly, 100 pL (96-w) or 50 pL (24-w) of blocking buffer (Phosphate buffered saline with 2% FBS and 1 pg / mL anti-human CD32 antibody (clone IV.3) was added to each well of the resultant harvested cells as described above. For the 96-w plate format, cells were centrifuged at 1200 rpm for 5 min and the cell pellet was gently mixed in a residual volume of about ~20 pL. For flow cytometry, a sufficient volume from each well (2 pL & 10 pL for the 96-w & 24-w formats respectively) was removed and combined for each unstained and fluorescence minus one (FMO) control. Appropriate antibody and FMO cocktails were added to each sample well and corresponding FMO controls respectively, and the plate was incubated at room temperature for 30 min, protected from light. After adding 200 pL of PBS+2% FBS to all sample and FMO-containing wells and centrifuging at 1200 rpm for 5 min, 100 pL of Propidium Iodide (PI) solution (1 pL of 1 mg / ml PI per 1 mL PBS+2% FBS) was added to each well. Stained samples were analysed on a CytoFLEX Flow Cytometer (Beckman Coulter).

[0101] Cell phenotypes were identified by flow cytometry using either a comprehensive or a simplified panel of marker combinations as shown in Table 1 .Table 1 : Markers for assessing erythroid, myeloid and lymphoid lineage cells by f ow cytometry.

[0102] Antibodies used for the comprehensive flow cytometry panel comprised:CD45 (Clone HI30), CD34 (Clone 581), CD235a (Clone HI264), CD41 (clone HIP8), CD33 (clone WM53), CD19 (clone SJ25C1), CD56 (clone CSSMB), CD11b (clone M1 / 70), CD15 (clone HI98), CD14 (clone M0P9), CD11c (clone 3.9) and FCeRla (clone AER-37) sourced from Biolegend, BD Biosciences or ThermoFisher. To identify lymphoid progenitors or T lymphocytes, FCeRla and CD11c antibodies were replaced with CD5 (clone L17F12) and CD7 (clone M-T701) sourced from Biolegend and BD Biosciences respectively. Antibodies used for the simplified flow cytometry panel comprised: GPA (clone HIR2), CD41 (clone HIP8), CD33 (clone HIM3- 4), CD15 (clone MC-480), CD19 (clone HIB19), CD56 (clone HCD56), CD45 (clone HI30) and CD34 (clone 581), sourced from Biolegend.

[0103] Exemplary flow cytometry gating strategies using a comprehensive or a simplified marker panel are shown in Figure 1A and 1 B, respectively. For initial processing, CD34+cells were gated based on: granularity and size (Fig. 1A & 1 B-i); single cells to exclude cell clumps / clusters (Fig. 1A & 1 B-ii); and viable cells based on PI staining to exclude debris and dead cells (Fig. 1A & 1 B -iii). For the comprehensive strategy, live cells were further gated to identify CD34+cells based on CD45 and CD34 expression (Fig. 1A-iv). The CD34+cells were further gated based on expression of the markers specified in Table 1 to identify various erythroid, myeloid or myeloid lineages (Fig. 1A v-xi, and Table 1).

[0104] For the simplified strategy, live cells were further gated based on the following marker expression: CD33 / CD15, CD19 / CD56 and GPA / CD41 to identify myeloid, lymphoid and erythroid lineages, respectively (Fig 1 B iv-vi). Thus, it is possible to effectively identify lymphoid, erythroid and myeloid lineage cells based on a reduced set of cell type-specific markers, thereby reducing reagent costs, complexity and time required to analyze EML assay output.Example 4: Effect of serum and serum-free conditions on EML assay output

[0105] To test the effect of serum on EML assay output, an EML assay was performed essentially as described in Examples 1 and 2 in a 96-well plate, except for a few modifications outlined below. Three serum-containing media formulated with IL-2 were tested, namely Alpha-MEM with nucleosides (STEMCELL Technologies), MyeloCult™ H5100 (STEMCELL Technologies) and Iscove's Modified Dulbecco's Medium (IMDM), IMDM and alpha-MEM comprising FBS titration (1%, 2.5%, 5%, 7.5%, 10% and 12.5%). Viability, cell numbers and flow cytometry assessments using the comprehensive panel (as described above) were performed at weeks 2, 3 and 4, as described in Example 3. For each medium tested, higher FBS concentrations (7.5% & higher) led to better EML output than lower FBS concentrations (data not shown). Therefore, an FBS concentration of 12.5% was used in further experiments. Total counts of CD34+, erythroid, myeloid and lymphoid lineage cells at week 3 showed comparable EML output for all 12.5% FBS-containing media formulations tested (Figure 2A), demonstrating that each of erythroid, myeloid and lymphoid lineages could be simultaneously output in an EML assay using various serum-containing media.

[0106] Next, EML assay output was tested in different serum-free media formulations, essentially as described in Examples 1 and 2 in a 96-well plate using StemSpan™ SFEM or StemSpan™ SFEMII (each STEMCELL Technologies) supplemented with EPO, IL-2, L- glutamine, and mercaptoethanol. IMDM ± 12.5% FBS was used as a positive control. Viability, cell number and comprehensive flow cytometry assessments were performed at weeks 2, 3 and 4 as described in Example 3. Total counts of CD34+, erythroid, myeloid and lymphoid lineage cells at week 3 showed that all assessed cell types could be detected (Figure 2B). Although lymphoid cell lineage counts were reduced by about 85%(SFEMII) and about 50% (SFEM) in serum-free formulations, erythroid lineage counts were increased by about 3-fold (SFEMII). CD34+and myeloid counts were comparable for all conditions tested. These data demonstrate that the disclosed EML assay effectively outputs erythroid, myeloid and lymphoid lineages under serum-free media conditions.Example 5: Cytokine and growth factor effects on EML assay output

[0107] A medium formulation as described above (Alpha-MEM with nucleosides (STEMCELL Technologies) + FBS + 2-mercaptoethanol + L-glutamine + EPO) was further supplemented with various cytokines and growth factors (IL-2, IL-3, IL-6, IL-15, Granulocyte colonystimulation factor (G-CSF), or stem cell factor (SCF)), and an EML assay starting from cord blood-derived CD34+cells was performed essentially as described in Example 2, and the cell lineage outputs were assessed as described in Example 3.With regard to IL-2, its exclusion resulted in no NK cell output (Figure 3A-vi), but a presence or absence of IL-2 did not have a significant impact on myeloid, dendritic and neutrophil cells (Fig. 3A-ii, Hi, vii). However, B cell and erythroid counts were lowered by about 70% and 84% respectively in the presence of IL-2 (Fig. 3A-iv, & v). To assess whether a reduced concentration of IL-2 could improve B cell and erythroid cell counts, IL-2 was tested at a lower concentration ranging from between about 5 ng / ml and 15 ng / ml (IL-2-low), or at a higher concentration ranging from between about 35 ng / ml and 45 ng / ml (IL-2-high) (Fig. 3B). Low and high concentrations of IL-2 appeared to result in similar output of all cells assayed (Figure 3B), which may suggest donor-specific differences compared to the results shown in Figure 3A. Nevertheless, it was observed that cell output was largely unaffected when reducing IL-2 concentration. Similar cell outputs at both tested IL-2 concentrations were observed for CD34+, dendritic, and megakaryocyte cells as well, while slightly lower monocyte output was observed in low IL-2 (data not shown).

[0108] This experiment also tested if IL-15 could be substituted for IL-2, and the results show that this cytokine may be interchangeable with IL-2 for supporting NK cell output. While B cell output was reduced by about 50% and about 20% compared to the IL-2 low and IL-2 high conditions respectively and erythroid output was reduced by about 47% and about 20% compared to the IL-2 low and IL-2 high conditions respectively, neutrophil output was comparable (Figure 3B). Counts of monocytes were about 100% higher in IL-15 or IL-2 high conditions compared to the IL-2 low condition (data not shown). In addition, no appreciable synergy was observed when combining IL-2 and IL-15 (data not shown).

[0109] With regard to IL-3 or G-CSF, EML assay output was mostly comparable to an EML media not supplemented with either cytokine (data not shown). Particularly, % of viable CD34+cells (10-20%) and monocytes (3-7%) observed at week 3 was comparable between EML medium + IL-3 or G-CSF vs EML medium - IL-3 or G-CSF, while addition of either G-CSF or IL-3 reduced erythroid output by about 30% (40% vs 6% of viable erythroid cells in EML - IL- 3 or G-CSF vs EML + IL-3 or G-CSF, respectively). Addition of either G-CSF or IL-3 also reduced B-cell output from about 10% to about 5% compared to EML media without these components. When EML medium was supplemented with both IL-3 and G-CSF, % of viable erythroid and B cells at week 3 was reduced by about 4.9% and about 1% respectively than when adding only IL-3 or G-CSF (data not shown).

[0110] With regard to supplementing EML media with IL-6, B cell, erythroid and dendritic cell output was reduced from about 7%, 30%, and 5% to about 0.7%, 4% and 2% respectively at week 3 (data not shown) and no NK cells were obtained. Addition of IL-6 increased monocytes and neutrophils from about 2% and 10% to about 7% and 40% respectively (data not shown).Addition of IL-6 to an EML media comprising IL-2 exaggerated the observed reductions in erythroid and B-cell outputs when adding IL-6 alone (as described above) but gave comparable NK cell output (about 0.8% to about 3%) to EML media comprising IL-2 without IL-6 (data not shown).

[0111] Overall, these results show that addition of G-CSF, IL-3 or IL-6 reduced erythroid and B-cell output, and NK cell output is abolished in the absence of IL-2. Interestingly, addition of IL-2 or IL-15 supported NK cell output without abrogating B cell and erythroid output.

[0112] Next, EML media (comprising IMDM as a basal medium, FBS, 2-mercaptoethanol and L-glutamine and supplemented with IL-2, SCF, EPO) in the presence or absence of IL-7, erythropoietin (EPO) or SCF were evaluated in an EML assay, essentially as described in Example 2 as assessed essentially as described in Example 3.

[0113] HSPCs derived from cord blood samples produced a similar percentage of viable CD34+(about 8%), erythroid (about 10%), myeloid (about 40%) and lymphoid (about 10%) cells in the presence or absence of IL-7 (but otherwise including IL-2, EPO and SCF) (data not shown). As expected, no erythroid output was observed in the absence of EPO (but in the presence of IL-2, IL-7, and SCF), demonstrating the essentiality of EPO in an EML assay medium (data not shown). Surprisingly, each of erythroid, myeloid and lymphoid lineages were output in EML medium lacking SCF (but otherwise including IL-2, IL-7 and EPO). Notably, levels of viable lymphoid and erythroid lineage cells were increased by about 2-fold in EML medium lacking SCF (data not shown).

[0114] To confirm the effects observed regarding SCF, an EML assay was performed as described in Examples 1 and 2 with cord blood-derived cells cultured in an EML medium comprising or excluding SCF, but otherwise comprising 7.5% FBS, 2-mercaptoethanol, L- glutamine, IL-7, EPO and IL-2. The results using EML medium lacking SCF were confirmed, whereby at week 3 each of erythroid, myeloid and lymphoid lineages were output (Fig. 4). Notably, absence of SCF in EML medium improved erythroid and megakaryocyte outputs by about 10-fold (Fig. 4-ii & iii), and improved B and NK (Fig. 4-iv & v) output by about 43% and 100% respectively. CD34+ cell outputs were similar (Fig. 4-i). However, myeloid, neutrophil, and monocyte output were reduced by about 66%, 75% and 68% respectively in the absence of SCF (Fig. 4-vi, vii & viii). Nevertheless, about 30-40 % of viable output cells at week 3 corresponded to myeloid lineage cells in the absence of SCF (data not shown).

[0115] Further supplementation of EML medium (comprising IMDM as a basal medium, FBS, 2-mercaptoethanol and L-glutamine and supplemented with IL-2, EPO and IL-7) with myeloid- associated cytokines such as IL-3, GCSF and GMCSF to enhance myeloid output in the absence of SCF was also tested. In the absence of SCF, monocyte output was about 3%,which addition of IL-3, GCSF or GMCSF increased to about 5%, 12% and 15% respectively (data not shown). In the absence of SCF, neutrophil output was about 6%, which addition of IL-3, GCSF or GMCSF increased to about 20%, 50% and 30% respectively (data not shown). In the absence of SCF, B-cell output was about 3%, which addition of IL-3, GCSF or GMCSF decreased to about 1.5%, 1.5% and 0.8% respectively (data not shown). Overall, these results surprisingly showed that an EML assay could output all assayed cell types from cord blood- derived CD34+HSPCs even in the absence of a common hematopoietic cytokine, SCF.

[0116] In order to output specific lymphoid progenitors / T lymphocytes (e.g. CD5+CD7+), a notch ligand (e.g DLL4-coated bead) was included in an EML medium comprising a basal medium (e.g. IMDM), FBS, 2-mercaptoethanol, L-glutamine, erythropoietin, IL-7 and IL-2 and lacking an SCF). Different tested concentrations of DLL4-coated beads (2.7 pmol / mL, 9 pmol / mL, & 27pmol / mL) had a similar impact on the output % of viable cells (data not shown). An increase of about 40% in a % of viable lymphoid progenitors / T lymphocytes (CD5+CD7+) was observed when including a notch ligand in the EML assay (Fig. 8B) compared to an EML assay without a notch ligand (Fig. 8A). B cell output was decreased by about 2% in the presence of the notch ligand (Fig. 8A & 8B). Erythroid lineage cell output was reduced by about 45% while MK cell output diminished in the presence of notch ligand (Fig. 8A & 8B). Therefore, these results demonstrated that B cells and lymphoid progenitors / T lymphocytes could be simultaneously obtained using an EML assay as described herein, in addition to myeloid and erythroid lineage cells.Example 6: Effect of using fresh vs cultured HSPCs on an EML assay

[0117] Whether or not fresh or cultured HSPC perform similarly in a functional assay was further assessed. Cord blood-derived HSPCs isolated in accordance with Example 1 were either used fresh or were pre-cultured in StemSpan™ SFEM II with StemSpan™ CD34+Expansion Supplement (STEMCELL Technologies) for 7 days. An EML assay was performed with such cells in an EML medium (comprising alpha-MEM with nucleosides (STEMCELL Technologies), FBS, 2-mercaptoethanol, L-glutamine and supplemented with IL-2 and EPO, in accordance with Example 2 (24-w format), and the output cells were assessed as in Example 3.

[0118] Fresh cells remained viable longer than pre-cultured HSPCs during the EML assay. While pre-cultured HSPCs exhibited >80% viability only for 3 weeks, fresh cells exhibited >80% viability for 5 weeks (data not shown). While slightly higher but more-or-less comparable counts of myeloid cells, B cells, NK cells, erythroid cells, and monocytes could be obtained from fresh or cultured HSPCs (Fig. 5- Hi, iv, vi, vii, & viii), output of CD34+cells, neutrophils and MK cells was reduced by about 98%, 83% and 100% respectively whenstarting from cultured cells (Fig. 5-i, ii & v). Further, when starting from cultured HSPCs, output of several lineages such as megakaryocytes, B cells, and to some extent NK cells, dropped earlier than for corresponding cells when obtained starting from fresh HSPCs, especially from week 4 onward (data not shown).Example 7 EML output potential of HSPCs derived from various sources

[0119] The suitability of HSPCs isolated (as per Example 1) from various sources such as cord blood (CB), adult mobilized peripheral blood (mPB), bone marrow (BM) or as derived from PSCs was assessed in an EML assay essentially as described in Example 2, using an EML medium (comprising IMDM as a basal medium, FBS, 2-mercaptoethanol and L- glutamine and supplemented with IL-2, SCF, EPO and IL-7), and the output cells was assessed by flow cytometry as described in Example 3.

[0120] For CB-derived HSPCs, the output cell lineages displayed different kinetics depending on the culture duration. Erythroid lineage cells (erythroid cells and MK cells) peak earliest around week 2 and decline thereafter. Lymphoid lineage cells are present at very low levels at week 2 and continue to increase over time peaking around week 4. Myeloid lineage cells display a steady increase over time, with monocytes typically being the majority remaining cell type at week 6 (Data not shown). mPB-derived HSPCs were found to retain high viability and CD34+cell output for at least 4 weeks (data not shown) and produced multilineage EML outputs in vitro at weeks 3 and 4. Although the lymphoid output was low (0.25%) at week 3 in the EML assay for mPB-derived HSPCs, it was still detectable and continued to rise to about 0.6% at Week 4 and about 2.1% at Week 5. For BM-derived HSPCs, lymphoid, erythroid and myeloid lineage outputs were detectable at weeks 3 and 4 and the output of lymphoid lineage cells increased from about 7% to about 12% between week 3 and week 4 (data not shown). As with cord blood, erythroid lineage cells declined between week 3 and week 4 and myeloid lineage cells increased over the same period for both mPB and BM derived HSPCs (data not shown).

[0121] Nevertheless, CB, mPB and BM-derived HSPCs simultaneously produced erythroid, myeloid, and lymphoid lineage cells as shown in Table 2. While higher monocyte and neutrophil cell counts were observed starting from BM- or mPB-derived HSPCs, higher B and NK cell counts were observed with CB-derived HSPCs.an EML assay starting from the indicated cell types (CB, mPB or BM-derived HSPCs). % represents the proportion / frequency of the indicated lineage among total viable single cells obtained after appropriate gating, as described in Example 3. 11 , 5 and 6 samples were tested for CB, mPB and BM-derived HSPCs, respectively.

[0122] With regard to PSC-derived CD34+HSPCs, two iPSC lines SCTi003-A or STiPS-F016 were differentiated as described in Example 1 , and the output cells after culture demonstrated an ability to simultaneously produce erythroid, myeloid, and lymphoid lineage cells as shown in Table 3.Table 3: Flow cytometry-based frequencies of cell types corresponding to erythroid, myeloid and lymphoid lineages from EML assay performed with PSC-derived HSPCs at Week 3. % represents the proportion / frequency of the indicated lineage among total viable single cells after appropriate gating as described in Example 3. *NE - Not Examined

[0123] I nterestingly, EML assay output of PSC-derived HSPCs was cell line-dependent. While the levels of MK cells, erythroid, B cells and NK cells were higher when starting from F016 cells, the levels of monocytes and neutrophils were higher when starting from 3A cells. Such observed variations could be attributed to factors such as cell line origin and / or reprogramming methods used. Nevertheless, these results show that an EML assay can be used to identify varying multilineage differentiation potential of PSC-derived CD34+cells.

[0124] Overall, the foregoing results demonstrated that an EML assay can be initiated using cells obtained / isolated from a wide variety of sources.Example 8: EML assay using single cord blood-derived HSPCs

[0125] Single cord blood-derived HSPCs were assessed by an EML assay, essentially as described in Example 2 (96-w), to evaluate differentiation potential at the single cell level. Single CD45midCD34+cells were seeded per well into 50 pL of EML medium comprising a- MEM, FBS, glutamine and beta-mercaptoethanol and supplemented with EPO, G-CSF and IL-2. Rather than a half-medium change at weeks 1 and 2, 50 pL and 100 pL of fresh medium was added to each well after weeks 1 and 2, respectively. Cells were harvested and analyzed by flow cytometry after either 3 weeks or 4 weeks, essentially as described in Example 3, with additional markers to identify NK cells (CD5 CD56+), NK-T cells (CD7+CD56+), Early T cell progenitors / LP cells (CD5+CD7+) and neutrophils (CD33+CD14 CD15+).

[0126] On a cell-by-cell basis, single HSPCs were found to have full lineage potential, as shown in Table 4. At week 3, 3 / 55 single cell clones showed EML potential (representative flow cytometry data not shown), 12 / 55 clones showed myeloid-only potential, 2 / 55 clones showed erythroid-only potential, while none of the clones showed lymphoid-only potential. At week 4, 7 / 60 single cell clones showed EML potential (representative flow cytometry data not shown), 3 / 60 clones showed myeloid-only potential, 3 / 60 clones showed erythroid-only potential, and 2 / 60 clones showed lymphoid-only potential.Table 4: Flow cytometry-based total counts at week 3 and week 4 of single cord blood-derived HSPCs and associated differentiation potential in an EML assay.

[0127] Therefore, these results demonstrated that an EML assay could output erythroid, myeloid, and lymphoid lineage cells, and combinations thereof, when starting from a single HSPC.Example 9: EML assay starting from limiting numbers ofcord blood-derived HSPCs

[0128] While an EML assay could be performed starting from single HSPCs, as described in Example 8, it may be time-consuming and inefficient to plate multiple single cells from low clonogenic HSPC populations (with a lower propensity of forming erythroid, myeloid and / or lymphoid lineage cells). Therefore, an in vitro limiting dilution method was developed starting from limiting numbers of HSPCs, instead of single HSPCs. An in vitro limiting dilution method may comprise measuring a probability of obtaining lymphoid lineage cells, and one or both of myeloid lineage cells and erythroid lineage cells from one or more HSPCs after culturing the one or more HSPCs in a culture medium.

[0129] Briefly, cord blood-derived HSPCs were isolated in accordance with Example 1 , and 40 replicates of different “cell doses” (2, 4, or 10 cells) were deposited in either 50 pL or1 OOpL of EML media comprising IMDM (STEMCELL Technologies), FBS, 2-mercaptoethanol, L- glutamine and supplemented with IL-2, IL-7 and EPO, into wells of a 96-well plate, and cultured essentially as described in Example 2. Following 4 weeks of culture, output cells were assessed as described in Example 3. At week 1 , a half-medium change or media addition was performed to bring all wells to 100uL. At week 2, 100uL of additional EML medium was added to each well. At week 3, a half medium change was performed. At week 4, wells were assessed visually for positive growth, harvested and stained for flow cytometric analysis. As an initial plating volume of either 50 pL or 100 pL was not observed to impact a clonogenicfrequency, cells from both plating conditions were treated as a single condition for further analysis. Wells were assigned as positive if >200 viable cells (propidium iodide negative) were detected by flow cytometry and at least 25 events in one cell type gate. Wells containing >25 events in multiple cell gates were assigned as positive for each such cell type.

[0130] The cell dose, no. of positive wells and trials were input into a limiting dilution analysis software (Extreme Limiting Dilution Analysis, ELDA) to determine a probability of cells within an input population of one or more HSPCs that are capable of producing a particular lineage (erythroid, myeloid and lymphoid) (Table 5).Table 5: Limiting dilution analysis to determine the probability a given cell dose (2, 4, and 10 cells) will output a cell lineage of interest. 40 replicates (Trials) were tested for each cell dose, and the number of positive wells (positives) were identified by flow cytometry. ELDA software calculated for a given population the probability estimate that an EML assay will output a cell lineage of interest with a 95% confidence interval (Cl), lower and higher Cl ranges are shown.

[0131] Based on the limiting dilution analysis, a probability of obtaining erythroid, myeloid and lymphoid lineage cells from an input population of cord blood-derived HSPCs was estimated as 13.2%, 39.4% and 28% respectively (Table 5). Therefore, an input population of cord blood derived HSPCs could be predicted to output an output population of cells comprising about 28% of cells that are capable of producing lymphoid lineage cells, 39.4% of cells that are capable of producing myeloid lineage cells, and 13.2% of cells that are capable of producing erythroid lineage cells.

[0132] These results demonstrate that an EML assay can be performed starting from limiting numbers of HSPCs to quantitatively assess lineage potential of an input population reflective of the composition of its heterogenous HSPCs and allow for comparison to a different input population (e.g. derived from a different source or a donor) to effectively predict multilineage differentiation potential (e.g. lymphoid, myeloid and erythroid lineage cells).Example 10: EML assay using HSPCs obtained from different donors

[0133] Donor-dependent differences in multilineage differentiation potential were assessed by an EML assay starting from HSPCs derived from cord blood samples (11 donors) or bone marrow samples (6 donors), essentially as described in Example 2 and assessments performed at week 3 essentially as described in Example 3.

[0134] Donor-dependent variation was observed in EML assay output (Figure 6). For instance, when starting from cord blood-derived HSPCs, donor 1 HSPCs output the highest % of viable lymphoid lineage cells (about 17-20%); equivalent % of myeloid lineage cells (about 40%) and lower % of erythroid lineage cells (about18-20%), as compared to other donors (Fig. 6A). Donor 6 HSPCs output the highest % of myeloid lineage cells (about 80%) and a relatively lower output of erythroid and lymphoid lineage cells (< about 10%) (Fig. 6A) as compared to other donors. HSPCs from donors 8-10 output the highest % erythroid lineage cells (about 40-50%) and lower output of myeloid lineage cells (about 30-40%) and lymphoid lineage cells (about 10-15%) as compared to other donors (Fig. 6A).

[0135] When starting from bone marrow-derived HSPCs, donors 1-4 output consistently high levels of myeloid lineage cells (about 40-60%) and consistently low erythroid levels (about 20%), while the opposite was true for donors 5 and 6 (about 50% erythroid and about 30% myeloid). While lymphoid output was more variable among the donors, donors 1 , 5 and 6 output low levels of lymphoid cells (Fig. 6B). Thus, it appears that comparatively lower erythroid potential correlates with comparatively higher myeloid potential, and vice versa, for BM-derived HSPCs, while lymphoid potential does not appear to correlate with erythroid and myeloid potential.

[0136] Therefore, these results demonstrate that an EML assay can effectively screen the multilineage potential of HSPCs from different donors / samples. Such an assay may be beneficial to identify / select an appropriate donor when a particular output is desired / required, such as for expansion / differentiation workflows or even clinical applications.Example 11: EML assay starting from HSPCs as exposed to various test conditions

[0137] Modulation of multilineage differentiation of HSPCs was assessed among cord blood- derived CD34+HSPCs (isolated as described in Example 1) pre-cultured for 7 days in an appropriate medium (e.g StemSpan™ SFEM II) supplemented with SCF, TPO and Flt3L (denoted as STF) and further supplemented with one or more chemical compounds (e.g. small molecule): (i) UM729; (ii) a cocktail comprising a histone deacetylase inhibitor (HDACi), a histone demethylase inhibitor (HDMi) and a histone methyltransferase inhibitor (HMTi); (iii) an inhibitor of Enhancer of Zeste Homolog 2 (EZH2) (denoted as EZH2i); and (iv) an 5- hydroxytryptamine 1 receptor (5-HT1 F-R) agonist. Fresh cord blood-derived HSPCs not precultured as described above were controls. Approximately 4000 cells were plated into EML medium comprising IMDM (STEMCELL Technologies), FBS, 2-mercaptoethanol, and L- glutamine supplemented with IL-2, IL-7 and EPO and the assay was performed essentially as described in Examples 2 and 3.

[0138] In general, fresh cells performed well in the EML assay for all cell types assessed (Figure 7), and in particular for CD34+cells and MK cells compared to the pre-cultured conditions. Regarding erythroid cells and monocytes, the combination of STF + EZH2i or 5- HT1 F-R agonist reduced output in comparison to fresh cells while all other tested conditions yielded more or less comparable or higher output. Regarding neutrophils, only pre-culture with STF+UM729 or STF+HDACi+HDMi+HMTi could either approach or meet, respectively, the output potential of fresh HSPCs. Regarding B cells, only slight differences in output potential were observed where fresh cells or pre-cultured cells were input into an EML assay. Addition of UM729 slightly increased the output of erythroid lineage cells, neutrophils, B cells and monocoytes compared to STF alone (Fig. 7-ii, Hi, iv, v). Addition of STF + HDACi + HDMi + HMTi led to an increased output of erythroid cells, neutrophils, B cells, monocytes and megakaryocyte cells compared to addition of STF alone (Fig. 7-ii-vi). Addition of EZH2i reduced the output of lymphoid, myeloid and erythroid lineages. While inclusion of a 5-HT1 F- R agonist led to a lowered output of erythroid cells and monocytes compared to STF alone (Fig. 7-ii, v), it increased the output of B cells (Fig. 7-iv).

[0139] Thus, an EML assay can effectively assess impacts of chemical compounds or other medium additives on the multilineage differentiation potential of HSPCs, such as into lymphoid, erythroid and / or myeloid cells.

[0140] While the present disclosure has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the disclosure is not limited to the disclosed examples. To the contrary, the disclosure is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[0141] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Claims

CLAIMS1) A method for detecting multilineage differentiation potential of one or more hematopoietic stem and progenitor cells (HSPCs), the method comprising:(i) providing the one or more HSPCs; and(ii) culturing the one or more HSPCs in a culture medium for a sufficient time to obtain an output population of cells comprising a lymphoid lineage cell, and one or both of a myeloid lineage cell and an erythroid lineage cell.2) The method of claim 1 , wherein the one or more HSPCs: a) are derived from an input population of cells, wherein the input population of cells is cord blood-derived, bone marrow-derived, peripheral blood-derived, mobilized peripheral blood-derived or pluripotent stem cell-derived (PSC); and / or b) are depleted of red blood cells (RBCs).3) The method of claim 1 or 2, wherein the one or more HSPCs are CD34+.4) The method of any one of claims 1 to 3, wherein culturing the one or more HSPCs is in the absence of feeder or stromal cells.5) The method of any one of claims 1 to 4, wherein the culture medium comprises: a) one or any combination of IL-2, IL-15, and erythropoietin; and / or b) serum at a concentration ranging from between about 1% and 30%.6) The method of any one of claims 1 to 5, further comprising obtaining the output population of cells comprising the lymphoid lineage cell, and one or both of the myeloid lineage cell and the erythroid lineage cell after culturing the one or more HSPCs in the culture medium for the sufficient time.7) The method of any one of claims 1 to 6, wherein the sufficient time elapses 6 weeks or less.8) The method of claim 6 or 7, wherein the output population of cells comprising the lymphoid lineage cell, and one or both of the myeloid lineage cell and the erythroid lineage cell is obtained within 2 weeks, 3 weeks, or 4 weeks of culturing the one or more HSPCs in the culture medium.9) The method of any one of claims 6 to 8, wherein the: a) erythroid lineage cell comprises an erythroblast and / or a megakaryocyte; b) myeloid lineage cell comprises a myeloblast cell, a basophil, a neutrophil, an eosinophil, a monocyte, a dendritic cell, and / or a mast cell; andc) lymphoid lineage cell comprises a natural killer (NK) cell, a lymphoid progenitor, a T lymphocyte, and / or a B lymphocyte.10) The method of any one of claims 1 to 8, wherein the output population of cells comprises: a) one of or any combination of a natural killer (NK) cell, a lymphoid progenitor, a T lymphocyte, and a B lymphocyte; and b) one or both of an erythroblast and a megakaryocyte; and / or c) one of or any combination of a myeloblast cell, a mast cell, a promyelocyte, a myelocyte, a basophil, a neutrophil, an eosinophil, a monocyte, and a dendritic cell.11) The method of any one of claims 1 to 10, further comprising comparing the output population of cells when the provided one or more HSPCs are isolated from a first donor or individual and at least a second donor or individual to identify donor- or individual-dependent differences in multilineage differentiation potential.12) The method of any one of claims 1 to 11 , further comprising exposing the one or more HSPCs to a test condition comprising one or more compound prior to or while culturing the one or more HSPCs in the culture medium to assess an effect of the compound on the multilineage differentiation potential of the one or more HSPCs.13) The method of claim 12, wherein the one or more compound comprises a small molecule, a chemical compound, a protein, a peptide, a nucleic acid or a nanoparticle.14) The method of any one of claims 1 to 13, further comprising quantifying a probability of obtaining lymphoid lineage cells, and one or both of myeloid lineage cells and erythroid lineage cells among the output population of cells.15) The method of any one of claims 1 to 14, wherein the culturing is performed in liquid phase.

Images