Methods for differentiating stem cells in culture

Abstract

The present disclosure relates to in vitro cell culture methods and supplements for differentiating a population of cells taken from an earlier culture environment. The methods and supplements relate to differentiating a population of stem or progenitor cells, such as starting from pluripotent stem cells, into hematopoietic lineage cells. The methods and supplements also relate to differentiating a population of stem or progenitor cells, such as starting from pluripotent stem cells, into renal lineage cells or compositions of renal cells. The methods and supplements may include one or more small molecules, which may improve downstream differentiation of target cells.

Description

METHODS FOR DIFFERENTIATING STEM CELLS IN CULTURECROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of United States Provisional Patent Application No. 63 / 735,372, filed December 18, 2024, the entire contents of which is hereby incorporated by reference in its entirety.TECHNICAL FIELD

[0002] This disclosure relates to cell culture applications, and more specifically to cell culture applications using stem or progenitor cells, and still more specifically to cell culture applications related to mammalian PSCs.BACKGROUND

[0003] Cells arising from the mesodermal germ layer give rise to a diverse set of tissues, including mesenchyme, hematopoietic system, muscle, bone, kidney, reproductive tract and heart. Among hematopoietic derivatives, NK cells, B cells and T cells are lymphocytes that provide defense against pathogens and tumors. NK cells play a critical role in innate immunity, capable of secreting proinflammatory cytokines as well as killing cancerous or virus-infected cells. T cells are cells of the adaptive immune system that detect diverse targets via their antigen-specific T cell receptors (TCRs) and perform effector functions such as cytokine secretion and cytotoxic killing of target cells. B cells are a critical player in humoral immunity.

[0004] Kidneys are complex organs that are responsible for many functions, including the filtering waste products and minerals from blood, maintenance of fluid and acid-base balance, clearance and reabsorption, and producing the erythropoietin that is essential for production of red blood cells.

[0005] Primary immune cells such as NK, B, and T cells, and as well kidney cells derived from primary cells are often heterogeneous and can vary in quality from donor to donor. Furthermore, primary cell sources are limited in number.

[0006] Pluripotent stem cells (PSCs) such as embryonic stem cells and induced pluripotent stem cells, on the other hand, provide a limitless supply of differentiated cells. PSCs therefore represent a valuable platform for generating homogenous, customizable, large-scale populations of differentiated immune cells (e.g., NK, B, and T cells) and kidney cells. PSC- derived kidney organoids, renal epithelial cells, and lymphocyte lineages have significant utility in applications such as regenerative medicine, disease modeling, toxicity testing, and high- throughput drug screening. Moreover, PSC-based systems enable standardized, reproducible manufacturing of differentiated cells for therapeutic use. such as lymphocytes and kidney cellsfor clinical applications. Kidney cells and organoids derived from PSCs would enable organ regeneration, disease modelling, and drug screening in vitro.

[0007] PSCs and PSC-derived cells are routinely grown as adherent cell culture, but adherent culture does not tend to be suitable for the generation of large amounts of cells and / or for large scale differentiation to cells for clinical applications. In contrast, suspension culture systems such as stirred-tank bioreactors, orbital shaker platforms, and other dynamic culture vessels — offer the potential for significantly greater scalability, improved nutrient diffusion, and more efficient production of differentiated cells. Nevertheless, most existing differentiation protocols and reagents were developed for adherent systems, and therefore substantial process optimization is needed to adapt PSC culture and differentiation, particularly lymphocyte and kidney lineages, to scalable suspension-based methods.

[0008] Thus, there is a need for improved and efficient differentiation protocols for producing lymphocytes and other mesoderm-derived cell types from PSCs within suspension culture systems and / or non-adherent culture system. Such methods would address limitations associated with primary cell sourcing, enhance scalability and reproducibility, and enable production of clinically relevant cell populations suitable for therapeutic development.SUMMARY

[0009] In one aspect of this disclosure are provided methods of differentiating a population of cells, such as stem or progenitor cells. In a related aspect, such methods may comprise differentiating a population of stem cells, such as stem or progenitor cells, in a culture environment different from an earlier or a following culture environment.

[0010] In one aspect, methods of this disclosure may comprise seeding a population of stem or progenitor cells in a first culture environment. Cells may be seeded in the first culture environment as single cells or as (small) clumps / clusters of cells (e.g. of about 5, 10, 25, 50, 100, or 200 cells).

[0011] In one aspect, methods of this disclosure may comprise seeding a population of stem or progenitor cells in a first culture environment, and culturing the population of stem or progenitor cells for a sufficient time in the first culture environment. In one aspect, methods of this disclosure may comprise seeding a population of stem or progenitor cells, culturing the population of stem or progenitor cells for a sufficient time in a first culture environment, and further differentiating (or biasing the differentiation of) the population of stem or progenitor cells toward a downstream lineage of cells. A sufficient time of culturing a population of stem or progenitor cells in a first culture environment may range from about 1 day to about 17 days.

[0012] A first culture environment of this disclosure may comprise a first culture vessel and a first culture medium. A first culture medium of this disclosure may be supplemented with one or more stage-specific cytokine(s) and / or growth factor(s). More specifically, a first culture medium may be supplemented with, in addition to one or more cytokine(s) and / or growth factor(s), one or both of an inhibitor of cytoskeleton modification and an inhibitor of stress response.

[0013] An inhibitor of cytoskeleton modification may be a Rho-associated protein kinase inhibitor. In one embodiment, a Rho-associated protein kinase inhibitor is Y-27632 or a functional equivalent thereof. An inhibitor of stress response may be an integrated stress response inhibitor or a functional equivalent thereof. In one embodiment, an integrated stress response inhibitor is an inhibitor of protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) signaling.

[0014] Methods of this disclosure may comprise a population of stem or progenitor cells derived from or cultured in an earlier culture environment, and such earlier culture environment may be different from a first culture environment.

[0015] Methods of this disclosure may comprise generating a downstream lineage of cells. More specifically, methods of this disclosure may comprise generating a downstream lineage of cells, wherein the downstream lineage of cells is differentiated at a higher efficiency compared to when a population of stem or progenitor cells is cultured in a first culture environment that does not comprise one or both of an inhibitor of cytoskeleton modification and the inhibitor of stress response. In some embodiments, a population of stem or progenitor cells in a first culture environment comprising both of an inhibitor of cytoskeleton modification and an inhibitor of stress response of this disclosure exhibits a higher differentiation efficiency compared to a population of stem or progenitor cells cultured in a first culture environment that does not comprise both of the inhibitor of cytoskeleton modification and the inhibitor of stress response.

[0016] Methods of this disclosure may comprise culturing a population of stem or progenitor cells in a first culture environment that is a 3D (e.g., suspension) culture. In some embodiments, methods of this disclosure may comprise culturing a population of stem or progenitor cells under non-adherent and / or suspension culture.

[0017] Methods of this disclosure may comprise exposing a starting population of stem or progenitor cells to a stressor either when transitioning the cells to the first culture environment or in the first culture environment. In some embodiments, the stressor is transient. In one embodiment, the stressor is a physical stressor (e.g. a turbulent mixing force). In oneembodiment, the stressor is a chemical stressor (e.g. a solution for dissociating cells). In one embodiment, the stressor is a combination of a physical stressor and a chemical stressor.

[0018] In methods where a population of stem or progenitor cells is exposed to a stressor, such upon transition to or in a first culture environment, such cells may exhibit a higher differentiation efficiency when cultured in the first culture environment (comprising one or both of an inhibitor of cytoskeleton modification and an inhibitor of stress response) compared to when the population of stem or progenitor cells is exposed to the stressor and cultured in a first culture environment that does not comprise one or both of the inhibitor of cytoskeleton modification and the inhibitor of stress response. In some embodiments, a population of stem or progenitor cells exposed to a stressor in a first culture environment comprising one or both of an inhibitor of cytoskeleton modification and an inhibitor of stress response (or upon transition thereto) exhibits a higher differentiation efficiency compared to a population of stem or progenitor cells exposed to the stressor in a culture environment does not comprise one or both of the inhibitor of cytoskeleton modification and the inhibitor of stress response.

[0019] Methods of this disclosure may comprise reducing exposure of a population of stem or progenitor cells to a first culture medium as a sufficient time elapses, such as by exchanging partially or completely the first culture medium with a different culture medium. In embodiments, the added culture medium is supplemented with at least one or more (stagespecific) cytokine(s) and / or growth factor(s), but not one or both of an inhibitor of cytoskeleton modification and an inhibitor of stress response.

[0020] Methods of this disclosure may further comprise transitioning a downstream lineage of cells (e.g., hematopoietic- or renal-fated cells) from a first culture environment to a second culture environment. Methods of this disclosure may further comprise transitioning a downstream lineage of cells from a first culture environment to a second culture environment, and culturing such downstream population of cells for a sufficient time in the second culture environment.

[0021] A second culture environment may comprise a second culture vessel (that is the same or different from the first culture vessel). A second culture environment may comprise a second culture medium (that is the same or different from the first culture medium). A second culture medium may be supplemented with at least one or more cytokine(s) and / or growth factor(s). In some embodiments, a second culture medium is supplemented with at least one or more cytokine(s) and / or growth factor(s) that are the same as or different from the at least one or more cytokine(s) and / or growth factor(s) present in a first culture medium.

[0022] Methods of this disclosure may further comprise transitioning an arising population of cells (e.g., lymphoid progenitor cells) from a second culture environment to a third cultureenvironment. Methods of this disclosure may further comprise transitioning an arising population of cells (e.g., lymphoid progenitor cells) from a second culture environment to a third culture environment, and culturing such arising population of cells for a sufficient time in the third culture environment.

[0023] A third culture environment may comprise a first culture vessel, a second culture vessel or a third culture vessel. A third culture environment of this disclosure may comprise a third culture medium (that is the same or different from the first or the second culture medium). A third culture medium may be supplemented with at least one or more cytokine(s) and / or growth factor(s). In some embodiments, a third culture medium is supplemented with at least one or more cytokine(s) and / or growth factor(s) that are the same as or different from the at least one or more cytokine(s) and / or growth factor(s) present in a second (or a first) culture medium.

[0024] A second culture environment may comprise a substrate (or a coating substrate) that interacts with an arising population of cells of a first culture environment. A third culture environment may also comprise a substrate (or a coating substrate) that interacts with an arising population of cells of a second culture environment.

[0025] A sufficient time of culturing in a second culture environment and / or a third culture environment may range from about 7 days to about 28 days.

[0026] A downstream lineage of cells (which may be differentiated from a first, second or third culture environment) may comprise mesoderm lineage cells, such as hematopoietic lineage cells ora composition comprising renal lineage cells. In some embodiments, the hematopoietic lineage cells comprise one or more lymphocyte populations or progenitors thereof. In one embodiment, the one or more lymphocyte populations or progenitors comprises T cells or NK cells. In one embodiment, the composition comprising renal lineage cells comprises epithelial, endothelial, mesenchymal, and neural cells (as may be organized into one or more organoids).

[0027] A population of stem or progenitor cells of this disclosure may comprise a pluripotent stem cell line.BRIEF DESCRIPTION OF THE DRAWINGS

[0028] 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.

[0029] Figures 1A and 1 B show bar graphs comparing differentiation efficiency of human PSCs (hPSCs) to CD56+NK cells. H9 ESC and SCTi003-A (3A) iPSC were cultured for 12-days in suspension culture, whether seeded as clumps or as single cells (sc). Frequency and yield (A), and associated fold changes (B), of CD56+NK cells per input CD34+cell when hPSCs were cultured initially (~2 days) either in the presence of both an inhibitor of integrated stress response (ISRi) and inhibitor of cytoskeleton modification (CSMi) or only CSMi. Shown are results from n=2.

[0030] Figures 2A and 2B show bar graphs comparing differentiation efficiency of hPSCs to T cells. hPSCs were cultured for 12-days in suspension culture, whether seeded as clumps or as single cells (sc). Frequency and yield of CD4+CD8+double positive (DP) T cells per input H9-derived CD34+cell (A), and frequency and yield of CD8+single positive (SP) T cells per input 3A-derived CD34+cell (B), when cultured initially (~2 days) either in the presence of both an inhibitor of integrated stress response (ISRi) and an inhibitor of cytoskeleton modification (CSMi) or only CSMi (n=1).

[0031] Figures 3A-C show differentiation efficiency of hPSCs to compositions of renal-like tissue (e.g. organoids). hPSCs were seeded as single cells and cultured for 21 days, and the number of successful differentiation experiments to yield kidney organoids when hPSCs were cultured initially (~1 day) in the presence of both an inhibitor of cytoskeleton modification (CSMi) and an inhibitor of integrated stress response (ISRi) or only CSMi (A) (n=11 -12). An image of an exemplary successful differentiation of renal-like tissue (e.g. organoids) showing convoluted tubular structures (white arrows) that resemble the structure and segmentation of a developing nephron (B). Representative immunofluorescence images (alone or merged) showing expression of different renal markers and cell types (white arrows) after a successful differentiation (C). Stained renal epithelium cells include podocytes (podocalyxin, “PODXL”), proximal tubules (lotus tetragonolobus lectin, “LTL”), and distal tubules (E-cadherin, “ECAD”). Stained endothelial cells express platelet endothelial cell adhesion molecule (PECAM-1 , or “CD31”). Stained mesenchymal cells express vimentin (VIM). Stained neural cells express beta-tubulin 3 (TUJ1). Nuclear strain DAPI. Scale bar = 100 pm.DETAILED DESCRIPTION

[0032] This disclosure relates to media compositions and / or supplements to be added into a medium, and to methods for differentiating a population of cells, such as a population of stem or progenitor cells. More specifically, this disclosure relates to methods of differentiating a population of stem or progenitor cells in a 3D (e.g., suspension) culture to a downstream lineage of cells using (stage-specific) differentiation media.

[0033] Where used herein, the term "stem or progenitor cell" refers to a cell that is capable of self-renewal and / or differentiating into a more specialized cell. Stem or progenitor cells may be derived from pluripotent stem cells (PSC), such as induced pluripotent stem cells (iPSC),embryonic stem cells (ESC), naive stem cells, extended stem cells, or the like. Stem or progenitor cells may be, whether primary or PSC-derived, HSPCs, preferably mammalian HSPCs, and still more preferably human HSPCs. Stem or progenitor cells may comprise kidney stem or progenitor cells which may include cells from stages of formation of a kidney or a functional unit thereof, such as stem or progenitor cells of late primitive streak, intermediate mesoderm, metanephric mesoderm, pretubular aggregates, and renal vesicles. Stem or progenitor cells when cultured in a suitable culture environment (comprising a suitable culture media and / or supplements) may differentiate into a downstream lineage of cells (e.g., mesoderm or endoderm lineage cells). A stem or progenitor cell of this disclosure may be maintained in well-known conditions, such as in the case of hematopoietic lineage cells in a StemSpanTM-branded medium (STEMCELL T echnologies) ; or in the case of PSC in a T eSRTM- branded medium (STEMCELL Technologies).

[0034] Where used herein, the term "pluripotent stem cell" or "PSC" refers to a cell that is capable of self-renewal and / or differentiating to any cell type of any of the three embryonic germ layers. ESC may be isolated from a blastocyst, while iPSC may be derived from any cell type by the forced expression of certain pluripotency genes, such as Oct4, Nanog, Sox2, Klf4, etc.

[0035] Where used herein, the term “hematopoietic stem and progenitor cell" or "HSPC" refers to a cell of the hematopoietic lineage that is capable of self-renewal and / or differentiating into a more specialized cell of the hematopoietic lineage. HSPC may be obtained from bone marrow (BM), umbilical cord blood (CB), embryonic through to adult peripheral blood (PB), thymus, peripheral lymph nodes, gastrointestinal tract, tonsils, gravid uterus, liver, spleen or any other tissue having localized populations of HSPC. HSPC may also be differentiated from PSC. A hallmark of HSPC is the expression of the transmembrane phosphoglycoprotein CD34, thus HSPC may be referred to as CD34+cells. Human HSPCs may be further defined by expression of CD45 and CD34, and may be still further defined by combinations of markers such as CD38, CD43, CD45RO, CD45RA, CD 10, CD49f, CD59, CD90, CD109, CD1 17, CD133, CD166, HLA-DR, CD201 , and integrin-alpha3 which may be used to distinguish HSPC subsets.

[0036] Where used herein, the term "lymphoid progenitor" refers to a cell type that is more specialized than a HSPC but is capable of further differentiating into one or more lymphoid cell types, such as B cells, T cells, or NK cells. A lymphoid progenitor cell may be a direct descendant of a HSPC or may be further removed from a HSPC. Further, a lymphoid progenitor cell may differentiate into a downstream lymphoid cell type directly or may undergo one or more further steps of differentiation before becoming a lymphoid cell type. One example of a lymphoid progenitor cell is a cell that is positive for the phenotypic markers CD7 and CD5.In another example, a lymphoid progenitor cell may be positive for CD7 but negative for CD5, or vice versa. In another example, a lymphoid progenitor cell may be negative for both CD7 and CD5. Other phenotypic markers that may be expressed by lymphoid progenitor cells include CD10, CD45RA, CD34, CD38, CD161 , CD122, CD117, CD127, CD1a and / or integrin37. While a lymphoid progenitor may be capable of differentiating into any type of lymphoid cells, in some embodiments a lymphoid progenitor may be more restricted in its differentiation capacity, and may only differentiate into one or more, but not all, types of lymphoid cells. The term “lymphoid progenitor” may encompass a primary cell, or a PSC- derived cell that phenotypically / functionally resembles a primary lymphoid progenitor.

[0037] Where used herein, the term “compositions of renal-like tissue” or “kidney cell aggregates” refers to a cellular structure grown in vitro that contains multiple cell types typically present in a mammalian kidney or in a functional unit thereof (e.g. nephrons). A kidney organoid may refer to a composition comprising multiple cell types of a nephron arranged in a patterned geometry. Kidney cells or aggregates (organoids) are characterized by the presence of epithelial, mesenchymal, and endothelial cell types. Among the epithelial cell types, kidney cells or aggregates (organoids) may comprise podocytes (NPHS1 , PODXL), proximal tubules (LTL, CUBN, AQP1), loop of Henle (CDH1 , UMOD), and distal tubules (CDH1 , GATA3, AQP2, ECAD). Other cell types such as stromal / interstitial mesenchymal (VIM), neural cells (TUJ1), and endothelial cells (CD31) may also be present.

[0038] Where used herein, the term “culture environment” refers to culture conditions or culture parameters in which a cell is cultured (e.g., a differentiation culture environment). Depending on the type of cell to be cultured, such culture conditions or parameters may include, among other things, types of culture (non-adherent and adherent cultures, etc), culture media and / or supplements, culture vessels, and culture temperature.

[0039] Where used herein, the term “suspension culture” or “non-adherent culture”, used interchangeably herein, may refer to a cell culture system wherein cells (single cells or small aggregates / clusters of cells) either do not or only a small fraction thereof adhere / attach to a surface of a cell culture vessel or a container. A non-adherent culture may also refer to a 2.5D culture wherein cells (single cells or small aggregates / clusters of cells) are embedded in a polymerized extracellular matrix, extracellular matrix protein, or hydrogel. In suspension culture, cells or cell aggregates under influence of an energy input (e.g. shear, orbital rotation, etc.) may be suspended in a cell culture medium with minimal or no contact to a cell culture vessel surface. Cells in suspension culture may multiply and / or differentiate in an agitated cell culture medium. Suspension culture may be contrasted with adherent culture, in which cells are cultured (e.g. differentiated) attached to a substrate (e.g. a wall of a cell culture container or a particle / bead), which may be coated with one or more extracellular matrix (ECM) proteins.Whether used for coating a well or for embedding cells, a concentration of an ECM or an ECM protein may range between about 0.1 pg / mL to about 1 mg / ml, or between about 1 pg / ml to about 500 pg / ml, or between about 3 pg / ml to about 200 pg / ml, or between about 5 pg / ml to about 150 pg / ml or between about 10 pg / ml to about 100 pg / ml.

[0040] Suspension culture of this disclosure may comprise culturing cells in a culture vessel of choice. Non-limiting examples of a suitable culture vessel for culturing cells in a suspension culture may include a bioreactor, a spinner flask, a suspension culture plate or any tissueculture plates (e.g., 6-well, 24-well or 96-well plates) that promote growth of stem cells in suspension. Optionally, a suspension culture system may be a closed culture vessel

[0041] Where it is desired to culture stem or progenitor cells in suspension, the cells may be seeded as clumps of cells (e.g. from 2 to 1000 cells) or as single cells (in a suspension). Otherwise, it may be desirable to pre-aggregate stem or progenitor cells using micro-well plates or any other such method to form aggregates of cells that are substantially uniform in terms of average diameter. Still further, it may be desirable to culture stem or progenitor cells on or in particles / beads / microcarriers in suspension culture conditions.

[0042] Where used herein, the term “culture media” refers to liquids that contact cells and that support and / or differentiate at least one cell type comprised in a culture environment of this disclosure. Culture media of this disclosure may comprise an appropriately supplemented basal medium. Indeed, basal media are well known in the art and are routinely formulated to include one or more of salt(s), amino acid(s), carbohydrate(s), buffer(s), trace element(s), vitamin (s), insulin, serum / albumin / serum replacement, protein(s), lipid(s), etc.

[0043] Culture media and / or supplements to be added to culture media may comprise (at least one) cytokine(s) and / or growth factor(s). Examples of cytokines and / or growth factors include, but are not limited to, IL-2, IL-3, IL-6, IL-7, IL-11 IL-15, SCF, FLT3L, TPO, TGF-beta, FGF-2, agonist of BMP signaling (e.g., BMP4), agonist of vascular endothelial growth factor (VEGF) signaling (e.g., VEGFA), and IGF-1 or IGF-2. A concentration of one or more cytokines and / or growth factors in any culture medium of this disclosure may range from about 0.05 ng / mL to about 5 pg / ml, or about 0.1 ng / mL to about 1 g / mL, or about 1 ng / ml to about 500 ng / ml, or about 10 ng / ml to about 250 ng / ml, or about 15 ng / ml to about 100 ng / ml, or about 20 ng / ml to about 50 ng / ml.

[0044] Culture media of this disclosure may be additionally supplemented with one or more small molecules. In some embodiments, the one or more small molecules comprise one or more small molecule inhibitors. Culture media (and / or supplements) of this disclosure may comprise an agonist of Wnt signaling (e.g., CHIR99021).

[0045] A small molecule that may be included in a culture environment (e.g. medium) of this disclosure may be an agent that inhibits modification / disruption of cytoskeletal structure. An exemplary such agent may be an inhibitor of a Rho-associated protein kinase, such as a Rho / Rock kinase inhibitor. Examples of Rho / Rock kinase inhibitors include, but are not limited to, Y-27632, Thiazovivin, Fasudil (HA-1077), GSK429286A, RKI-1447, H-1152 dihydrochloride, Azaindole 1 (TC-S 7001). In one embodiment, the Rho / Rock kinase inhibitor is Y-27632. A concentration of such an agent that inhibits modification / disruption of cytoskeletal structure may range from about 1 nM to about 1 mM, or about 20 nM to about 500 pM, or about 50 nM to about 250 pM, or between about 100 nM to about 100 pM. In one embodiment, a concentration of an agent that inhibits modification / disruption of cytoskeletal structure as used in methods of this disclosure is about 50 ± 10 pM, 25 ± 5 pM, or 10 ± 2 pM.

[0046] Another small molecule that may be included in a culture environment (e.g. medium) of this disclosure may be an agent that modulates a stress response of a cell. An exemplary such agent may be an inhibitor of a stress response, such as an inhibitor of protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) signaling. In one embodiment, an agent that modulates a stress response of a cell is an integrated stress response inhibitor, such as a symmetric bisglycolamide ISRIB (Integrated Stress Response inhibitor), whether as the cis or trans diastereomers, or a mixture of both. In a specific embodiment, an agent that modulates a stress response of a cell is trans-ISRIB (N,N'-trans-1 ,4-cyclohexanediylbis[2-(4- chlorophenoxy)acetamide]). A concentration of an agent that modulates a stress response of a cell as may be used in this disclosure may range from about 0.001 pM to about 10 pM, or about 0.005 pM to about 5 pM, or about 0.01 pM to about 1 pM. In one embodiment, a concentration of an agent that modulates a stress response of a cell as used in methods of this disclosure is about 10 pM ± 1 pM, 5 pM ± 1 pM, 0.7 pM ± 0.2 pM.Methods

[0047] In one aspect of this disclosure are provided methods of differentiating cells to a downstream lineage of cells. In other words, methods are provided for differentiating a starting or input population of cells (e.g., a population of stem or progenitor cells) into a downstream lineage of cells.

[0048] In another aspect of this disclosure are provided methods of differentiating a population of stem or progenitor cells into a downstream lineage of cells, comprising culturing a population of stem or progenitor cells for a sufficient period of time in a first culture environment, the first culture environment comprising a first culture vessel and / or a first culture medium.

[0049] In another aspect of this disclosure are provided methods of differentiating a population of stem or progenitor cells to a downstream lineage of cells, comprising culturing a population of stem or progenitor cells for a sufficient period of time in a first culture environment, the first culture environment comprising at least a first culture medium, wherein the first culture medium comprises one or more small molecules.

[0050] In another aspect of this disclosure are provided methods of differentiating a population of stem or progenitor cells to a downstream lineage of cells, comprising culturing a population of stem or progenitor cells for a sufficient period of time in a first culture environment, the first culture environment comprising at least a first culture medium, wherein the first culture medium comprises one or both of an inhibitor of cytoskeleton modification and an inhibitor of stress response.

[0051] In another aspect of this disclosure are provided methods of generating a downstream lineage of cells wherein the downstream lineage of cells is differentiated at a higher efficiency.

[0052] In another aspect ofthis disclosure are provided methods of generating a downstream lineage of cells wherein the downstream lineage of cells is differentiated at a higher efficiency in the presence of one or both of an inhibitor of cytoskeleton modification and an inhibitor of stress response.

[0053] In another aspect of this disclosure are provided methods of generating a downstream lineage of cells wherein the downstream lineage of cells is differentiated at a higher efficiency compared to a population of stem or progenitor cells cultures in a culture environment (e.g., a first culture environment) that does not comprise one or both of an inhibitor of cytoskeleton modification and an inhibitor of stress response.

[0054] In another aspect of this disclosure are provided methods of improving differentiation efficiency of a population of stem or progenitor cells into a downstream lineage of cells, comprising culturing a population of stem or progenitor cells for a sufficient period of time in a first culture environment.

[0055] In another aspect of this disclosure are provided methods of improving differentiation efficiency of a population of stem or progenitor cells to a downstream lineage of cells, comprising culturing a population of stem or progenitor cells for a sufficient period of time in a first culture environment comprising one or more small molecules.

[0056] In another aspect of this disclosure are provided methods of improving differentiation efficiency of a population of stem or progenitor cells to a downstream lineage of cells, comprising culturing a population of stem or progenitor cells for a sufficient period of time in afirst culture environment comprising one or both of an inhibitor of cytoskeleton modification and an inhibitor of stress response.

[0057] In another aspect of this disclosure are provided methods of exposing a population of stem or progenitor cells to a stressor.

[0058] In another aspect of this disclosure are provided methods of exposing a population of stem or progenitor cells to a stressor either when transitioning the cells to a first culture environment or a first culture environment.

[0059] In another aspect of this disclosure are provided methods wherein a population of stem or progenitor cells exposed to a stressor in a culture environment (e.g., a first culture environment) exhibit a higher differentiation efficiency.

[0060] In another aspect of this disclosure are provided methods wherein a population of stem or progenitor cells exposed to a stressor exhibit a higher differentiation efficiency when cultured in a first culture environment compared to the population of stem or progenitor cells exposed to the stressor and cultured in a culture environment (e.g., a first culture environment) that does not comprise one or both of the inhibitor of cytoskeleton modification and the inhibitor of stress response.

[0061] In another aspect of this disclosure are provided methods of improving differentiation efficiency (to a downstream lineage of cells) of a population of stem or progenitor cells exposed to a stressor, comprising culturing a population of stem or progenitor cells (having been or as exposed to a stressor) for a sufficient period of time in a first culture environment, the first culture environment comprising at least a first culture medium, wherein the first culture medium comprises one or both of an inhibitor of cytoskeleton modification and an inhibitor of stress response.

[0062] In any aspect, methods of this disclosure may comprise providing / seeding a population of stem or progenitor cells (e.g., PSCs) in a first (differentiation) culture environment, the first (differentiation) culture environment comprising a first culture vessel and a first culture medium.

[0063] Cells are not particularly limited, but are preferably mammalian. A cell of this disclosure may be a stem or a progenitor cell, or a population thereof. An exemplary stem cell or progenitor cell of this disclosure may be a pluripotent stem cell (PSC). If the PSC is a cell line, it may be an ESC line (e.g., H9) or an iPSC line (e.g., SCTi003-A, “3A”; SCTi004-A, “4A”). Another exemplary stem cell or progenitor cell of this disclosure may be a cell type downstream of PSC, but nevertheless still retaining the ability to self-renew and differentiateto at least one (but not all) downstream cell lineage, such as stem progenitor cells fated to a particular germ layer (e.g. HSPC, renal stem cells, epithelial stem cells).

[0064] A cell, or a population thereof, to be cultured in a first culture environment may have been derived / cultured in or transitioned from an earlier culture environment. In the context of a differentiation protocol, a first culture environment and an earlier culture environment may be different.

[0065] A culture environment of this disclosure (e.g. an earlier culture environment, a first culture environment, or any subsequent environment herein) is the set of conditions under which a cell or cells are seeded, and in which such cell or cells are subsequently cultured or incubated or differentiated. Methods of this disclosure may comprise at least one culture environment, or may comprise more than one culture environment. Where a successive culture environment is different from a preceding environment, they may differ in terms of one or more of: types of cells introduced / cultured; containers in which cells are introduced / cultured; environmental conditions under which cells are introduced / cultured; duration of culture; how cells are cultured; or culture media and / or culture supplements used, or any combination thereof.

[0066] Any culture environment of this disclosure is not limited in terms of containers in which cells are cultured, provided that they accommodate a sufficient volume of culture medium, taking into consideration seeding density and growth rate. A container may accommodate changing volumes of cell culture medium, beginning from a relatively small volume at the time of seeding which increases as the culture progresses and more medium is added.

[0067] Any culture environment of this disclosure is not limited in terms of environmental conditions under which cells are cultured (e.g. temperature, darkness, humidity, and pressure, etc.), provided that they are permissive of the intended culture or incubation or differentiation. Any culture environment may comprise exposing cells to an incubation environment that supports differentiation and / or organoid formation, and may further comprise incubation parameters that are standard in the art. Standard incubation parameters, such as in terms of oxygen levels, carbon dioxide levels, atmospheric pressure, and temperature, are well known.

[0068] Any culture environment of this disclosure is not limited in terms of cell culture medium, provided it is appropriate for the culturing task at hand. In a specific embodiment, a cell culture medium used in a culture environment is a differentiation medium. Thus, a cell differentiation medium may be formulated as appropriate to support and / or direct differentiation of cells (e.g. a population of stem or progenitor cells, or intermediate downstream progeny thereof) toward a target downstream cell lineage, such as by including protein, chemical, and / or small molecule factors therein.

[0069] Any culture environment of this disclosure is not limited in terms of duration of culture, provided that the duration is not too long that a culture of cells begins to senesce or die and that the duration is not long enough to yield target (differentiated) cells.

[0070] Any culture environment of this disclosure is not limited in terms of how the cells are cultured, more particularly whether they are cultured adhered to a substrate or under nonadherent conditions (e.g. in suspension culture).

[0071] If cultured under non-adherent conditions (e.g. in suspension), cells may be freely suspended in a cell culture medium or may be adhered to a suspension substrate, such as a microparticle, bead, or the like. Regardless, culture under non-adherent conditions (e.g. in suspension) may comprise exposing cells in a container to a stressor / force, such as a physical stressor. Turbulent mixing (e.g. agitation) of a cell culture to distribute cells throughout a cell culture medium may be performed in any of numerous known ways, such as by placing a container on an orbital shaker, using roller bottles and associated apparatus, rotating an impeller or a magnetic stirrer, etc.

[0072] In addition or in the alternative, cells may be cultured in an environment where a culture substrate is either coated or uncoated. A substrate of this disclosure may comprise a physical surface such as a bead or culture vessel wall, a chemical surface such as a polymer or synthetic hydrogel, or a biological surface, such as a macromolecule or another cell. If uncoated, a substrate may be an ultra-low attachment plate, or the like. Or, an uncoated container may comprise a feeder cell- or stromal cell-free system. If coated, a coating may render one or more surfaces of a container less amenable to adhesion by the cells, such as with a commercially available surfactant. Other examples of coatings may comprise an extracellular matrix or an extracellular matrix protein. In some embodiments, a substrate may be coated with an agent that stimulates differentiation and / or maturation.

[0073] In the specific context of a first culture environment, a population of stem or progenitor cells (e.g. PSC) may be provided / seeded in a first culture vessel and a first culture medium either as single cells or as clumps of cells (e.g. 1-200, or 1-500, or 1-1000 cells), whether as an adhered (e.g. monolayer) or a non-adhered (e.g. suspension) culture.

[0074] A first culture medium of a first culture environment, may comprise one or more cytokines and / or growth factors, as specified herein. A first culture medium of a first culture environment may comprise one or more small molecules, as specified above. A first culture medium of a first culture environment may comprise one or more cytokines and / or growth factors and one or more small molecules, each as specified herein. In an embodiment, a first culture medium of a first culture environment, comprises one or more of TPO, SCF, FLT3L, FGF2, BMP4, VEGFA, and TGF-beta. In an embodiment, a first culture medium of a firstculture environment, comprises one or both of an inhibitor of cytoskeleton modification and an inhibitor of stress response. In an embodiment, a first culture medium of a first culture environment, comprises i) one or more of TPO, SCF, FLT3L, FGF2, BMP4, VEGFA, and TGF- beta, and ii) one or both of an inhibitor of cytoskeleton modification and an inhibitor of stress response.

[0075] As indicated above, provided / seeded cells (in a first culture environment) may have been transitioned from an earlier culture environment, and the earlier culture environment may be different from the first culture environment, such as in terms of culture container, culture (environmental conditions), cell culture medium, and type of culture.

[0076] An earlier culture environment may comprise stem or progenitor cells in maintenance culture conditions, and transition to a first culture environment may comprise exposure to differentiation conditions. In an earlier culture environment, stem or progenitor cells (e.g. PSC) may be aggregated using any known approach into compositions comprising 10 or more, 50 or more, 100 or more, 150 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, or 1000 or more cells. For example, aggregates of cells may be formed by depositing a desired number of cells into the bottom of a tube or a well of a cell culture plate. Or, aggregates may be formed by depositing a desired number of cells into a well of an Aggrewell™ microwell device, to ensure the efficient and reproducible formation of uniformly sized aggregates. In one embodiment, the number of cells used to form an aggregate ranges from about 1 and 150,000, or about 5 and 100,000, or about 10 and 10,000, or about 100 and 1 ,000.

[0077] In one embodiment, stem or progenitor cells (e.g. PSCs) formed as aggregates are dissociated into a single cell suspension or into a plurality of relatively smaller clumps such as by trituration and / or treatment with a dissociation reagent (e.g. a trypsin solution-based or Gentle Cell Dissociation Reagent (STEMCELL Technologies), transitioned into a first culture environment.

[0078] Cells when they are transitioned into non-maintenance culture (e.g. differentiation) they may experience significant stress. In addition, when stem or progenitor cells are passaged, they may also experience significant stress, such as becoming dissociated to single cells. In many cases, cells that are seeded as single cells or at clonal densities may undergo mass cell death shortly after plating. Accordingly, a differentiation efficiency may be very low or fail altogether among single cells in a suspension when exposed to differentiation culture conditions. Thus, culture environments (e.g. culture medium) of this disclosure may comprise one or more small molecules that reduce stress and / or promote cell survival thereby benefiting downstream differentiation.

[0079] Transitioning cells from an earlier culture environment to a first culture environment may of itself comprise a stressor, such as through dissociation and re-seeding, whether as clumps or single cells and whether at normal or clonal cell densities (e.g. <1000 cells / cm2, or <1000 cells / well).

[0080] Following providing / seeding a population of stem or progenitor cells in a first culture environment, methods of this disclosure may comprise culturing the population of cells for a sufficient time in the first culture environment.

[0081] A sufficient period of (culture) time in a first culture environment may be at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, or at least 17 days.

[0082] A sufficient period of (culture) time in a first culture environment (of suspension culture) may range from about 1 to about 17 days, about 3 to about 15 days, about 5 to about 13 days, or about 7 to about 10 days.

[0083] As a population of cells is cultured in a first culture environment (in a first culture medium, as described above), methods of this disclosure may comprise reducing exposure to a first culture medium as a sufficient period of (culture) time elapses. By way of non-limiting example, a first culture medium may comprise i) one or more cytokines or growth factors, ii) one or more small molecules, or iii) any combination of the foregoing, and such first culture medium may be periodically exchanged as a sufficient period of time elapses, whether partially or completely, with a differently formulated culture medium. In one embodiment, a first culture medium comprises one or more cytokines or growth factors and one or more small molecules, and partial or complete medium changes during the sufficient period of time are carried out with a culture medium comprising one or more cytokines or growth factors but not one or more small molecules. In one embodiment, a first culture medium comprises one or more cytokines or growth factors and more than one small molecule, and partial or complete medium changes during the sufficient period of time are carried out with a culture medium comprising one or more cytokines or growth factors but only one or none of the small molecules. In one embodiment, a first culture medium comprises one or more cytokines and / or growth factors, and one or both of an inhibitor of cytoskeleton modification and an inhibitor of stress response, but a replenishment medium comprises one or more cytokines and / or growth factors, but not one or both of an inhibitor of cytoskeleton modification and an inhibitor of stress response.

[0084] A sufficient period of (culture / differentiation) of cells in a first culture environment of reducing exposure to one or more small molecules of this disclosure may be measured in days, such as about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, or longer. However, a culture of cells in a first culture environment may be exposed to a maximal concentration of one or more small molecules in a first culture environment for a shorter period of time, such as about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days, or ranging from about 0-4 days, or from about 1 -3 days.

[0085] Following culturing a population of stem or progenitor cells for a sufficient time in a first culture environment, methods of this disclosure may comprise differentiating (or biasing the differentiation of) a population of stem or progenitor cells toward a downstream lineage of cell.

[0086] In the specific context of culturing a population of stem or progenitor cells with the desire of differentiating to a mesoderm lineage, culturing in a first culture environment may bias or differentiate a population of stem or progenitor cells toward a hematopoietic lineage (fate), or any other mesodermal lineage, provided that a first culture environment is appropriately selected. Thus, a population of stem or progenitor cells seeded in a first culture environment may differentiate directly to or toward, or through one or more intermediates to or toward, a mesodermal (e.g. hematopoietic) lineage upon a sufficient period of culture time. If differentiation passes through more than one intermediate, then arising cells of a first culture environment may be transitioned from a first culture environment to a second culture environment.

[0087] In the specific context of culturing a population of stem or progenitor cells with the desire of differentiating to a different mesoderm lineage, culturing in a first culture environment may bias or differentiate a population of stem or progenitor cells toward a mesodermal (e.g. renal) lineage (fate), or any other endodermal lineage, provided that a first culture environment is appropriately selected. Thus, a population of stem or progenitor cells seeded in a first culture environment may differentiate directly to or toward, or through one or more intermediates to or toward, a mesodermal (e.g. renal) lineage upon a sufficient period of culture time. If differentiation passes through more than one intermediate, then arising cells of a first culture environment may be transitioned from a first culture environment to a second culture environment.

[0088] Methods of this disclosure may further comprise transitioning an arising population of cells from a first culture environment to a culture environment (e.g., a second culture environment) that is different from the first culture environment.

[0089] Following transition from a first culture environment to a downstream (second) culture environment, methods of this disclosure may comprise culturing an arising population (e.g. downstream lineage) of cells from a first culture environment for a sufficient time in a second culture environment to further differentiate the arising cells. A second culture environment maybe different from a first culture environment in terms of one or more of culture vessel, culture (environmental) conditions, culture medium, culture duration, how the cells are cultured, etc.

[0090] Where a second culture environment comprises a second culture vessel and a second culture medium, the second culture vessel may be the same type or different from a first culture vessel, and / or a second culture medium may be the same or different from a first culture medium

[0091] A second culture vessel may comprise a substrate coating (e.g., a culture vessel coating) that interacts with an arising population of cells (of a first culture environment) in a second culture environment. Where a second culture environment comprises a substrate coating, the substrate coating may be an extracellular matrix or extracellular matrix protein. Non-exhaustive examples of coatings may include fibronectin, gelatin, collagen, an immobilized Notch ligand, or commercially available coatings such as StemSpan™ Lymphoid Differentiation Coating Supplement (STEMCELL Technologies) or Matrigel™ (Corning). In one embodiment, a second culture vessel does not comprise a coating that interacts with an arising population of cells (of a first culture environment) in a second culture environment.

[0092] Regardless, the nature of the cells transitioned from a first culture environment into a second culture environment may be different compared to the cells originally seeded and cultured in the first culture environment.

[0093] In the specific context of differentiating a population of stem or progenitor cells to a mesodermal lineage (e.g. hematopoietic lineage), cells arising from a first culture environment (e.g. CD34+HSPC, or CD34+-like cells) may be transitioned into a second culture environment to further differentiate the arising population of cells (e.g. into lymphoid progenitors). Thus, a second culture environment may comprise culturing an arising population in a basal medium (e.g., StemSpan™ SFEM II, STEMCELL Technologies) supplemented with one or more cytokines and / or growth factors such as SCF, TPO, FLT3L, and IL-7, and differentiating in a culture vessel coated with a coating material (e.g. StemSpan™ Lymphoid Differentiation Coating Material, STEMCELL Technologies).

[0094] In the specific context of differentiating a population of stem or progenitor cells to another mesoderm al lineage (e.g. renal cells), cells arising from a first culture environment (e.g. cavitated PSC spheroids or spheroids-like cells) may be transitioned into a second culture environment to further differentiate the arising population of cells (e.g. into multilineage tissue compositions). An exemplary multilineage tissue composition may be a kidney organoid, and in such context differentiation in a second culture environment, may undergo a stepwise differentiation of a population of cavitated spheroids to or through one or more of late primitive streak, intermediate mesoderm, metanephric mesoderm, pretubular aggregates, andrenal vesicles, which may ultimately form kidney organoids. Thus, a second culture environment may comprise culturing an arising population (e.g., cavitated hPSC spheroids) in a basal medium (e.g., STEMDidiff™ Kidney Basal Medium, STEMCELL Technologies) comprising a Wnt agonist (e.g., CHIR99021) (e.g., STEMdiff™ Kidney Supplement SG and Supplement DM (STEMCELL Technologies)).

[0095] Indeed, differentiation may be arrested at any desired stage in the event of multistage differentiation.

[0096] If culturing in a second culture environment is necessary to differentiate a target downstream lineage of cells, an arising population of cells from the second culture environment may be ready for downstream use. If not ready for downstream use (after culture in a second culture environment), an arising population of cells from the second culture environment may be transitioned into a third culture environment. Thus, methods of this disclosure may further comprise transitioning an arising population of cells from a second culture environment to a culture environment (e.g., a third culture environment) that is different from the second culture environment.

[0097] Following transition from a second culture environment to a downstream (third) culture environment, methods of this disclosure may comprise culturing an arising population (e.g. downstream lineage) of cells from a second culture environment for a sufficient time in a third culture environment to further differentiate the arising cells. A third culture environment may be different from a second culture environment in terms of one or more of culture vessel, culture (environmental) conditions, culture medium, culture duration, how the cells are cultured, etc.

[0098] Where a third culture environment comprises a third culture vessel and / or a third culture medium, the third culture vessel may be the same type (but pre-treated differently, such as by presence or absence of a coating) or different from a second culture vessel, and / or a third culture medium may be the same or different from a second (or a first) culture medium.

[0099] A third culture vessel may comprise a substrate coating (e.g., a culture vessel coating that interacts with an arising population of cells (of a second culture environment) in a third culture environment. Where a third culture environment comprises a substrate coating, the substrate coating may be an extracellular matrix or an extracellular matrix protein. Non- exhaustive examples of such culture vessel coatings may include fibronectin coatings, gelatin coatings, collagen coatings, an immobilized Notch ligand, or commercially available coatings such as StemSpan™ Lymphoid Differentiation Coating Supplement (STEMCELL Technologies) or Matrigel™ (Corning). In one embodiment, unlike a second culture vessel, athird culture vessel does not comprise a coating that interacts with an arising population of cells (of a second culture environment) in a third culture environment.

[0100] Regardless, the nature of the cells transitioned from a second culture environment into a third culture environment may be different compared to the cells originally seeded and cultured in the second culture environment.

[0101] In the specific context of differentiating a population of stem or progenitor cells to a mesodermal lineage (e.g. hematopoietic lineage), cells arising from a second culture environment (e.g. lymphoid progenitors) may be transitioned into a third culture environment to further differentiate the arising population of cells (e.g. into lymphocytes or lymphocyte-like cells, such as T or NK cells).

[0102] In one embodiment, an arising population of cells from a third culture environment comprises T cells. If an output population comprises T cells, such cells may be CD4+CD8+double positive T cells, CD8+single positive T cells, or CD4+single positive T cells. Thus, a third culture environment may comprise culturing an arising population (from a second culture environment) in a basal medium (e.g., SFEM II (STEMCELL Technologies)) supplemented with one or more cytokines and / or growth factors such as FLT3L, IGF-1 , and IL-7 and differentiating in a culture vessel coated with a coating material (e.g. StemSpan™ Lymphoid Differentiation Coating Material (STEMCELL Technologies)).

[0103] In one embodiment, an arising population of cells from a third culture environment comprises NK cells. Thus, a third culture environment may comprise culturing an arising population (from a second culture environment) in a basal medium (e.g., SFEM II (STEMCELL Technologies) supplemented with one or more cytokines and / or growth factors such as FLT3L, IGF-1 , and IL-7 and differentiating in a culture vessel coated with a coating material (e.g. StemSpan™ Lymphoid Differentiation Coating Material, STEMCELL Technologies).

[0104] In a second culture environment and / or a third culture environment, a sufficient period of (culture) time may be at least 3 days, at least 5 days, at least 7 days, at least 10 days, at least 12 days, at least 14 days, at least 16 days, at least 18 days, or longer. More specifically, a sufficient period of (culture) time may range from about 3 days to about 21 days, about 5 to about 17 days, or about 7 to about 14 days. In one embodiment, a sufficient period of (culture) time ranges from about 7 to about 18 days, or about 12 to about 16 days.

[0105] Transitioning between successive culture environments of this disclosure (e.g. from an earlier culture environment to a first culture environment, or from a first / second culture environment to a second / third culture environment) may comprise a physical transition of cells arising from a first / second culture vessel and / or medium to a second / third culture vessel and / or medium. Or, transitioning from a first / second culture environment to a second / thirdculture environment may comprise a transition from a second culture medium to a third culture medium, but continuing culture in a second culture vessel.

[0106] Methods of this disclosure may result in improved differentiation efficiency of a population of stem or progenitor cells when cultured in a first culture environment (comprising a first culture medium comprising one or both of i) one or more cytokines and / or growth factors, and ii) one or both of an inhibitor of cytoskeleton modification and an inhibitor of stress response) compared to a population of stem or progenitor cells cultured in a culture environment different from the first culture environment (e.g. a culture environment that does not comprise one or both of an inhibitor of cytoskeleton modification and an inhibitor of stress response). Differentiation efficiency may be measured in terms of frequency of a target cell type, and / or differentiation efficiency may be measured in terms of yield of a target cell type per input cell. Differentiation efficiency may be measured in terms of a successful differentiation (meeting certain design criteria) compared to a failed differentiation (as determined by objective criteria). Objective criteria for a successful differentiation may be based on: expression of one or more markers; presence / absence of one or more cell types; morphology of output cells or cell compositions, or any combination thereof.

[0107] Accordingly, methods of this disclosure may comprise obtaining a differentiated population of cells with a higher efficiency when culturing cells in a first culture environment in comparison to a culture environment different from the first culture environment. In addition or in the alternative, methods of this disclosure may comprise improving differentiation efficiency of a population of stem or progenitor cells when a first culture environment comprises one or both of an inhibitor of cytoskeleton modification and an inhibitor of stress response. A differentiation efficiency of stem or progenitor cells (e.g. PSCs) to a target downstream cell type may be improved when a culture environment (e.g. a first culture environment) comprises both of an inhibitor of cytoskeleton modification and an inhibitor of stress response compared to a culture environment not comprising both of an inhibitor of cytoskeleton modification and an inhibitor of stress response, such as only one or none. In one embodiment, a population of stem or progenitor cells in a first culture environment comprising one or both of an inhibitor of cytoskeleton modification and an inhibitor of stress response exhibit a higher differentiation efficiency compared to a population of stem or progenitor cells in a culture environment that does not comprise one or both of an inhibitor of cytoskeleton modification and an inhibitor of stress response.

[0108] A target cell arising or generated (directly or indirectly) from a differentiation with improved efficiency may correspond to the mesoderm lineage, or more specifically to a mesoderm lineage cell type. In one embodiment, a target cell arising or generated (directly orindirectly) from a differentiation with improved efficiency corresponds to a hematopoietic lineage cell.

[0109] A differentiation efficiency of stem or progenitor cells (e.g., PSCs) when cultured in a first culture environment of this disclosure, may be reflected in terms of a frequency of arising target cells, such as NK cells (e.g. CD56+NK cells,) or T cells (e.g. CD4+CD8+T cells, CD8+T cells, CD4+T cells, etc). A frequency of arising target cells (when stem or progenitor cells are cultured in a first culture environment of this disclosure) may improve by about 1% or more, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. More specifically, a frequency of arising target cells may be improved by about 1% or more, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more when stem or progenitor cells are cultured in a first culture environment comprising at least (or one or both of) i) one or both of an inhibitor of cytoskeleton modification and ii) an inhibitor of stress response, compared to when cultured in a culture environment that does not comprise at least (or one or both of) an inhibitor of cytoskeleton modification and an inhibitor of stress response.

[0110] A differentiation efficiency of stem or progenitor cells (e.g., PSCs) when cultured in a first culture environment of this disclosure, may be reflected in terms of a fold increase of arising target cells, such as NK cells (e.g. CD56+NK cells,) or T cells (e.g. CD4+CD8+T cells, CD8+T cells, CD4+T cells, etc). A fold increase of arising target cells (when stem or progenitor cells are cultured in a first culture environment of this disclosure) may comprise about 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, 10-fold or more, 15-fold or more, 20-fold or more, 25-fold or more, 30- fold or more, 40-fold or more, or 50-fold or more. More specifically, a fold increase of arising target cells may comprise about 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, 10-fold or more, 15-fold or more, 20-fold or more, 25-fold or more, 30-fold or more, 40-fold or more, or 50-fold or more arising target cells when stem or progenitor cells are cultured in a first culture environment comprising at least (or one or both of) i) one or both of an inhibitor of cytoskeleton modification and ii) an inhibitor of stress response, compared to when cultured in a culture environment that does not comprise at least (or one or both of) an inhibitor of cytoskeleton modification and an inhibitor of stress response.

[0111] A differentiation efficiency of stem or progenitor cells (e.g., PSCs) when cultured in a first culture environment of this disclosure, may be reflected in terms of a yield of arising target cells per input cell, such as NK cells (e.g. CD56+NK cells,) or T cells (e.g. CD4+CD8+T cells, CD8+T cells, CD4+T cells, etc). An improved yield (per input cell) of arising target cells (whenstem or progenitor cells are cultured in a first culture environment of this disclosure) may comprise about 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 125 or more, 150 or more, 175 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, or 1000 or more cells per input cell (e.g. per input CD34+cell). More specifically, an improved yield of arising target cells may comprise about 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 125 or more, 150 or more, 175 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 or more, or 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, or 1000 or more arising target cells (e.g. per input CD34+cell) when stem or progenitor cells are cultured in a first culture environment comprising at least (or one or both of) i) one or both of an inhibitor of cytoskeleton modification and ii) an inhibitor of stress response, compared to when cultured in a culture environment that does not comprise at least (or one or both of) an inhibitor of cytoskeleton modification and an inhibitor of stress response.

[0112] A target cell arising or generated (directly or indirectly) from a differentiation with improved efficiency may correspond to the mesoderm lineage, or more specifically to a mesoderm lineage cell type such as renal lineage cells. In one embodiment, a target cell arising or generated (directly or indirectly) from a differentiation with improved efficiency corresponds to a renal lineage cell (e.g. a renal cell or a composition of renal cells). In one embodiment, the renal lineage cells may comprise epithelial, endothelial, mesenchymal, and neural cells. In one embodiment, the renal lineage cells may comprise one or more of epithelial, endothelial, mesenchymal, and neural cells.

[0113] A differentiation efficiency of stem or progenitor cells (e.g. PSCs) when cultured in a first culture environment of this disclosure, may be reflected in terms of increased differentiation success rate of arising target cells, such as renal cells or compositions of renal cells (e.g. organoids). A differentiation success rate of arising target cells (when stem or progenitor cells are cultured in a first culture environment of this disclosure) may improve by about 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. More specifically, a differentiation success rate of arising target cells may be improved by about 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more when stem or progenitor cells are cultured in a first culture environment comprising at least(or one or both of) i) one or both of an inhibitor of cytoskeleton modification and ii) an inhibitor of stress response, compared to when cultured in a culture environment that does not comprise at least (or one or both of) an inhibitor of cytoskeleton modification and an inhibitor of stress response.

[0114] A target cell arising or generated (directly or indirectly) from a differentiation with improved efficiency may correspond to the endoderm, or more specifically to a endoderm lineage cell type.

[0115] In any of the foregoing methods, a population of stem or progenitor cells may be exposed to a stressor in a first culture environment or on transition to a first culture environment (such as from an earlier culture environment). Thus, in certain aspects, methods of this disclosure may comprise improving differentiation efficiency of a population of stem or progenitor cells exposed to a stressor in a first culture environment or on transition to a first culture environment (such as from an earlier culture environment), wherein the first culture environment comprises (one or both of) an inhibitor of cytoskeleton modification and an inhibitor of stress response, and increased differentiation efficiency is obtained after culturing in the first culture environment compared to when a population of stem or progenitor cells exposed to the stressor is not cultured in the presence of (one or both of) an inhibitor of cytoskeleton modification and an inhibitor of stress response.

[0116] The following non-limiting examples are illustrative of the present disclosure.ExamplesExample 1: PSC maintenance

[0117] H9 ES cells, and SCTi003-A (3A) and SCTi004-A (4A) iPSC cells were maintained on Matrigel™ coated plates in either mTeSR™1 (STEMCELL Technologies), eTeSR™ (STEMCELL Technologies), or mTeSR™ Plus (STEMCELL Technologies) media for 6-8 days, in accordance with the manufacturer's recommendations. Complete media changes were performed daily. PSC colonies were clump passaged onto freshly coated Matrigel™ plates in maintenance culture.Example 2: Preparing PSC aggregates in the presence of one or more small molecules

[0118] Single cell or clump suspensions of H9 and 3A PSCs, maintained in accordance with Example 1 , were cultured in a first culture environment of this disclosure. Briefly, PSCs were seeded into one or more wells of a non-tissue culture treated plate in a derivation medium (e.g. EB Formation Medium), such as STEMdiff™ Hematopoietic — EB Basal Medium + STEMdiff™ Hematopoietic — EB Supplement A (STEMCELL Technologies), including either i) 10 pM of an inhibitor of cytoskeleton modification (CSMi) (e.g., Rho kinase inhibitor such as Y-27632), or ii) 10 pM of an inhibitor of cytoskeleton modification (CSMi) and 0.7 pM of aninhibitor of stress response (ISRi) (e.g., Trans-ISRIB). If using a non-tissue culture treated 6- well plate, 2.5 mL of EB Formation Medium was initially added per well and 2.5 mL of a cell suspension (~1.4x106cells / mL) in EB Formation Medium was added thereto.

[0119] Seeded hPSCs were cultured on an orbital shaker at approximately 70 rpm at 37°C for 2 days. On day 3, when mesoderm or mesoderm-like cells emerged, half media changes were performed with STEMdiff™ Hematopoietic — EB Basal Medium + STEMdiff™ Hematopoietic — EB Supplement B (STEMCELL Technologies) and every 2-3 days thereafter, to differentiate the mesodermal cells to CD34+hematopoietic progenitors. After a total of 12 days, floating PSC-derived CD34+cells were harvested for downstream differentiationExample 3: Deriving lymphoid progenitors from differentiated hematopoietic progenitors

[0120] CD34+HSPC cells produced as described in Example 2 were differentiated to lymphoid progenitors (LPs) in a second culture environment of this disclosure using StemSpan™ Lymphoid Progenitor Expansion Medium (STEMCELL Technologies). Prior to seeding the cells, non-tissue culture treated cultureware was coated with the StemSpan™ Lymphoid Differentiation Coating Material (STEMCELL Technologies) diluted to 1 * in PBS, according to the manufacturer's recommendations, then rinsed with PBS to remove unbound coating material.

[0121] After 3-4 days in culture, an equal volume of StemSpan™ Lymphoid Progenitor Expansion Medium was added to each well, and a half-medium change was performed each 3-4 days thereafter taking care to not disrupt the cells. After approximately 7 days, PSC- derived CD5+CD7+lymphoid progenitor cells emerged.Example 4: Deriving NK cells from differentiated lymphoid progenitors

[0122] PSC-derived CD5+CD7+lymphoid progenitors (LPs) of Example 3 were differentiated to NK cells in a third culture environment of this disclosure. Briefly, LPs were seeded in 0.5 mL of StemSpan™ NK Cell Differentiation Medium (StemSpan™ SFEM II supplemented with StemSpan™ NK Cell Differentiation Supplement, STEMCELL Technologies) per well of an uncoated 24-well plate at 1 *105cells per mL, and cultured for approximately 2 weeks at 37° C, 5% CO2, relative humidity. After the first 3-4 day period, an equal volume of StemSpan™ NK Cell Differentiation Medium was added to each well, and every 3-4 days thereafter halfvolume media changes were performed taking care to not disrupt the cells.

[0123] After the 2-week culture period, the arising CD56+NK cells were harvested and analyzed by flow cytometry using anti-human CD56 antibody (STEMCELL Technologies). Figure 1A shows the frequency and yield of day-28 CD56+NK cells (per input CD34+cell) derived from H9 and 3A PSC lines (as initially cultured for the first ~2 days in the presence ofISRi and CSMi or in CSMi alone). Figure 1 B shows the fold change in frequency or yield of day-28 PSC-derived CD56+NK cells cultured in the presence of ISRi and CSMi compared to in CSMi alone.

[0124] When hPSCs are seeded as clumps in the presence of CSMi and in the absence of ISRi and then differentiated as described in Examples 2-4, a frequency of >80% of PSC- derived CD56+NK cells was differentiated, but the yield of such cells per input CD34+cell was relatively low (~30 NK cells). When hPSCs are seeded in the same conditions but as single cells, and then differentiated as described in Examples 2-4, no PSC-derived CD56+NK cells were output in the CSMi only condition (data not shown). In contrast, when hPSCs were seeded as single cells in the presence of both CSMi and ISRi, a comparable frequency of PSC-derived CD56+NK cells to clump-based differentiation in the presence of only CMSi was obtained, but surprisingly a yield of >600 PSC-derived CD56+NK cells per input CD34+cell was obtained (Figure 1 A). Based on the foregoing data, a nearly 25-fold change in yield of NK cells is obtained in the presence of both CSMi and ISRI as compared to only in the presence of CSMi (Figure 1 B).

[0125] These results indicate a beneficial, and possibly rescuing role of ISRi when used at early stages of hPSC differentiation to NK cells, particularly when seeding hPSC as single cells for differentiation to CD34+cells. Thus, culture conditions at early stages of differentiation exhibited a beneficial role in downstream differentiation (e.g. CD34+to LP and then LP to NK cells).Example 5: Deriving T cells from differentiated lymphoid progenitors

[0126] PSC-derived CD5+CD7+lymphoid progenitors (LPs) of Example 3 were differentiated to T cells in a third culture environment of this disclosure. Briefly, LPs were seeded in 0.5 mL of StemSpan™ T Cell Progenitor Maturation Medium (StemSpan™ SFEM II supplemented with StemSpan™ T Cell Progenitor Maturation Supplement, STEMCELL Technologies) per well of a 24-well plate (coated as described in Example 3) at 1 *105cells per mL, and cultured for approximately 2 weeks at 37° C, 5% CO2, relative humidity. After the first 3-4 day period, an equal volume of StemSpan™ T Cell Progenitor Maturation Medium was added to each well, and every 3-4 days thereafter half-volume media changes were performed taking care to not disrupt the cells.

[0127] After the 2-week culture period, the arising T cells were harvested and analyzed by flow cytometry using fluorochrome-conjugated antibodies against CD3, CD4, CD8a, CD8|3, and TCRap. When starting from H9 cells, differentiation went through CD4+CD8+(DP) T cells, and frequency and yield of DP T cells is shown in Figure 2A. However, 3A iPS cells are derivedfrom T cells, so this differentiation by-passed the double-positive T cell stage, and frequency and yield of 3A-derived CD8+(SP) T cells is shown in Figure 2B.

[0128] When H9 hPSCs are seeded as clumps in the presence of CSMi and in the absence of ISRi and then differentiated as described as described in Examples 2, 3, and 5, a frequency of ~80% of PSC-derived DP T cells was differentiated, and the yield of such cells per input CD34+cell was less than 50 (Figure 2A). When H9 hPSCs are seeded in the same conditions but as single cells, and then differentiated as described in Examples 2, 3, and 5, no PSC- derived T cells were output in the CSMi only condition (data not shown). In contrast, when H9 hPSCs were seeded as single cells in the presence of both CSMi and ISRi, a comparable frequency of PSC-derived DP T cells to clump-based differentiation in the presence of only CMSi was obtained, but surprisingly a yield of >100 PSC-derived DP T cells per input CD34+cell was obtained (Figure 2A).

[0129] When SCT-3A hPSCs are seeded as clumps in the presence of CSMi and in the absence of ISRi and then differentiated as described in Examples 2, 3, and 5, a frequency of ~12% of PSC-derived SP T cells was differentiated, and the yield of such cells per input CD34+cell was less than 1 (Figure 2B). When SCT-3A hPSCs are seeded in the same conditions but as single cells, and then differentiated as described in Examples 2, 3, and 5, no PSC-derived T cells were output in the CSMi only condition (data not shown). In contrast, when SCT-3A hPSCs were seeded as single cells in the presence of both CSMi and ISRi, surprisingly a ~4x increase in frequency of PSC-derived SP T cells to clump-based differentiation in the presence of only CMSi was obtained, and a yield of >13 PSC-derived SP T cells per input CD34+cell was obtained (Figure 2B).

[0130] These results indicate a beneficial, and possibly rescuing role of ISRi when used at early stages of hPSC differentiation to T cells, particularly when seeding hPSC as single cells for differentiation to CD34+ cells. Thus, culture conditions at early stages of differentiation exhibited a beneficial role in downstream differentiation (e.g. CD34+to LP and then LP to T cells).Example 6: Deriving kidney organoids from PSC

[0131] Single cell or clump suspensions of H9 and 4A PSCs maintained in mTeSR™1 in accordance with Example 1 were cultured to form cavitated PSC spheroids and then differentiated to kidney organoids using the STEMdiff™ Kidney Organoid Kit (STEMCELL Technologies).

[0132] Briefly, hPSCs were seeded at a range of single-cell densities (e.g., 750, 1000, 2000, and 3000 single cells) into each well of a Matrigel-coated 96-well plate and cultured at 37° C, 5% CO2, relative humidity for 1 day in mTeSR™ 1 medium including either i) 10 pM CSMi, orii) 10 pM of CSMi and 0.7 pM of ISRi, as described in Example 2. On the next day, adherent cells were overlaid with an additional layer of Matrigel™ to form cavitated PSC spheroids within the following 48 hours or so. On day 3, further differentiation was initiated by transitioning the cultures to STEMdiff™ Kidney Organoid Kit media (STEMCELL Technologies). During the next 18 days of differentiation the cells were directed in a stepwise manner through one or more stages of late primitive streak, posterior intermediate mesoderm, and metanephric mesoderm to give rise to pretubular aggregates, then renal vesicles that ultimately form kidney organoids. As an alternative approach, to form kidney organoids in 3D day-11 cells were harvested and reaggregated in an AggreWell™ (STEMCELL Technologies) plate and cultured for approximately 10 more days in STEMdiff™ Kidney Basal Medium supplemented with STEMdiff™ Kidney Supplement DM to form kidney organoids in 3D.

[0133] Exposing hPSC to both ISRi and CSMi during culture initiation resulted in successful differentiation of kidney organoids in nearly all experiments. In comparison, only about half of the differentiation experiments were successful when hPSCs are exposed to only CSMi during culture initiation (Figure 3A). Determination of a successful versus failed differentiation of hPSCs to kidney organoids in the presence of CSMi+ISRi versus CSMi alone, was based on at least the generation of convoluted tubular structures (shown by white arrow in Figure 3B) that resemble the structure and segmentation of the developing nephron, and by immunostaining for renal epithelium cells, endothelial cells, neural and mesenchymal cells (shown by white arrows in Figure 3C). Renal epithelium cells typically stain for podocalyxin “PODXL” (e.g podocytes), lotus tetragonolobus lectin “LTL” (e.g. proximal tubules), and E- cadherin “ECAD” (e.g. distal tubules). Endothelial cells typically stain for platelet endothelial cell adhesion molecule “CD31”. Mesenchymal cells typically stain for vimentin “VIM”. Neural cells typically stain for beta-tubulin 3 “TUJ1”. Under CSM only condition, differentiation either failed to form a kidney organoid, or a formed kidney organoid showed a different morphology or cell type expression pattern (data not shown).

[0134] These results indicate a beneficial, and possibly rescuing role of ISRi when used in combination with CMSi at early stages of hPSC differentiation to kidney organoids, particularly when seeding hPSCs at low or clonal cell densities. Thus, culture conditions at early stages of differentiation exhibited a beneficial role in downstream differentiation.

Claims

Claims:1 . A method of differentiating a population of stem or progenitor cells, comprising: seeding the population of stem or progenitor cells in a first culture environment; culturing the population of stem or progenitor cells for a sufficient time in the first culture environment, the first culture environment comprising a first culture vessel and a first culture medium supplemented with i) one or more cytokines and / or growth factors, and ii) one or both of an inhibitor of cytoskeleton modification and an inhibitor of stress response; and differentiating the population of stem or progenitor cells toward a downstream lineage of cells.

2. The method of claim 1 , wherein the population of stem or progenitor cells was cultured in an earlier culture environment different from the first culture environment.

3. The method of claim 1 or 2, further comprising generating the downstream lineage of cells, wherein the downstream lineage of cells is differentiated at a higher efficiency compared to when a population of stem or progenitor cells is cultured in a first culture environment that does not comprise one or both of the inhibitor of cytoskeleton modification and the inhibitor of stress response.

4. The method of any one of claims 1-3, wherein the first culture environment comprises nonadherent and / or suspension culture.

5. The method of any one of claims 1-4, wherein the inhibitor of cytoskeleton modification is a Rho-associated protein kinase inhibitor.

6. The method of claim 5, wherein the Rho-associated protein kinase inhibitor is Y-27632 or a functional equivalent thereof.

7. The method of any one of claims 1-6, wherein the inhibitor of stress response is an integrated stress response inhibitor or a functional equivalent thereof.

8. The method of any one of claims 1 -7, wherein the population of stem or progenitor cells are seeded as single cells in the first culture environment.

9. The method of any one of claims 1-8, further comprising exposing the population of stem or progenitor cells to a stressor either when transitioning the cells to the first culture environment or in the first culture environment.

10. The method of claim 9, wherein the population of stem or progenitor cells exposed to the stressor exhibit a higher differentiation efficiency when cultured in the first culture environment compared to when the population of stem or progenitor cells is exposed to the stressor andcultured in a first culture environment that does not comprise one or both of the inhibitor of cytoskeleton modification and the inhibitor of stress response.

11. The method of any one of claims 1-10, wherein the sufficient time ranges between about 1 and about 17 days.

12. The method of any one of claims 1-11 , further comprising reducing exposure to the first culture medium as the sufficient time elapses by exchanging, partially or completely, the first culture medium with a culture medium supplemented with at least i) one or more cytokines and / or growth factors, but not ii) one or both of the inhibitor of cytoskeleton modification and the inhibitor of stress response.

13. The method of any one of claims 1-12, further comprising: transitioning the downstream lineage of cells from the first culture environment to a second culture environment; and culturing the downstream lineage of cells for a sufficient time in the second culture environment, the second culture environment comprising the first culture vessel or a second culture vessel, and / or a second culture medium, the second culture medium supplemented with at least one or more cytokines and / or growth factors.

14. The method of claim 13, further comprising: transitioning an arising population of cells from the second culture environment to a third culture environment; and culturing the arising population of cells for a sufficient time in the third culture environment, the third culture environment comprising the first culture vessel, the second culture vessel, or a third culture vessel, and / or a third culture medium, the third culture medium supplemented with at least one or more cytokines and / or growth factors.

15. The method of claim 13 or 14, wherein a) the second culture environment comprises a substrate coating that interacts with the arising population of cells of the first culture environment, and / or b) the third culture environment comprises a substrate coating that interacts with the arising population of cells of the second culture environment.

16. The method of any one of claims 13-15, wherein the sufficient time in the second culture environment and / or the third culture environment ranges between about 7 and about 28 days.

17. The method of any one of claims 1-16, wherein the downstream lineage of cells comprises mesoderm lineage cells.

18. The method of claim 17, wherein the mesoderm lineage cells comprise hematopoietic lineage cells or a composition comprising renal lineage cells.

19. The method of claim 18, wherein a) the hematopoietic lineage cells comprise one or more lymphocyte populations or progenitors thereof, or b) the composition comprising renal lineage cells comprises epithelial, endothelial, mesenchymal, and neural cells.

20. The method of any one of claims 1-19, wherein the population of stem or progenitor cells comprises a pluripotent stem cell line.

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