Functionalized hydrogels and methods of use in t cell differentiation

Abstract

There is provided a hydrogel, such as a functionalized hydrogel, for use in the expansion and / or differentiation of cells, that are encapsulated within the three-dimensional structure of the hydrogel. In some aspects of the invention, the hydrogel better mimics the thymus microenvironment than prior hydrogels for T cell expansion and differentiation and can be used in the expansion and differentiation of HSPCs, HSCs or PCs or combinations thereof in the production of T cells, T-cell committed precursors or progenitor cells and in methods for producing mature T cells. Methods of making the hydrogels, methods of culturing the cells in the hydrogel and uses of the hydrogels and cells produced are also described.

Description

FUNCTIONALIZED HYDROGELS AND METHODS OF USE IN T CELL DIFFERENTIATIONRelated Applications

[0001] This application is related to and claims priority and the benefit from US Provisional Patent Application No. 63 / 603,323, filed November 28, 2023, entitled “Functionalized Hydrogels and Methods of Use”, which is incorporated herein by reference.Field of Invention

[0002] The present invention relates to novel hydrogels such as functionalized hydrogels, to compositions comprising components of said hydrogels, to the components of said compositions and to methods for making and using any of the foregoing. In some aspects, the invention is used for T cell differentiation. In other aspects, the invention can be used for the generation of T cells, including progenitor, precursor and mature T cells.Sequence Listing - Incorporation by Reference of Material Submitted Electronically

[0003] The Sequence Listing, which is a part of the present disclosure, is submitted concurrently with the specification in accordance with WIPO Standard 26 (ST26). The file was created on November 28, 2024 and uploaded in XML compliant format directly to the PCT Receiving Office on filing and is 8kb, the name of the file is T8486105WO.XML. The subject matter of the Sequence Listing is incorporated herein in its entirety by reference.

[0004] It is hereby stated that the information recorded in computer readable form is identical to the sequence listings referred to in this description.Background of the Invention

[0005] T cells are the major components of the adaptive immune system responsible for mediating cellbased immune responses. They are a type of white blood cell that help the body fight infection and disease. Various T cell therapies have been and are being developed. This includes, T cell immunotherapy, including T cell transfer therapy, and adoptive T cell therapy, such as chimeric antigen receptor (CAR) T cell immunotherapy and T cell receptor (TCR)-T cell therapies. T cell therapies have been used in the treatment of various cancers, including to restore T cell levels after chemotherapy or other treatment-associated depletion, and modified T cells have been used to treat cancers such as in CAR-T therapy. CAR-T therapy has been approved for the treatment of various cancers and is an emerging treatment for autoimmune disease.

[0006] CAR-T cell therapy uses a patient’s T cells that are genetically modified in the laboratory to recognize and specifically attack cancer cells (Levine et al., 2017). Treatment with CAR-T cells has led to promising clinical responses in patients with B cell leukemia and lymphoma (Sterner & Sterner, 2021). Unlike traditional cancer therapies, recent data has demonstrated that CAR T cell therapy can induce prolonged remissions and, in some cases, cure patients with B cell malignancies such as B cell acute lymphoblastic leukemia (Cappell & Kochenderfer, 2023).

[0007] While CAR-T cell therapy has strong potential to treat the types of cancer that currently have no cure, the short supply of donor T cells remains a critical barrier (Tang et al., 2022). Additionally, the total treatment cost is estimated to reach up to $500,000 per patient, posing a significant financial burden on the healthcare system (Choi et al., 2022). As the process involves harvesting T-cells from the patient, if the patient is not ready to receive the therapy once prepared within its shelf life, or if something happens to the prepared batch of modified T cells, or there is insufficient yield, additional patient samples may be required to produce the CAR-T cell batch, at additional time (delay) and cost.

[0008] As such, the potential of such therapies is hampered by the short supply of donor T cells. To make the treatment more accessible, efficient and cost effective, there is a need for new strategies for T cell production that enhance yield and which are more efficient.

[0009] T cells are developed from bone marrow stem cells, i.e., hematopoietic stem cells (HSCs). During T cell lineage development, HSCs migrate to the thymus where they predominantly develop into T cells as opposed to cells of the myeloid lineage. The natural thymic microenvironment is a unique and controlled environment that facilitates T cell differentiation (Thapa & Farber, 2019). Further, central to T cell maturation in the native thymus is Notch signaling, activated by pulling forces by Notch ligands of thymic epithelial cells exerted on the Notch 1 receptor of HSCs and hematopoietic progenitor cells (HPCs, collectively HSPCs or blood progenitor cells) (Zuniga-Pflucker, 2004).

[0010] Attempts have been made to facilitate the differentiation of therapeutic T cells from stem cells by culturing stem cells on platforms intended to recreate the thymic microenvironment. However, thymic models to date have been unable to recapitulate the crucial elements of the thymic microenvironment required for T cell maturation.

[0011] One approach in in vitro T cell manufacturing relies on immobilized Notch ligands to drive differentiation. For instance, HSCs can be cultured on a feeder cell layer of murine fibroblasts or bone marrow stromal cells transduced to express the Notch ligands Delta-like 1 or 4 (DLL1 , DLL4) in vitro (Mohtashami et al., 2013; Schmitt & Zuniga-Pflucker, 2002). However, these methods are xenogeneic and use serum-supplemented media, making them not easily translatable for clinical settings.

[0012] To address these limitations, a two-dimensional (2D) plate-based approach was developed by immobilizing Notch ligand DLL4 and vascular cell adhesion molecule 1 (VCAM1), an adhesion molecule found in the thymus (Edgar et al., 2022; Michaels et al., 2022). The system generated mature functional T cells from both cord blood (CB)- and induced pluripotent stem cell (iPSC)-derived HSPCs. Another method involves presenting Notch ligand to HSPCs on the surface of polystyrene beads (Trotman-Grant et al., 2021). The method can be adapted for scalable cell differentiation in a dynamic system, like a bioreactor. However, these methods are limited to the in vitro generation of T cells on a 2D surface and are therefore unable to recreate many crucial elements of the thymic microenvironment required for optimizing T cell development, such as the thymus’s three-dimensional (3D) architecture and complex extracellular matrix (ECM) composition which determines ECM stiffness and intricate cell-cell and cell- ECM interactions.

[0013] Biomaterial-based platforms have also been explored as reservoirs for the delivery of T cells, platforms for activating and expanding a patient’s mature T cells or as in vivo differentiation systems for transplanted HSCs. For example, studies have demonstrated that T cells delivered using biomaterials in vivo can have increased anti-tumor effects compared to intravenously administered T cells (Hu et al., 2021 ; Stephan et al., 2015). Others have shown that HSC transplantation along with subcutaneous injection of DLL4-functionalized bone marrow cryogels in mice enhances progenitor T cell engraftment in the thymus, where they give rise to mature cytotoxic T cells (Shah et al., 2019). However, this approach relied on attracting cells to migrate into the cryogel rather than using the cryogel as a delivery reservoir. Current methods are limited either by the initial availability of the patient’s mature T cells or reliance on thymus engraftment to produce mature T cells (Shah et al., 2019; Chin et al., 2020; Hu et al., 2021).

[0014] The use of 3D hydrogels in attempts to mimic tissue microenvironments involves using a variety of materials to reconstruct aspects of the native ECM. However, hydrogels used to date have not sufficiently mimicked the thymus microenvironment. For instance, in some, the cells are on the surface of a functionalized hydrogel, while in others, the properties of stiffness or 3D architecture are not optimized. See for example Kratzer et al., 2019, which describes the use of a PEG-based hydrogel functionalized with DLL1 (immobilized using gold), again, wherein the cells are on the surface of the hydrogel, failing to mimic the microenvironment of the thymus, which is hypoxic under normal physiological conditions.

[0015] Therefore, there remains an unmet need to optimize in vitro T cell differentiation and T cell production yields from HSPCs (such as umbilical cord blood-, bone marrow-, peripheral blood- andiPSC-derived HSPCs) in a manner that produces functional T cells suitable for various uses, such as regenerative therapies and immunotherapies.Summary of the Invention

[0016] In some aspects, the present invention provides a new three-dimensional (3D) hydrogel that can be functionalized with various immobilized thymic ECM components such as Notch ligands for producing mature human T cells and their progenitors in vitro (Figure 1). The invention also provides methods for making the 3D hydrogel and for developing mature functional T cells and their progenitors from HSCs, HPCs or HSPCs cultured in the hydrogel composition.

[0017] In some embodiments, the invention provides a three-dimensional (3D) hydrogel comprising: a first biocompatible polymer that is a polysaccharide polymer; at least one second biocompatible polymer, wherein the first and second biocompatible polymers are crosslinked (directly or indirectly, or in one embodiment modified to be connected using click chemistry as described herein); and at least one signalling molecule immobilized directly or indirectly to the first and / or second biocompatible polymer (or in one embodiment modified to be connected using click chemistry, as described herein), or in some embodiments on the first biocompatible polymer wherein in some aspects of the invention the at least one of the signalling molecules is: one that promotes hematopoietic stem and progenitor cell (HSPC), hematopoietic stem cell (HSC) and / or hematopoietic progenitor cell expansion and differentiation to progenitor and precursor T cells and / or mature T cells; such as in some embodiments is a Notch ligand, such as in some embodiments is selected from one or more of: DLL1 , DLL3, DLL4, JAG1 , and JAG2, or from DLL1 or DLL4 or is DLL4; and / or alone or in addition to “(i)” promotes cell adhesion or is an integrin receptor ligand, such as in some embodiments VCAM-1 , or a molecule with an RGD integrin receptor site, wherein in some aspects the signalling molecule is a human or human recombinant molecule. In some embodiments the signalling molecule is present in the hydrogel at a range of about 50 to about 200, or from about 50 to about 100 micrograms per mL of hydrogel.

[0018] In some aspects of the invention the first biocompatible polymer of the hydrogel is selected from the group consisting of: alginate, chitosan, cellulose, pectin, hyaluronic acid (HA), agarose, chondroitin sulfate, carrageenan, inulin, starch, salts or esters of any of the foregoing and any combinations of the foregoing, or selected from the group consisting of alginate, chitosan, cellulose, pectin, hyaluronic acid (HA), salts or esters of any of the foregoing and any combinations of the foregoing, or is hyaluronic acid or salts or esters thereof, such as sodium hyaluronate, potassium hyaluronate, magnesium hyaluronate and calcium hyaluronate. In some embodiments, the first polymer is from about 150 - about 300 kDa.

[0019] In some embodiments, the at least one second biocompatible polymer is selected from one or more of a natural polymer and / or a synthetic polymer, such as a synthetic polymer that is inert to degradation from cellular enzymes. In some other embodiments the at least one second biocompatible polymer is a synthetic polyvinyl alcohol, polyethylene glycol (PEG), sodium polyacrylate, acrylate polymers and copolymers, salts or esters thereof, or an about 5 to about 6 kDa synthetic polymer of any of the foregoing.

[0020] In some further embodiments, at least one of the at least one second biocompatible polymer is a crosslinker and one or more of a hydrogel stabilizer, a stiffening agent, a cell adhesion molecule, a RGD - based polymer that promotes cell adhesion and / or migration, and / or a polymer that has an integrin receptor binding cite, such as RGD. In some other aspects of the invention, the at least one of the second biocompatible polymers is a gelatin, such as an about a 40 - about 70 kDa or in some other aspects about a 60kDa gelatin.

[0021] In yet some other embodiments, the at least one second biocompatible polymer is a PEG, a gelatin or combination thereof.

[0022] In some aspects of the invention a person of skill in the art would understand that the selection of the second biocompatible polymer may depend on the degree it is subject to degradation from cellular enzymes and that this may vary depending on the source of the cells within the hydrogel. In hydrogels where the exposure to such cellular enzymes affects the stability of the hydrogel, a synthetic polymer may need to be added that is more inert to said degradation. For instance, when the hydrogel is seeded with cord blood derived cells, the second biocompatible polymer can be a natural (e.g. a gelatin alone), synthetic (e.g. PEG) or both. In other embodiments when the hydrogel is seeded with iPSCs, the second biocompatible polymer is a preferably a combination of a natural (e.g. gelatin) and a synthetic (e.g. PEG) polymer. In other embodiments it can be any polymer with an RGD or integrin binding site combined with a polymer that is inert (or more inert or sufficiently inert to degradation from cellular enzymes to which it may be exposed).

[0023] In some aspects, the 3D hydrogel of the invention forms a 3D matrix. In some embodiments, the hydrogel of the invention is in a liquid form (such as prior to gelation). In yet some other embodiment the hydrogel is a 3D gel.

[0024] In some embodiments, the 3D hydrogel of the invention is seeded with hematopoietic stem and progenitor cells (HSPC), hematopoietic stem cells (HSC) and / or hematopoietic progenitor cells (HPC) or combinations thereof, which are encapsulated within the 3D hydrogel. In some aspects the cells are added to the liquid form of the hydrogel and when the hydrogel gels, the cells and cells derived from said seeded cells are encapsulated within or by the hydrogel. In some other aspects the hydrogel with the cells are cultured under conditions that promote cell expansion and differentiation. In some other aspects spheroids comprising said cells are formed in the hydrogel.

[0025] In some aspects, the cells are derived from any source of HSPC, HSC or HPC or from derived from cord blood or induced pluripotent stem cell (iPSC) derived cells or in other aspects the aforementioned cells are human cells.

[0026] In some aspects of the invention, the first biocompatible polymer, the second biocompatible polymer and the signalling molecule of the hydrogel are connected using click chemistry. In some further aspects, the signalling molecule is an Fc-fusion protein signalling molecule, and bound to an Fc fusion protein binding agent, such as Protein A (ProA) or Protein G (ProG) or ProG. In some other aspects, the first and second biocompatible polymer and Fc fusion protein binding agent are modified to enable such conjugation and / or crosslinking, as the case may be through click chemistry.

[0027] In some aspects, the methods and products of the invention, including the differentiation process are non-xenogeneic and use serum-free media. This facilitates the clinical use of the resulting cells. In some other aspects, the present invention provides a differentiation process that more closely mimics the crucial elements of the thymic microenvironment required for T cell development, namely the thymus’s 3D architecture and complex ECM composition which determine stiffness and intricate cellcell and cell-matrix interactions. In some other aspects, the differentiation platform and its components are biocompatible and suitable for both in vitro and in vivo use, such as an injectable hydrogel in a patient for in vivo T cell delivery and in situ T cell generation, thereby being used as both a development and delivery system of T cell therapies.

[0028] Generally, in some aspects, the invention provides a hydrogel comprising: a first biocompatible polymer that is a polysaccharide polymer; a second biocompatible polymer; a Fc fusion protein binding agent; and at least one Fc fusion protein ligand conjugated to the Fc fusion protein binding agent, wherein the first biocompatible polymer is (i) crosslinked to the second biocompatible polymer and (ii)conjugated to the Fc fusion protein binding agent through click chemistry. The first and second biocompatible polymers and Fc fusion protein binding agent are modified to enable such crosslinking and conjugation through click chemistry. In some aspects, the hydrogel can be in a liquid and / or a gelled state. In some other aspects, the hydrogel is in a gelled state, wherein the hydrogel polymers are crosslinked to form a 3D hydrogel matrix. In yet some other embodiments, the hydrogel is seeded with at least one cell, which is encapsulated within the hydrogel in a 3D structure.

[0029] In some aspects, the 3D hydrogel may be seeded with HSCs, HPCs or HSPCs and used to promote and / or enhance the expansion and differentiation of the seeded cells to progenitor and precursor T cells and mature T cells. In some other aspects, the 3D hydrogel is seeded with about 500,000 cells per 1 ml of hydrogel. Other ranges include but are not limited to (about 300,000 - about 2,000,000 cells per 1 mL of hydrogel. In the preferred embodiment of the invention, the seeded cells are HSCs, HPCs or combinations thereof (i.e. , HSPCs). In some other aspects, the cells are derived from cord blood using methods known in the art. In yet other aspects, the cells are derived from iPSCs using methods known in the art.

[0030] The present invention further comprises methods of making the 3D hydrogels as described herein. In one aspect, the method of making the 3D hydrogel of the present invention comprises (i) a crosslinking step, wherein a first biocompatible polymer is crosslinked with at least one second biocompatible polymer to form a 3D hydrogel matrix, and wherein the crosslinking may be achieved using chemical means, such as click chemistry, or physical means, such as photo-crosslinking; and (ii) a signalling molecule immobilization step, wherein at least one signalling molecule is immobilized within the 3D hydrogel matrix by directly or indirectly conjugating the at least one signalling molecule to the first biocompatible polymer and / or the at least one second biocompatible polymer.

[0031] The entire document is intended to be related as a unified disclosure, and it should be understood that the foregoing summary is not intended to define every aspect of the invention. Other features, objects and advantages of the present invention are apparent in the detailed description that follows. However, it will be understood that the detailed description, while indicating embodiments of the present invention, is given by way of illustration only, not limitation.

[0032] All combinations of features described herein are contemplated, even if the combination of features are not found together in the same sentence paragraph or section of this document. In addition, the invention includes, as an additional aspect, all embodiments of the invention narrower in scope in any way than the variations specifically mentioned herein.Brief Description of Drawings

[0033] Figure 1 is a schematic representation of a hydrogel-based method to mimic the thymic microenvironment for T cell production, where (a) depicts endogenous CD34(+) HSCs generated in bone marrow migrating to the thymus where the Notch ligands including DLL4 presented by thymic epithelial cells engage the Notchl receptor on HSCs triggering T cell differentiation, and (b) depicts the approach of the present invention comprising a serum-free feeder-free system using a hydrogel functionalized with thymic components such as Notch ligands including DLL4 ligand to create a 3D thymic-like niche encapsulating HSCs in a media that triggers T cell differentiation.

[0034] Figure 2 illustrates the synthesis of HA-norbornene. (a) Schematic representation of HA- norbornene synthesis, (b) Typical1H NMR (500 MHz, D2O, 5) of HA-norbornene showing successful substitution of norbornene.

[0035] Figure 3 illustrates a method for immobilization of thymic components in a HA hydrogel using Protein G. (a) Protein G (ProG) is modified with methyltetrazine (Tz). (b) ProG-Tz conjugated to HA- norbornene binds Fc-fused proteins including DLL4-Fc for Notch activation of encapsulated cells. The HA is a polymer that comprises many such norbornene sites for conjugation of ProG-Tz and for crosslinking with methyltetrazine-modified crosslinkers, such as PEG-Tz and / or gelatin-Tz. The figure depicts only one such norbornene site, (c) HA-norbornene is crosslinked with PEG-Tz and / or gelatin- Tz using norbornene-tetrazine click chemistry to form a hydrogel matrix. Again, HA is a polymer that comprises many such norbornene sites for gelatin or PEG crosslinking.

[0036] Figure 4 illustrates the extent of modification of Protein G and gelatin with methyltetrazine, (a) Modification of Protein G with methyltetrazine is verified using electrospray ionization mass spectrometry, (b) Ultraviolet-visible (UV-Vis) spectroscopy absorbance at 524 nm is used to quantify the degree of gelatin modification with methyltetrazine.

[0037] Figure 5 illustrates the synthesis of PEG-Tz using an amide coupling reaction, (a) Schematic of 4-arm PEG-Tz synthesis from 4-arm PEG amine, (b) Typical1H NMR (500 MHz, D2O, 5) of 4-arm PEG- Tz showing successful substitution.

[0038] Figure 6 illustrates cell encapsulation in the hydrogels, (a) Microscopy of spheroids formed in the hydrogel, wherein cells were stained for nuclei (DAPI) and actin (GFP) and merged with bright field. Z-stack of one spheroid is shown demonstrating that the spheroids are not hollow. Scale bar = 10 pm. (b) Microscopy of hydrogel with formed cell spheroids. Scale bar = 100 pm. (c) 3D rendering and transmitted detection of spheroids generated in the hydrogels.

[0039] Figure 7 illustrates the generation of lineage-specific progenitor T (Pro-T) cells and lineage- committed precursor T (Pre-T) cells in DLL4-functionalized HA hydrogel, (a) Different stages of T cell maturation include CD7(+) early T cell progenitors (ETPs), CD7(+)CD5(+) T cell lineage-specific Pro-T cells, CD7(+)CD5(+)CD1a(+) T cell lineage-committed Pre-T cells, initial single positive (ISP) CD4(+) cells, double positive (DP) CD4(+)CD8(+) cells and mature single positive (SP) CD4(+) or CD8(+) T cells, (b) Schematic overview and timeline to assess the DLL4-functionalized HA hydrogel’s capability to generate Pro-T and Pre-T cells over 2 weeks, (c) Flow cytometry analysis of cells expressing CD5, CD7 and CD1a generated in DLL4-functionalized HA hydrogel for 2 weeks using media with a lymphoid progenitor expansion supplement containing cytokines SCF, IL7, TPO and Flt3L. Flow cytometry is representative of three replicates, (d) Frequency of Pro-T cells generated in the HA-based hydrogels with Protein G only (HA-ProG), with non-immobilized DLL4-Fc only (HA-DLL4-Fc) and with immobilized DLL4 (HA-ProG-DLL4-Fc) on day 14 (n=3). (e) Frequency of Pro-T and Pre-T cells generated in hydrogels with 50, 100 and 200 pg of DLL4-Fc per 1 mL of HA-ProG hydrogel. Data represent mean ± SD for 3 biological replicates. * p < 0.05; ** p < 0.01 ; *** p < 0.001. The HA hydrogel in the description of Figures 7(b)-(d) also includes PEG, i.e. HA-norbornene crosslinked with PEG-Tz using click chemistry.

[0040] Figure 8 illustrates the ability of HSPCs to generate T cells in HA-ProG-DLL4 3D hydrogels crosslinked with gelatin (HA-ProG-DLL4-G wherein G represent gelatin) compared to the inventors’ previously verified DLL4+VCAM1 functionalized 2D substrate, (a)-(c) Flow cytometry analysis of CD5(+)CD7(+) Pro-T cells, CD1a(+)CD7(+) Pre-T cells, CD33(+) myeloid cells, CD56(+) natural killer cells and CD34(+) HSPCs generated after 2 weeks. Flow cytometry is representative of 3 replicates, (a) Flow cytometry analysis of cells generated using negative controls (hydrogels without DLL4). (b) Flow cytometry analysis of cells generated using positive controls (DLL4- and VCAM1 -coated 2D plate, (c) Flow cytometry analysis of cells generated in HA-ProG-DLL4-G hydrogels.

[0041] Figure 9 illustrates the influence of gelatin and VCAM1 on T cell commitment of CB-derived CD34(+) HSCs in HA-ProG-DLL4 hydrogel, wherein gelatin acts as a crosslinker, a stiffening agent and a cell adhesion molecule containing RGD sequences that interacts with cell surface receptors, (a) Gelatin is modified with methyltetrazine (Tz). (b) Frequencies of Pro-T and Pre-T cells generated in HA- ProG-DLL4 hydrogel with increasing gelatin-Tz concentrations (0, 2, 5, 10%) (n=3). In (b) to (e), PEG- Tz is used in the 0% gelatin-Tz condition, whereas PEG-Tz is absent when 2%, 5% or 10% gelatin-Tz is used, (c) Quantification of Young moduli (kPa) of HA-ProG-DLL4 hydrogel with increasing gelatin-Tz concentrations (0, 2, 5, and 10%) (n=3). (d) Frequencies of ETPs, Pro-T cells, Pre-T cells and myeloid cells generated from CB-derived CD34(+) HSCs encapsulated for 2 weeks in various formulations of hydrogels with and without gelatin-Tz (HA-ProG-DLL4-G and HA-ProG-DLL4 respectively) and with orwithout VCAM1-Fc (n=3). (e) Yield of combined Pro-T and Pre-T cells generated per 1 CD34(+) HSC input in the presence and absence of gelatin and / or VCAM1. Data represent mean ± SD for 3 biological replicates. * p < 0.05; ** p < 0.01 ; *** p < 0.001.

[0042] Figure 10 illustrates that cells migrating out of the engineered hydrogel can continue T cell differentiation in the presence of ligands that enhance Notch signaling, (a) Schematic representation of HSC-encapsulating hydrogel in a transwell placed above a well coated with DLL4 and VCAM1. (b) Flow cytometry analysis assessing CD5 and CD7 expression in cells remaining inside the hydrogel for 2 weeks, cells migrated out of the gel into wells coated with DLL4 and VCAM1 and cells migrated into uncoated wells. Flow cytometry is representative of three replicates, (c) Frequencies of CD5(+)CD7(+) Pro-T cells generated within the hydrogel, migrated out of the hydrogel in the presence of DLL4 and VCAM1 and migrated outside the hydrogel in the absence of DLL4 and VCAM1. Data represent mean ± SD for 3 biological replicates. * p < 0.05.

[0043] Figure 11 illustrates T cell differentiation kinetics in the presence and absence of VCAM1 upon switching from media with a lymphoid progenitor expansion supplement containing cytokines SCF, IL7, TPO and Flt3L to media with a maturation supplement containing IL-7 and Flt3L (but not TPO or SCF). (a-b) Frequencies of Pro-T and Pre-T cells generated in hydrogels with and without VCAM1 over 4 weeks of culture. Cytokines TPO and SCF were removed from media at week 2 (n=2). (c-d) Frequencies of Pro-T and Pre-T cells generated in hydrogels with and without VCAM1 over 5 weeks of culture. Cytokines TPO and SCF were removed from media at week 3 (n=2).

[0044] Figure 12 illustrates that mature functional T cells develop from CB-derived CD34(+) HSCs in HA-ProG-DLL4-G hydrogel. The HA-ProG-DLL4-G hydrogel in this figure does not include PEG. (a) Schematic overview and timeline to assess the hydrogel’s capability to generate functional T cells, (b) Frequencies of ETPs, myeloid cells, Pro-T cells and Pre-T cells generated over 4 weeks in HA-ProG- DLL4-G hydrogel (n=3). (c) Flow cytometry analysis of cells expressing CD4, CD8b, CD3 and TCRap generated in HA-ProG-DLL4-G hydrogel on day 35. Flow cytometry is representative of three stimulation replicates, (d) Frequencies of CD4(+) ISP, CD4(+)CD8(+) DP and CD3(+)TCRaP(+) T cells generated in HA-ProG-DLL4-G hydrogel on day 35 (n=3). (e) Flow cytometry analysis of cells expressing CD4, CD8b, CD3 and TCRap generated in HA-ProG-DLL4-G hydrogel on day 84 (n=3). Flow cytometry is representative of three stimulation replicates, (f) Frequencies of mature CD8(+) SP and CD3(+)TCRaP(+) T cells generated in HA-ProG-DLL4-G hydrogel on day 84 (n=3). (g) Flow cytometry analysis of cells expressing CD4, CD8b, CD3 and TCRap generated in HA-ProG-DLL4-G hydrogel for 35 days and activated with CD3 and CD28 Immunocult reagent for an additional 14 days. * p < 0.05; ** p < 0.01 ; *** p < 0.001. (h) Frequencies of CD4(+) SP, CD8(+) SP, CD4(+)CD8(+) DP andCD3(+)TCRaP(+) T cells generated in HA-ProG-DLL4-G hydrogel for 35 days and activated with CD3 and CD28 Immunocult reagent for an additional 14 days (n=3). Data represent mean ± SD for 3 biological replicates, (i-j) Following 14 days of activation and expansion, CD3(+) cells were assayed for production of effector proteins in the absence (i) or presence (j) of nonspecific phorbol 12-myristate 13- acetate (PMA) / ionomycin stimulation. Intracellular flow cytometry is representative of three stimulation replicates.

[0045] Figure 13 illustrates Pro-T cell development from iPSC-derived HSPCs in the hydrogel with immobilized DLL4. (a) Schematic overview of the procedure of generating T cells from iPSCs, including hemogenic induction in aggrewells, CD34(+) cell enrichment using magnetic separation, endothelial-to- hematopoietic transition of CD34(+) cells using DLL4+VCAM1 coated plates and encapsulation of budded HSPCs into hydrogels with immobilized DLL4. (b) Flow cytometry analysis of cells expressing CD34, CD184, CD43 and CD73 generated in aggrewells after 8 days of hemogenic induction. Flow cytometry is representative of 3 stimulation replicates, (c) Flow cytometry analysis of CD5(+)CD7(+) Pro-T cells generated after budded HSPCs are cultured on DLL4+VCAM1 coated plates. Flow cytometry is representative of 3 replicates, (d) Schematic overview and timeline of media composition. HSPCs generated from PSCs were encapsulated into the hydrogels with immobilized DLL4 and cultured for 2 weeks. Lymphoid progenitor expansion media containing IL-7, Flt3L, TPO and SCF was either used for 2 weeks of culture or replaced after 1 week of culture with maturation media containing only IL-7 and Flt3L. (e) Flow cytometry analysis of cells expressing CD5 and CD7 generated in hydrogels with immobilized DLL4 after 2 weeks of HSPC encapsulation in the hydrogels. Pro-T cells are identified by CD5 and CD7 co-expression. Flow cytometry is representative of three replicates, (f) Frequencies of CD5(+)CD7(+) Pro-T cells and CD5(-)CD7(+) ETPs generated after 2 weeks of HSPC encapsulated in the hydrogels. Cells cultured for 1 week in the lymphoid progenitor expansion media followed by 1 week of maturation media were compared to cells cultured for 2 weeks in the lymphoid progenitor expansion media (n=3). Data represent mean ± SD for 3 biological replicates. * p < 0.05; ** p < 0.01 ; *** p < 0.001. The examples using iPSCs use both PEG and gelatin crosslinkers, wherein gelatin acts as a crosslinker, a stiffening agent and a cell adhesion molecule containing RGD sequences that interacts with cell surface receptors.

[0046] Figure 14 illustrates that culturing iPSC-derived HSPCs in HA-ProG-DLL4 hydrogel crosslinked with both PEG-Tz and gelatin-Tz (HA-ProG-DLL4-PEG-G) under hypoxic conditions enhances differentiation to Pro-T cells, (a) Schematic overview of timeline and media conditions used to assess the effects of DLL4 concentration and hypoxia on differentiation of PSC-derived HSPCs into CD5(+)CD7(+) Pro-T cells, (b) Brightfield images of cells generated after 2 weeks in HA-ProG-DLL4- PEG-G hydrogels with varying concentrations of DLL4 and in normoxic vs hypoxic conditions, (c) Flowcytometry analysis and (d) frequencies of CD5(+)CD7(+) Pro-T cells generated after 2 weeks in HA- ProG-DLL4-PEG-G hydrogels with varying concentrations of DLL4 and in normoxic vs hypoxic conditions.

[0047] Figure 15 illustrates that mature T cells develop from iPSC-derived HSPCs in HA-ProG-DLL4 hydrogel crosslinked with both PEG-Tz and gelatin-Tz (HA-ProG-DLL4-PEG-G). (a) Schematic overview and timeline of experiments to assess T cell phenotype and function, (b) Flow cytometry analysis of CD5(+)CD7(+) Pro-T cells generated after budded HSPCs are cultured in the HA-ProG- DLL4-PEG-G hydrogel system for 14 days. Flow cytometry is representative of three replicates, (c) Frequencies of CD5(+)CD7(+) Pro-T cells generated from different batches of HSPCs encapsulated in HA-ProG-DLL4-PEG hydrogel with and without VCAM1 (n=3). (d) Flow cytometry analysis of cells expressing CD4, CD8b, CD3 and TCRap generated in HA-ProG-DLL4-PEG-G hydrogel on day 21 . Flow cytometry is representative of three replicates, (e) Frequencies of cells expressing CD4, CD8b, CD3 and TCRap generated in HA-ProG-DLL4-PEG-G hydrogel on day 21 (n=3). (f) Flow cytometry analysis of cells expressing CD4, CD8b, CD3 and TCRap generated in HA-ProG-DLL4-PEG-G hydrogel on day 28. Flow cytometry is representative of three replicates, (g) Flow cytometry analysis of cells expressing CD4, CD8b, CD3 and TCRap generated in HA-ProG-DLL4-PEG-G hydrogel on day 21 followed by activation and expansion with CD3 and CD28 Immunocult reagent for an additional 14 days outside the hydrogel. Flow cytometry is representative of three replicates, (h) Flow cytometry analysis of mature CD8b(+) SP and CD3(+)TCRaP(+) T cells generated in HA-ProG-DLL4-PEG-G hydrogel on day 21 followed by activation and expansion with CD3 and CD28 Immunocult reagent for an additional 14 days outside the hydrogel (n=3). Data represent mean ± SD for 3 biological replicates. * p < 0.05; ** p < 0.01 ; *** p < 0.001.

[0048] Figure 16 illustrates that T cells generated in HA-ProG-DLL4-G-PEG-G hydrogel from PSC- derived HSPCs are functional and capable of cytokine secretion. CD4(+)CD8(+) DP T cells generated in HA-ProG-DLL4-G-PEG-G hydrogel were harvested on day 21 followed by activation and expansion with CD3 and CD28 Immunocult reagent for an additional 14 days outside the hydrogel. Resulting CD8(+) SP T cells were cultured with CD3 and CD28 Immunocult reagent for an additional 7 days. CD3(+) cells were subsequently assayed for production of effector proteins in the presence or absence of nonspecific PMA / ionomycin stimulation. Intracellular flow cytometry is representative of three stimulation replicates. Data represent mean ± SD for 3 biological replicates.Detailed Description of the Invention

[0049] Broadly, the present invention provides a novel approach to developing and using hydrogels (3D hydrogels) to mimic niche in vivo microenvironments and to direct cell differentiation and expansion. In some aspects of the invention, the hydrogel comprises a biocompatible polysaccharide polymer, preferably one that is present or mimics those found in the ECM of the tissue desired to be mimicked, conjugated to a suitable ligand (e.g. Notch ligand, integrin ligand or other desired ligand), optionally by using a fusion protein linker (such as Protein A or Protein G) through click chemistry (such as norbornene / methyltetrazine click chemistry), and wherein the biocompatible polysaccharide is also crosslinked with a biocompatible crosslinker polymer and / or stiffening agent through click chemistry (e.g. norbornene / methyltetrazine), wherein the crosslinker (e.g. PEG and / or gelatin and / or other suitable crosslinkers) and stiffening agent (e.g. gelatin) are the same or different. The components of the hydrogel can be mixed in solution with cells (such as HSPCs) so that when the hydrogel forms, the cells are encapsulated within the hydrogel. The HSPCs can be derived from a variety of sources, including cord blood (CB) and induced pluripotent stem cells (iPSCs). As the hydrogel is porous, it can be placed in suitable culture media for instance to promote differentiation and / or expansion and / or viability which will reach the encapsulated cell(s). In some aspects, the hydrogels of the invention promote the formation of cell spheroids within the hydrogel. Once the desired cells are produced, the cells can be recovered by removing the hydrogel, for example using enzymes and / or hydrolysis and recovering or selecting for the desired cell(s), such as by selecting cells with certain cell markers.

[0050] More particularly in certain aspects, the present invention provides a functionalized three dimensional (3D) hydrogel for promoting HSPCs to differentiate to T cells. In some aspects the invention provides a novel hydrogel (both in solution liquid form and in solid gel form) and hydrogel composition and methods for producing T cell precursor and / or progenitor cells and / or T cells, including mature human T cells in vitro. In contrast to existing platforms, the 3D hydrogel of the invention, in some aspects, allows for use of serum-free media and culturing the developing T cells without need for coculture with xenogeneic feeder cells.

[0051] In other aspects, the hydrogel platform is biocompatible and functionalized with critical components of the extracellular matrix required for T cell development. Encapsulation of cells in the 3 D hydrogel under these conditions also allows and results in the formation of 3D spheroids thus providing cell-cell interactions that promote T cell development. Further, the hydrogel - based platform in some aspects of the invention is formed in manner to more closely mimic the thymic microenvironment, that promotes the development of T cells (versus myeloid cells). As a result, the hydrogel-based platform of the invention mimics the thymic microenvironment, producing T cells and / orT cell progenitors and / or precursors that may be suitable for research and / or clinical use, such as generating CAR T cells for clinical use.

[0052] In yet other aspects, the invention provides an easy and efficient way to form functionalized hydrogels using click chemistry wherein one can make adjustments to the hydrogel components (clicking in ligands, cross-linkers, adjusting size and ratios) to achieve desired stiffness and other properties to mimic features of a physiological tissue.

[0053] Definitions

[0054] Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4thed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et ai, Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties.

[0055] As used herein “biocompatability” refers to the quality of not having toxic or injurious effects on biological systems.

[0056] As used herein “click chemistry” or “click reaction” refers to an approach to chemical synthesis that emphasizes efficiency, simplicity, selectivity, and modularity in chemical processes used to join molecular building blocks. It includes both the development and use of "click reactions", a set of simple, biocompatible chemical reactions that meet specific criteria like high yield, fast reaction rates, and minimal byproducts. Several reactions have been identified that fit the concept including: [3+2]cycloadditions, such as the Huisgen 1 ,3-dipolar cycloaddition, in particular the Cu(l)-catalyzed stepwise variant, which are are often referred to simply as Click reactions; Thiol-ene reaction; Diels-Alder reaction and inverse electron demand Diels-Alder reaction; [4+1] cycloadditions between isonitriles (isocyanides) such as norbornene and tetrazines such as methyltetrazines; nucleophilic substitution especially to small strained rings like epoxy and aziridines; carbonyl-chemistry-like formation of ureas but not reactions of the aldol type due to low thermodynamic driving force; addition reactions to carbon-carbon double bonds like dihydroxylation or the alkynes in the thiol-yne reaction; and Sulfur (VI) Fluoride exchange.

[0057] As used herein, “crosslinking” refers to forming a polymeric network or matrix in a hydrogel by either physical or chemical means. Hydrogels can be crosslinked using a variety of methods, including but not limited to chemical crosslinking using chemical agents as in click chemistry and photocrosslinking using photosensitive polymers such as methacrylate.

[0058] As used herein “functionalized hydrogels” are biomaterials that are created by combining hydrogels with other functional materials to mimic the properties of biological tissues. For example, in one aspect, the present invention provides a hydrogel that is fabricated to better mimic the ECM of the thymus to promote HSPC differentiation to commit to cells to T cell lineage by comprising immobilized DLL4. In some other aspects, the hydrogel has other features that promote T cell differentiation and production.

[0059] As used herein “gelatin” is a biopolymer which is prepared by thermal denaturalization of collagen, which is often sourced from animal skin and bones and connective tissue (such as cattle, chicken, pigs, and fish), in the presence of dilute acids. Gelatin consists of a large number of glycine, proline, and 4-hydroxy proline residues. It is an irreversibly hydrolyzed form of collagen and is often referred to as “hydrolyzed collagen”.

[0060] As used herein “hydrogel” refers to a three-dimensional (3D) structured networks of crosslinked hydrophilic polymer matrices capable of holding a large amount of water (> 10%by definition) and displaying useful characteristics such as softness, toughness, biocompatibility, stretchability, and deformability. The crosslinking among the hydrophilic functionalities facilitates their structural integrity and prevents their immediate dissolution in the aqueous environment. Their ability to entrap and preserve a substantial amount of water or biological solutions, and the unique combination of softness and flexibility are similar to natural soft tissues, and thus make them promising materials to mimic their properties. (Dodda et al., 2023). Hydrogels can transition from liquid to gel state under various conditions such as temperature, pH, light and pressure. Natural polymers for hydrogel preparation include hyaluronic acid, chitosan, heparin, alginate, gelatin and fibrin. Common synthetic polymers includepolyvinyl alcohol, polyethylene glycol, sodium polyacrylate, acrylate polymers and copolymers thereof. Whereas natural hydrogels are usually non-toxic, and often provide other advantages for medical use, such as biocompatibility, biodegradability, antibiotic / antifungal effect and improve regeneration of nearby tissue, their stability and strength is usually much lower than synthetic hydrogels. There are also synthetic hydrogels that can be used for medical applications, such as polyethylene glycol (PEG), polyacrylate, and polyvinylpyrrolidone (PVP). Chemical hydrogels - covalent bonds. Physical hydrogels usually have high biocompatibility, are not toxic, and are also easily reversible by simply changing an external stimulus such as pH, ion concentration (alginate) or temperature (gelatin).

[0061] As used herein, “process” and “method” are used interchangeably, as are “step” and “phase”.

[0062] As used herein, the cells used are cells of any subject (as defined below), including human cells. The invention is not necessarily limited to human cells, and other cell species that have similar cell differentiation and development may work.

[0063] As used herein, a “hemogenic endothelial cell”, “hemogenic endothelium cell” or “HEC” is a specialized subset of developing vascular endothelial cell that acquires hematopoietic potential and can give rise to multilineage hematopoietic stem and progenitor cells (HSPCs). In the present invention, the presence of HECs was identified by the following cell surface marker profile: CD34(+) and CD43(-).

[0064] As used herein, “pluripotent stem cells” or “PSCs” are cells that can self-renew. Self-renewal is the capacity of the stem cells to divide indefinitely, producing unaltered cell daughters maintaining the same properties of the parent cell. In particular conditions or under specific signals, a stem cell is able to exit from self-renewal and engage a program leading to differentiation into specialized cell types deriving from the three germ layers (ectoderm, endoderm, and mesoderm). In general, there are two types of PSCs, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). ESCs are derived from the inner cell mass (ICM) of preimplantation embryos and can be indefinitely maintained and expanded in the pluripotent state in vitro. Pluripotent stem cells can also be obtained by inducing dedifferentiation of adult somatic cells through an in vitro technology, known as cell reprogramming. Similar to ESCs, iPSC can be expanded indefinitely and are capable of differentiating in all the derivatives of the three germ layers.

[0065] As used herein, “stem cell(s)” are undifferentiated cells that can divide to produce some offspring cells that continue as stem cells and some cells that are destined to differentiate (become specialized). They can differentiate into more specialized cells but also have the capacity for selfrenewal. Stem cells are an ongoing source of the differentiated cells that make up the tissues and organs of animals and plants. Stem cells include pluripotent stem cells (PSCs) such as embryonic stem cells(ESCs) and induced pluripotent stem cells (iPSCs), multipotent stem cells such as cord blood (CB) stem cells and adult stem cells, which are found in various tissues.

[0066] As used herein, “progenitor cell(s)” descend from stem cells that then further differentiate into specialized cell types (one or more types of cells). They are more specific than a stem cell and can be pushed to differentiate into its "target" cell. There are many types of progenitor cells throughout the human body. Each progenitor cell generally is only capable of differentiating into cells that belong to the same tissue or organ and typically do not have the ability for self-renewal.

[0067] As used herein, a “blood progenitor” or “blood progenitor cell” is a hematopoietic cell that has properties of either a hematopoietic stem cell (HSC) or a hematopoietic progenitor cell (HPC, collectively HPSCs). “Blood progenitor cell” may be used interchangeably with the terms “hematopoietic stem / progenitor cell” or “hematopoietic stem and progenitor cell” (HSPC). In the present invention, the presence of blood progenitor cells I HSPCs was identified by the following cell surface marker profile: CD34(+)CD43(+). Such cells have multilineage developmental capacity and are capable of differentiating into cells belonging to multiple hematopoietic lineages including, but not limited to, myeloid cells, erythroid cells, megakaryocytes, lymphoid cells, mast cells, basophils and eosinophils. Such cells may or may not be capable of self-renewal.

[0068] As used herein, “precursor cell(s)” are an intermediate cell before they become differentiated after being a stem cell. Usually, a precursor cell has the capacity to differentiate into only one cell type.

[0069] As used herein, “progenitor T cells”, “precursor T cells” and “progenitor and precursor T cells” refer to the intermediate cell types derived (directly or indirectly) from a PSC, a CD34(+) HSPC or other stem cell that have the capacity to differentiate into one or more types of mature T cells. As used herein, progenitor and precursor T cells include the following cell types, listed according to the stages of T cell maturation from least to most differentiated (Figure 7a): a. “early T cell progenitors” or “early T cell progenitor cells” or “ETPs”, which were identified in the present invention by the following cell surface marker profile: CD7(+); b. “T cell lineage-specific progenitor cells” or “lineage-specific progenitor T cells” or “Pro-T cells”, which were identified in the present invention by the following cell surface marker profile: CD7(+)CD5(+); c. “T cell lineage-committed precursor cells” or “lineage-committed precursor T cells” or “Pre-T cells”, which were identified in the present invention by the following cell surface marker profile: CD7(+)CD5(+)CD1a(+);d. “initial single positive” or “ISP” T cells, which were identified in the present invention by the following cell surface marker profile: CD3(-)CD4(+)CD8(-); and e. “double positive” or “DP” T cells, which were identified in the present invention by the following cell surface marker profile: CD3(+)CD4(+)CD8(+).

[0070] As used herein, a “mature T cell” is a T cell that has developed its own T cell receptor (TCR) or expresses an engineered TCR on its cell surface. In the present invention, mature T cells were identified as CD3(+)TCRaP(+) cells that were “single positive” or “SP”, additionally displaying either one of the following cell surface marker profiles: CD4(+)CD8(-) or CD4(-)CD8(+). A mature SP T cell may also express a chimeric antigen receptor (CAR) and may be a CAR-expressing T cell engineered to lack TCR. Mature T cells may also be identified by the cell surface marker profile: CD8(+)CAR(+).

[0071] As used herein, “isolated” means non-naturally occurring cells or cell populations not themselves found in nature, for instance in a different cellular environment, culture or media that is not found in nature and that has utility in isolated form that is not present in naturally occurring non-isolated cells or cell populations. For instance, cells produced by the methods of the present invention such as the resulting or obtainable progenitor, precursor and / or mature T cells can, in one embodiment, be isolated by cell sorting / isolation methods known in the art, and / or by selecting for their characteristic one or more cell surface markers.

[0072] As used herein, “aggregating”, “aggregated” or “aggregation of” PSCs into 3D multicellular structures refers to PSCs that naturally, or when induced by culture media conditions, aggregate and form 3D multicellular structures. The groups of multiple cells adhere to each other but do not adhere to the cell culture vessel. In some embodiments, the aggregates are more than one, more than two, more than five or generally from 10-1000 cells. See for instance llngrin et al., 2008.

[0073] As used herein, “dissociating” cells means using mechanical or enzymatic methods to separate multicellular aggregates into single cells, for instance using a cell dissociation enzyme such as trypsin,

[0074] As used herein, “isolating or harvesting cells using genetic expression and / or cell surface markers that are characteristic of cell type to be isolated” includes, but is not necessarily limited to binding cells comprising the cell surface marker with antibodies conjugated to fluorescent molecules, or conjugated to molecules that can be bound by magnetic or paramagnetic beads, and subsequently selecting for cells with the desired cell surface marker, for instance by fluorescence-activated cell sorting or binding the cells to a substrate.

[0075] As used herein, “subjecting the HSPCs to a media formulation that directs the cells to differentiate to T cells, progenitor and / or precursor T cells” means the use of growth media that contains factors which lead to their differentiation, for instance the media described in the Examples.

[0076] As used herein, “culturing the cells within the 3D hydrogel under conditions that promote lymphoid specification (lymphopoiesis), differentiation into progenitor and precursor! cells and T cell maturation” means the use of growth media that contains factors sensed by hematopoietic cells which lead to their differentiation into progenitor and precursor T cells and / or mature T cells, for instance in some embodiments comprising or consisting of a common basal media such as IMDM, RPMI, aMEM, SFEM II or StemPro34 and supplemented with IL-7, Flt3L and one or more of the additional factors SCF, TPO, CXCL12, TNF-a, IL3 or other additional small molecules such as apoptosis inhibitors, metabolites, vitamins, bovine serum albumin, human serum albumin, or additional components.

[0077] When cells are cultured in “media” or “staged media”, the invention is not limited to the media and stages described herein. A component may be substituted or supplemented with another component or components of similar function and purpose within the media, and amounts and timing of stages can vary accordingly. Further, “base media” or “basal media” as used herein may be any base media that can be any media designed and optimized and suitable for growing and / or culturing the particular cell type(s) or known to support the culturing and / or growth of particular cell types.

[0078] As used herein, “xenogeneic” refers to denoting, relating to, or involving tissues or cells belonging to individuals of different species, whereas “non-xenogeneic” has the converse meaning.

[0079] As used herein, “feeder-free” refers to culture conditions that are free from cells of other sources, for instance in reference to culturing human pluripotent stem cells and derivatives and not adding cells to the media from other sources other than hematopoietic stem cells, and “serum-free " refers to culture conditions designed to grow a specific cell type or perform a specific application in the absence of serum and is non-xenogeneic.

[0080] As used herein, a "defined culture medium" refers to a chemically defined formulation comprised solely of chemically defined constituents. A defined medium may include constituents having known chemical compositions. Medium constituents may be synthetic and / or derived from known nonsynthetic sources. For example, a defined medium may include one or more growth factors secreted from known tissues or cells. However, the defined medium will not include the conditioned medium from a culture of such cells. A defined medium may include specific, known serum components isolated from an animal, including human serum components, but the defined medium will not include serum. Anyserum components provided in the defined medium such as, for example, bovine serum albumin (BSA), are preferably substantially homogeneous.

[0081] As used herein, “hyaluronic acid” includes hyaluronic acid and its salts, such as sodium hyaluronate, potassium hyaluronate, magnesium hyaluronate and calcium hyaluronate. Hyaluronic acid is a polymer of disaccharides, which are composed of D-glucuronic acid and N-acetyl-D-glucosamine, linked via alternating p-(1— >4) and p-(1— >3) glycosidic bonds. Hyaluronic acid can be 25,000 disaccharide repeats in length. Polymers of hyaluronic acid can range in size from 5,000 to 20,000,000 Da in vivo. In some preferred embodiments, the hyaluronic acid of the invention is from about 150 or 151 to about 300 kDa.

[0082] As used herein, a “signaling molecule” that “can activate or enhance activation of the Notch signaling pathway” includes but is not limited to Notch ligands DLL1 , DLL3, DLL4, JAG1 and JAG2 and integrin ligands. In a preferred embodiment of the invention, said ligands are selected from Notch ligands such as DLL4 and integrin ligands such as gelatin, VCAM1 and others. The Notch ligands such as DLL4 interact with and activate Notch receptors. The integrin ligands, which in some embodiments is VCAM1 , are cell adhesion molecules that bind to the integrin a4pi dimer, promoting interaction between the cell and the functionalized surface, thereby enabling additional interaction between the cell and the surface-bound Notch ligand such as DLL4. Thus, the Notch and integrin ligands together enhance activation of the Notch signaling pathway when they come into contact with the cell surface. The present inventors have previously shown synergistic effects of immobilized or adsorbed DLL4 and VCAM1 ligands in the production of HPSCs, progenitor T cells and mature T cells (Michaels et al., 2022).

[0083] As used herein, "Delta-like-4", "DL4", “DLL4” and "Notch ligand DL4" refer to a protein that in humans is encoded by the DLL4 gene. DL4 is a member of the Notch signaling pathway and is also referred to in the art as "Delta like ligand 4" and "DLL4". Herein, reference to DLL4 is not limited to the entire DL4 protein but includes at least the signaling peptide portion of DLL4. For example, a commercially available product (Sino Biological) comprising the extracellular domain (Met 1-Pro 524) of human DLL4 (full-length DLL4 accession number NP_061947.1 ; SEQ ID NO: 1) fused to the Fc region of human IgG 1 at the C-terminus is a DL4 protein suitable for use herein.

[0084] As used herein, "Vascular cell adhesion molecule 1", "VCAM-1" and “VCAM1” refer to a protein that in humans is encoded by the VCAM1 gene. VCAM1 is a cell surface sialoglycoprotein, a type I membrane protein that is a member of the Ig superfamily. VCAM1 is also referred to in the art as "vascular cell adhesion protein 1” and “cluster of differentiation 106” (CD106). Herein, reference to VCAM1 is not limited to the entire VCAM1 protein, but includes at least the signaling peptide portion of VCAM1 (QIDSPL (SEQ ID NO: 2) or TQIDSPLN (SEQ ID NO: 3)). For example, a commerciallyavailable mouse VCAM-1 Fc chimeric protein (R&D) that comprises (Phe25-Glu698) region of mouse VCAM-1 (full-length murine VCAM-1 accession number CAA47989; SEQ ID NO: 4) fused with the Fc region of human IgG 1 is a VCAM-1 protein suitable for use herein. Use of at least a portion of human VCAM-1 (full-length human VCAM-1 accession number P19320, NP001069, EAW72950; SEQ ID NO: 5) may also be suitable for use in the method provided herein.

[0085] As used herein, “integrin(s)” refer to a superfamily of cell adhesion receptors that bind to extracellular matrix (ECM) ligands, cell-surface ligands and soluble ligands. They are transmembrane op heterodimers and at least 18 a and eight subunits are known in humans, generating 24 heterodimers. On ligand binding, integrins transduce signals into the cell interior; they can also receive intracellular signals that regulate their ligand-binding affinity.

[0086] As used herein, the terms “administration”, “administering” and variants thereof refer to introducing a composition or agent (e.g., nucleic acids such as cDNA) into a subject and includes concurrent and sequential introduction of one or more compositions or agents. For example, administration can refer to therapeutic, pharmacokinetic, diagnostic, research, placebo and experimental methods. Administration also encompasses in vitro and ex vivo treatments. Administration may be carried out by any suitable route, including orally, pulmonarily, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally or subcutaneously), rectally, intralymphatically, intratumorally or topically. Administration includes self-administration and the administration by another. A suitable route of administration allows the composition or the agent to perform its intended function. For example, if a suitable route is intravenous, the composition is administered by introducing the composition or agent into a vein of the subject.

[0087] As used herein, “carrier” includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions.

[0088] The phrase “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce a toxic, an allergic or similar untoward reaction when administered to a host.

[0089] As used herein, the term “exogenous” is meant to refer to a substance present in a cell or organism other than its native source. The term “exogenous” as used herein can refer to a nucleic acid (e.g., encoding a polypeptide) or a polypeptide that has been introduced by a process into a biological system such as a cell or organism in which it is not normally found, and where one wishes to introducethe nucleic acid or polypeptide into such a cell or organism. Alternatively, “exogenous” can refer to a nucleic acid or a polypeptide that has been introduced by a process into a biological system such as a cell or organism in which it is found in relatively low amounts and one wishes to increase the amount of the nucleic acid or polypeptide in the cell or organism (e.g., to create ectopic expression or levels). In contrast, as used herein, the term “endogenous” refers to a substance that is native to the biological system or cell in which it is found.

[0090] The term “in vivo" refers to assays and processes that occur in or within an organism, such as a multicellular animal. In some of the aspects described herein, a method or use can be said to occur “in vivo" when a unicellular organism, such as a bacterium, is used. The term “ex vivo” refers to methods and uses that are performed using a living cell with an intact membrane that is outside of the body of a multicellular animal or plant (e.g., explants, cultured cells including primary cells and cell lines, transformed cell lines, extracted tissue or cells including blood cells, among others). In some preferred aspects of the invention, the methods of producing blood progenitor cells and cells derived therefrom, such as progenitor T cells and T cells, are done ex vivo.

[0091] The term “in vitro” refers to assays and methods that do not require the presence of a cell with an intact membrane, such as cellular extracts, and can refer to the introducing of a programmable synthetic biological circuit in a non-cellular system, such as a medium not comprising cells or cellular systems, such as cellular extracts.

[0092] The term “subject” as used herein refers to a human or animal to whom treatment, including prophylactic treatment, is provided. Usually, the animal is a vertebrate such as but not limited to a primate, rodent, domestic animal or game animal. Primates include but are not limited to chimpanzees, cynomolgus monkeys, spider monkeys and macaques (e.g., Rhesus). Rodents include but are not limited to mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include but are not limited to cows, horses, pigs, deer, bison, buffalo, feline species (e.g., domestic cat), canine species (e.g., dog, fox and wolf), avian species (e.g., chicken, emu and ostrich) and fish (e.g., trout, catfish and salmon). In certain aspects described herein, the subject is a mammal (e.g., a primate or a human). A subject can be male or female. Additionally, a subject can be an infant or a child. In some embodiments, the subject can be a neonate or an unborn subject (e.g., in utero). Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of diseases and disorders. In addition, the methods and compositions described herein can be used for domesticated animals and / or pets. A human subject can be of any age, gender, race or ethnic group (e.g., Caucasian (white), Asian, African, black, AfricanAmerican, African European, Hispanic, Mideastern, etc.). In some embodiments, the subject can be a patient or other subject in a clinical setting. In some embodiments, the subject is already undergoing treatment.

[0093] As used herein, the terms “suppress”, “decrease” “interfere”, “inhibit”, “reduce” and like terms generally refer to the act of reducing, either directly or indirectly, a concentration, level, function, activity or behavior relative to the natural, expected or average condition, or relative to a control condition.

[0094] As used herein, the terms “treat”, “treating” and “treatment” include abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical symptoms of a condition, substantially preventing the appearance of clinical symptoms of a condition and / or obtaining beneficial or desired clinical results. Treating further refers to accomplishing one or more of the following: (a) reducing the severity of one or more disorders; (b) limiting development of symptoms characteristic of one or more disorders being treated; (c) limiting worsening of symptoms characteristic of one or more disorders being treated; (d) limiting recurrence of one or more disorders in patients that have previously had the disorder(s); and (e) limiting recurrence of symptoms in patients that were previously symptomatic for one or more disorders.

[0095] “Beneficial or desired clinical results”, such as pharmacologic and / or physiologic effects, include but are not limited to the prevention of a disease, disorder or condition from occurring in a subject that may or may not be predisposed to the disease, disorder or condition but does not yet experience or exhibit symptoms of the disease (prophylactic treatment); alleviation of symptoms of a disease, disorder or condition; diminishment of extent of a disease, disorder or condition; stabilization (not worsening) of a disease, disorder or condition; preventing spread of a disease, disorder or condition; delaying or slowing progression of a disease, disorder or condition; amelioration or palliation of a disease, disorder or condition; and combinations thereof, as well as prolonging survival as compared to expected survival if not receiving treatment.

[0096] As used herein, an “effective amount” is an amount sufficient to produce a desired effect (e.g., enhance cell culture, differentiation into various cell types or inhibition of expression of a target gene or sequence). Suitable assays for measuring expression of a target gene or sequence include, for example, examination of protein or RNA levels using techniques known to those of skill in the art such as dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation and enzyme function, as well as phenotypic assays known to those of skill in the art.

[0097] As used herein, “therapeutic effect” refers to a consequence of treatment, the results of which are judged to be desirable and beneficial. A therapeutic effect can include, directly or indirectly, thearrest, reduction or elimination of a disease manifestation and / or the progression of a disease manifestation.

[0098] As used herein, the terms “therapeutic amount(s)”, “therapeutically effective amount(s)”, an “amount effective” and “pharmaceutically effective amount(s)” of an active agent are used interchangeably to refer to an amount that is sufficient to provide the intended benefit of a treatment. However, dosage levels are based on a variety of factors, including the type of injury, the age, weight, sex and / or medical condition of a patient, the severity of a condition, the route of administration and the particular active agent employed. Thus, the dosage regimen may vary widely but can be determined routinely by a physician or healthcare professional using standard methods. Additionally, the terms “therapeutic amount(s)”, “therapeutically effective amount(s)” and “pharmaceutically effective amount(s)” include prophylactic or preventative amounts of the compositions of the described invention. In prophylactic or preventative applications of the described invention, pharmaceutical compositions or medicaments are administered to a patient susceptible to or otherwise at risk of a disease, disorder or condition in an amount sufficient to eliminate or reduce the risk, lessen the severity or delay the onset of the disease, disorder or condition, including biochemical, histologic and / or behavioral symptoms of the disease, disorder or condition, its complications and intermediate pathological phenotypes presenting during development of the disease, disorder or condition. It is generally preferred that a maximum dose be used, that is, the highest safe dose according to some medical judgment. The terms “dose” and “dosage” are used interchangeably herein.

[0099] For any therapeutic agent described herein, a therapeutically effective amount may be initially determined from preliminary in vitro studies and / or animal models. A therapeutically effective dose may also be determined from human data. The applied dose may be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximal efficacy based on the methods described herein and other well-known methods is within the capabilities of the person of ordinary skill in the art. General principles for determining therapeutic effectiveness may be found in Chapter 1 of Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th Edition, McGraw-Hill (New York) (2001), which is incorporated herein by reference.

[0100] As used herein, the terms “comprising”, “comprise”, “contain” and variations thereof are used in reference to compositions, methods and respective components thereof that are essential to the method or composition, yet open to the inclusion of unspecified elements, whether essential or not.

[0101] As used herein, the term “consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment.

[0102] As used herein, “consisting of” refers to compositions, methods and respective components thereof that are exclusive of any element not recited in that description of the embodiment.

[0103] As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural references, such as “one or more” unless the context requires otherwise. For example, references to “the method” includes one or more methods and / or steps of the type described herein and / or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described herein. The abbreviation, “e.g.” is derived from the Latin exempli gratia and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example”. With respect to elements described as one or more within a set, it should be understood that all combinations within the set are contemplated. If aspects of the invention are described as “comprising” a feature, embodiments also are contemplated that include “consisting of or “consisting essentially of” the feature.

[0104] As used herein, the terms “such as”, “for example” and the like are intended to refer to exemplary embodiments and not to limit the scope of the present disclosure.

[0105] Unless otherwise defined herein, scientific and technical terms used herein shall have the meanings that are commonly understood by persons of ordinary skill in the art to which the invention and / or disclosure pertains.

[0106] Other than in the operating examples or where otherwise indicated, all numbers expressing quantities of ingredients, molecular weight, reaction conditions and so forth as described herein should be understood as modified in all instances by the term “about”. Unless otherwise indicated, the term “about” when used in connection with percentages can mean ±1%. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0107] Notwithstanding that the numerical ranges and parameters setting forth the scope of the invention are approximations, the numerical values set forth in the specific operating examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0108] Practical implementation may include any or all of the features described herein. These and other aspects, features and various combinations may be expressed as methods, apparatus, systems, means for performing functions, uses and in other ways, combining the features described herein.

[0109] Features, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example unless incompatible therewith. All of the features disclosed herein (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The invention is not restricted to the details of any examples or embodiments described herein. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings) or to any novel one, or any novel combination, of the steps of any method or process disclosed.I. Functionalized hydrogels

[0110] Hydrogels are crosslinked networks of hydrophilic polymers that have been extensively used for 3D cell culture (Oliveira & Reis, 2008). They have the potential to closely mimic ECM stiffness and intricate 3D cell-cell and cell-matrix interactions of native organs (Xue et al., 2022). In some aspects, present invention provides a hydrogel that is functionalized to mimic the microenvironment and ECM of the thymus.

[0111] In some aspects, the invention provides a 3D hydrogel to promote the differentiation and / or expansion of cells, such as HSPCs, HSCs, HPCs or combinations thereof in a method to produce progenitor and precursor T cells, or in other embodiments mature T cells.

[0112] Generally, the invention provides a hydrogel comprising: a first biocompatible polymer that is a polysaccharide polymer; a second biocompatible polymer; a Fc fusion protein binding agent; and at least one Fc-fusion protein ligand conjugated to the Fc fusion protein binding agent, wherein the first biocompatible polymer is conjugated to (i) the second biocompatible polymer and (ii) the Fc fusion protein binding agent through click chemistry. The first and second biocompatible polymer and Fc fusion protein binding agent are modified to enable such conjugation through click chemistry. In some aspects, the hydrogel can be in a liquid and / or a gelled state, wherein in the gelled state. In yet some other embodiments, the hydrogel is seeded with at least one cell, which is encapsulated within the hydrogel in a three dimensional structure. In some other aspects, the hydrogel is seeded with about 500,000 cells per 1 mL hydrogel. In some other aspects, the hydrogel is ceded with about 300,000 to about 2,000,000cells / ml of hydrogel. In a preferred embodiment of the invention, the seeded cells are HSPC, HSC, HPC or combinations thereof. In some other aspects, the cells are derived from cord blood (CB), using methods known in the art. In yet other aspects, the cells are derived from iPSCs, using methods known in the art.

[0113] Generally, in some aspects, the 3D hydrogel of the invention comprises a first biocompatible polymer that is a polysaccharide polymer, at least one second biocompatible polymer and at least one signalling molecule immobilized directly or indirectly to the first and / or second polymers, wherein the first biocompatible polymer is crosslinked to the at least one second biocompatible polymer through click chemistry, and wherein the first and second biocompatible polymers are modified to enable crosslinking through click chemistry.

[0114] In some aspects, the 3D hydrogel components are selected to mimic the 3D structure, microenvironment, and / or stiffness of the ECM of the thymus. In some aspects, the hydrogel stiffness is determined by the relative amounts of the biocompatible polymers and corresponds to a stiffness of a region of a human thymus. In some other aspects, the 3D hydrogel stiffness has a Young modulus of 2.5 to 3.5 kPa.

[0115] In some embodiments of the invention, the first polymer may comprise synthetic or natural polysaccharides including but not limited to alginate, chitosan, cellulose or hyaluronic acid (HA) or salts or esters thereof. HA is a natural polysaccharide that is found in thymic and bone marrow extracellular matrix (Goncharova et al., 2012; Lins, 2022). It is a commonly used polymer for hydrogel systems due to its biocompatibility, and abundance of functional groups, such as carboxyl, hydroxyl, and N-acetyl groups, for functionalization or crosslinking (An et al. , 2021). Moreover, studies have shown that addition of HA into hydrogel culture of HSPCs improves their proliferation, while gelatin-based 3D microgels have been used to co-culture OP9-DLL4 with murine HSCs to produce progenitor T cells (Vallmajo- Martin et al., 2020; Suraiya et al., 2020). As such, in a preferred embodiment the first polymer of the hydrogel is a hyaluronic acid (HA) or salt or ester thereof. In some aspects, the hyaluronic acid is a hyaluronic acid salt selected from the group consisting of sodium hyaluronate, potassium hyaluronate, magnesium hyaluronate and calcium hyaluronate. In some aspects, the first biocompatible polymer is a hyaluronic acid salt that is about 150 to about 300 kDa. In another embodiment it is a modified hyaluronic acid to enable linkage to a second polymer and an Fc fusion protein binding agent through click chemistry.

[0116] In some aspects, the at least one second biocompatible polymer is selected from one or more of a natural polymer and / or a synthetic polymer, such as a synthetic polymer that is inert to degradation from cellular enzymes. In some other aspects, the at least one second biocompatible polymer acts asboth (i) a crosslinker and (ii) one or more of a hydrogel stabilizer, a stiffening agent, a cell adhesion molecule, a RGD-based polymer that promotes cell adhesion and / or migration and a polymer that has an integrin receptor binding cite, such as RGD. In yet some other aspects, the at least one second biocompatible polymer is a synthetic polyvinyl alcohol, polyethylene glycol (PEG), sodium polyacrylate, acrylate polymers and copolymers, salts or esters thereof, an about 5 to about 6 kDA synthetic polymer, or a combination of any of the foregoing.

[0117] In some embodiments, the at least one second biocompatible polymer is a gelatin, a PEG or a combination thereof. In other embodiments, the at least one second biocompatible polymer is a gelatin, which may be about 40 to about 70 kDa. A person of skill in the art will understand that other biocompatible polymers with varying properties will be suitable.

[0118] In some embodiments, the signaling molecule may be an Fc-fusion protein, wherein the 3D hydrogel further comprises an Fc fusion protein binding agent, wherein the first biocompatible polymer is conjugated to the Fc fusion protein binding agent through click chemistry, and wherein the first biocompatible polymer and Fc fusion protein binding agent are modified to enable such conjugation through click chemistry. In some aspects, norbornene-tetrazine (e.g., methyltetrazine) click chemistry is used to crosslink the first and second biocompatible polymers. In some aspects, the first polymer is modified with at least two norbornenes and the second polymer and the Fc fusion protein binding agent are each modified with at least one methyltetrazine.

[0119] In some embodiments the click chemistry used in the formation of hydrogels of the invention is a norbornene (Nb)-tetrazine (such as methyltetrazine) ligation, wherein the first polymer (e.g. HA) is modified with norbornene (e.g. 5-norbornene -2 methyl amine) to produce HA-norbornene. As HA is a polymer, in some embodiments from 150 - 300KDa, there will be many norbornene molecules on the HA polymer (e.g. in some embodiments 1 .5 equivalents of norbornene as compared to HA), each being potential sites for norbornene-tetrazine (methyltetrazine) click chemistry for conjugating with either the second polymer or the Fc fusion protein binding agent, each of later, being modified with a tetrazine such as a methyl tetrazine such as methyltetrazine-sulfo-NHS ester. In some embodiments, about 24 ug ProG per 1 mg of HA is used. Thus the modified first polymer is conjugated through click chemistry to the modified Fc fusion protein binding agent though a norbornene-methyltetrazine linkage and also conjugated the modified second polymer through a norbornene-methyltetrazine linkage.

[0120] In some embodiments the second polymer is a cross-linking moiety. The crosslinking agent can be selected from a variety of natural or synthetic crosslinkers. In some embodiments the second polymer cross linker is a synthetic polymer, such as polyethylene glycol (PEG), including but not limited to about 5 to about 6 kDa PEG. A person skilled in the art will understand that broader ranges will be suitable. Insome embodiments it is a multi-arm PEG, such as a 4-arm PEG, e.g. a bioinert 4-arm PEG, or in some other embodiments a 6-arm PEG. In some aspects the PEG is a multi-arm PEG derivative selected from the group consisting of PEG-amine , PEG-azide, PEG-thiol, PEG-acrylamide, PEG-maleimide, PEG- NHS ester, PEG-hydroxyl, PEG-DBCO, PEG acid, PEG-aldehyde, methoxy-PEG, PEG-acrylate, biotin- PEG, cholesterol-PEG, PEG-epoxide, PEG-hydrazide, PEG diacrylate and salts thereof, or in other embodiments it is a PEG-amine or salt thereof.

[0121] In one embodiment, the second biocompatible polymer is PEG due to its convenience, flexibility, and ability to prevent protein adsorption and cell adhesion. This selection allows for the investigation of the consequences of introducing bioactive elements to the biomaterial (Moore & West, 2019). As such, in some preferred embodiments, the modified second polymer is a PEG-tetrazine (or PEG - methyltetrazine). In some embodiments the PEG is present is present from 0 to about 16.8 mg / mL hydrogel. In some other embodiments the modified 4-arm PEG is a 4-arm PEG-amine has a concentration of about 126 mg per 1 mL of 3D hydrogel. In yet other embodiments the PEG amine or salt thereof is modified with a methyltetrazine acid.

[0122] In some other embodiments, the second polymer may be a cross-linker. In some embodiments it may also be a stiffening agent, such as an adhesion molecule, including but not limited to gelatin, fibronectin and its derivatives, collagen and its derivatives, laminin and its derivatives and P selectin. Inclusion of adhesion molecules into T cell differentiation systems has been shown to enhance differentiation (Shukla et al., 2017a; Edgar et al., 2022; Iriguchi et al, 2021). Gelatin hydrogels found their application in tissue engineering due to their biocompatibility, cell adhesion features and ease of modification (K. Han et al., 2021). Moreover, gelatin is derived from denaturation of collagen, which is one of the most abundant ECM proteins in the human body and is responsible for activating pro-survival signaling pathways in various cell types (Castagnaro et al., 2018; Gendron et al., 2003). While gelatin has been shown to support murine HSC differentiation to T cells progenitors, gelatin has not been used for human T cell development (Suraiya et al., 2020). As such, in some preferred embodiments, the modified second polymer is a gelatin-tetrazine (or gelatine-methyltetrazine). In some embodiments, the gelatin is a about 60 kDa. In other embodiments it is from about 20 to about 100 kDa, in others from about 40 to about 70kDa. In some other embodiments the gelatin is present in the hydrogel from about 1 - about 10% w / v, in other embodiments from about 2 - about 10%. In yet some other embodiments from about 2 - about 5%, in yet some other embodiments at about 5% w / v of hydrogel.

[0123] In yet some other embodiments, the hydrogel may comprise more than one type of second polymer, such as it can comprise a synthetic polymer, a natural polymer or both, or more particularly in some embodiments, it can comprise a PEG, a gelatin or both, each being modified with a tetrazine (e.g.a methyltetrazine) to enable click linkage to the modified first polymer. In some embodiments, the one or more second polymer comprise separately or in combination from 0 to about 10% w / v of the hydrogel. The amount of natural (e.g. gelatin) and synthetic (e.g. PEG), second polymers can vary and be adjusted to obtain the desired stability and stiffness. In another embodiment when the HSPCs are derived from cord blood, the second polymer can be a PEG, a gelatin or both. In yet another embodiment when the HSPCs are derived from iPSCs, then the at least one second polymer comprises both gelatin and PEG.

[0124] In a preferred embodiment of the present invention for the generation of progenitor and precursor T cells and mature T cells, the stiffness of the hydrogel would mimic that of the ECM of the Thymus. In some embodiments the Young Modulus is about 2.5 - about 3.5 kPa, in some other embodiments about 2.8 -about 3.3, in yet some other embodiments about 3.0, in yet some other embodiments about 3.1 + / - 1.74kPa.

[0125] In some embodiments the Fc fusion protein binding agent is present to bind to a signalling ligand to immobilize the signalling ligand, such as a Notch signaling ligand or an integrin or adhesion ligand, selected to functionalize the hydrogel to achieve the desired cell differentiation and expansion. In a preferred embodiment, the Fc Fusion protein binding agent is Protein G (ProG) or Protein A (ProA), in some other embodiments it is ProG. As such the Fc Fusion Protein binding agent binds to a FC fusion protein such as at least one human Fc fusion signaling protein, which is a signaling protein fused to the human immunoglobulin Fc domain. The at least one signaling ligand is selected from the group consisting of one or more Notch ligands, one or more integrin ligands and a combination thereof, wherein the one or more Notch ligands are selected from the group consisting of DLL1 , DLL3, DLL4, JAG1 , JAG2 and a combination thereof, but more preferably DLL4 or recombinant human DLL4. In some other aspects the notch ligand (DLL4) is present at a range of about 50 - about 200 ug / mL of hydrogel, or in some embodiments from about 50 - about 100 ug / mL of hydrogel. In some other aspects, the one or more integrin ligands are selected from the group consisting of VCAM1 , ICAM1 , vitronectin, fibrinogen, fibronectin and its derivatives, collagen and its derivatives, laminin and combinations thereof.

[0126] The Fc fusion protein binding agent ProG is modified by tetrazine (methyltetrazine, or more specifically modified by methyltetrazine-sulfo-NHS ester or a suitable methyltetrazine ester) to conjugate through click chemistry to the modified first polymer (e.g. HA-norbornene). In some embodiments the ProG-Tz has a concentration of about 24 pg per 1 mg of hyaluronic acid.

[0127] Previous studies suggested that depending on cell type, specific ECM stiffness can enhance Notch signaling, which plays a crucial role in T cell development (Kretschmer et al., 2023; Safaee et al., 2017). Additionally, the inventors have previously shown VCAM1 to increase Notch signaling of differentiating HSCs in 2D culture systems and drive stem cells towards T cell fate (Shukla et al., 2017a,Michaels et al., 2022). Previous studies have explored the use of cell adhesion-promoting proteins such as VCAM1 and retronectin to enhance T cell differentiation from HSPCs (Iriguchi et al., 2021 ; Michaels et al., 2022). One embodiment of the present invention adopts a novel approach, incorporating gelatin with Notch ligand-functionalized hydrogels for accelerated T cell differentiation. While gelatin hydrogels are known to enhance differentiation of some encapsulated cells, including neural and angiogenic differentiation from mesenchymal stem cells (Lee et al., 2017; Yao et al., 2021), gelatin hydrogels have not been reported to affect human T cell development.

[0128] In yet some embodiments the hydrogel of the invention is in liquid or gelled form. In yet other embodiments in gelled form.

[0129] In yet some embodiment, the invention provides a compositions of various components and combination of components of the hydrogel, such as: the modified first polymer (e.g. HA-norbornene), the modified second polymer (e.g. PEG-Tz or gelatin-Tz), modified FC fusion protein binding agent (e.g. ProG-Tz), FC fusion protein binding agent and the FC fusion protein (e.g. ProG-Fc- DLL4) or modified FC fusion protein binding agent and the Fc fusion protein (e.g. mTz-ProG-Fc-DLL4). In other embodiments, HA-norbornene-ProG-Tz with or without DLL4-Fc and / or HA-norbornene conjugated to one or more modified second polymers. The components can be added to solution under conditions to form the hydrogel with desired properties. In yet other aspects, the invention provides a solution of the liquid hydrogel and seed cells (HSPCs), as well as the gelled hydrogel with encapsulated cells, and or spheroids.

[0130] Very generally, in some aspects the hydrogel comprises:First Polymer Second Polymer Immobilized ligandHA (or other Immobilized notch polysaccharide) Gelatin / collagen PEG ligand50-200 mg / mL hydrogel0.75 - 1.1 % w / v 1-10% w / v (or 2-10% or 2-5% or 5%) 0-2% w / v 0.005-0.02% w / v

[0131] The present invention further comprises methods of making the 3D hydrogels as described herein.

[0132] In one aspect, the method of making the 3D hydrogel of the present invention comprises (i) a crosslinking step, wherein a first biocompatible polymer is crosslinked with at least one secondbiocompatible polymer to form a 3D hydrogel matrix, and wherein the crosslinking may be achieved using chemical means, such as click chemistry, or physical means, such as photo-crosslinking; and (ii) a signalling molecule immobilization step, wherein at least one signalling molecule is immobilized within the 3D hydrogel matrix by directly or indirectly conjugating the at least one signalling molecule to the first biocompatible polymer and / or the at least one second biocompatible polymer.

[0133] In one aspect, the first biocompatible polymer and the at least one second biocompatible polymer are modified to enable the crosslinking step, to enable the signalling molecule immobilization step, or to enable both the crosslinking step and the signalling molecule immobilization step.

[0134] In another aspect, the signalling molecule is modified to enable the signalling molecule immobilization step. In one embodiment, a binding agent is used to immobilize the signalling molecule within the 3D hydrogel matrix, and the binding agent is modified to enable conjugation with the first biocompatible polymer and / or the at least one second biocompatible polymer. In yet another aspect, the signalling molecule is a human Fc-fusion protein and the binding agent is a protein that binds with high affinity to the Fc-fusion protein, such as Protein A or Protein G.

[0135] In one embodiment, the biocompatible polymers and / or the binding agent are modified with click chemistry reactive groups. In another embodiment, norbornene-tetrazine click chemistry is used. For example, in one embodiment, the first biocompatible polymer is modified with a norbornene group and the at least one second biocompatible polymer is modified with a methyltetrazine group. In another embodiment, the binding agent is modified with a click chemistry reactive group, such as a norbornene group or a methyltetrazine group.

[0136] In one aspect, the first biocompatible polymer, the at least one second biocompatible polymer and the signalling molecule are mixed in a solution to form the 3D hydrogel in a liquid state; the at least one cell may be seeded in the liquid state 3D hydrogel, the at least on cell selected from the group consisting of hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPCs) and a combination thereof (i.e., HSPCs); and then the 3D hydrogel becomes a gelled state, thereby encapsulating the at least one seeded cell.II. Method for producing T cells and their progenitors using the compositions and hydrogels of the invention

[0137] The invention provides a method for in vitro T cell development, such as human T cell development, comprising: providing the 3D hydrogel of the invention and seeding the 3D hydrogel with HSCs, HPCs or HSPCs. In some aspects of the invention the cells are human cells. In other aspectsthe cells are cord blood derived or iPSC derived cells. The hydrogel is in a form (liquid, gel or other) to enable it to encapsulate the cells in the 3D gelled form of the gel.

[0138] In some aspects of the invention the method involves culturing the cells within the 3D hydrogel under conditions that promote lymphoid specification (lymphopoiesis), differentiation into progenitor and precursor T cells and T cell maturation, generating [at least one cell type selected from the group consisting of] CD7(+) early T cell progenitors (ETPs), CD7(+)CD5(+) lineage-specific Pro-T cells, CD7(+)CD5(+)CD1a(+) lineage-committed Pre-T cells, initial single positive (ISP) CD4(+) cells, double positive (DP) CD4(+)CD8(+) cells, mature single positive (SP) CD4(+) or CD8(+) T cells capable of cytokine secretion and combinations thereof.

[0139] In some other aspects, the cells are seeded in the 3D hydrogel at a cell density of about 300,000 - about 2,000,000 cells per 1 mL of 3D hydrogel. In another aspect, the cells are seeded at a density of about 500,000 cells per 1 mL of 3D hydrogel. In yet another aspect, the cells are seeded in the 3D hydrogel when the hydrogel is in a liquid state.

[0140] In some aspects of the invention, the culturing conditions are non-xenogeneic, feeder-free and serum-free; and the conditions comprise a defined culture medium.

[0141] In some aspects, the conditions comprise culturing the cells in a basal media supplemented with at least one cytokine. In some other aspects the basal media is selected from the group consisting of IMDM, RPMI, aMEM, SFEM II, StemPro34, XVIVO 15 and any suitable media designed and optimized for culturing human stem cells; and the at least one cytokine is selected from the group consisting of IL- 7, Flt3L, SCF, TPO, CXCL12, TNF-a, IL3, other small molecules including apoptosis inhibitors, metabolites, vitamins, bovine serum albumin and human serum albumin and combinations thereof.

[0142] In some other aspects of the method the cells are human umbilical cord blood (CB)-derived hematopoietic stem cells (HSCs) and the method comprises culturing the cells within the 3D hydrogel, such as the gelled form of the hydrogel comprising: a. a first culture stage, wherein the conditions comprise a basal media supplemented with SCF, IL7, TPO and Flt3L for 2 to 3 weeks; and b. a second culture stage, wherein the conditions comprise a basal media supplemented with IL7 and Flt3L for an additional 3 to 10 weeks.

[0143] The method in some embodiments for CB derived cells, further comprising: dissolving the 3D hydrogel after the second culture stage using an enzyme solution and / or hydrolysis depending on thesecond polymer; isolating CD4(+)CD8(+) DP T cells generated after the second culture stage; transferring the CD4(+)CD8(+) DP T cells to a two-dimensional (2D) substrate coated with DLL4 and VCAM1 ; and culturing the CD4(+)CD8(+) DP T cells in a basal media supplemented with IL7, Flt3L, a CD3 / CD28 activator and IL- 15 for 2 weeks to generate mature CD8(+) SP T cells and CD3(+)TCRaP(+) T cells.

[0144] In some other embodiments the cells are pluripotent stem cell (PSC)-derived hematopoietic stem and progenitor cells (HSPCs). In other aspects they are human cells. In other aspects, wherein culturing the PSC-derived HSPCs within the 3D hydrogel comprises: a first culture stage, wherein the conditions comprise a basal media supplemented with SCF, IL7, TPO and Flt3L for about 1 to 2 weeks; and a second culture stage, wherein the conditions comprise a basal media supplemented with IL7 and Flt3L for an additional about 2 to 10 weeks. In other aspects the method, further comprising: dissolving the 3D hydrogel after the second culture stage using an enzyme solution; isolating CD4(+)CD8(+) DP T cells generated after the second culture stage; transferring the CD4(+)CD8(+) DP T cells to a 2D substrate coated with DLL4 and VCAM1 ; and culturing the CD4(+)CD8(+) DP T cells in a basal media supplemented with IL7, Flt3L, a CD3 / CD28 activator and IL-15 for 2 or more weeks to generate mature CD8(+) SP T cells and CD3(+)TCRaP(+) T cells.

[0145] In yet some other aspects, the cells are cultured under hypoxic conditions to enhance differentiation and / or expansion.III. Cells produced by / obtainable from the methods and use of the hydrogels of the present invention.

[0146] Culturing HSPCs, such as cord blood derived or iPSC derived HSPCs, encapsulated in the functionalized hydrogels of the present invention, wherein the hydrogels comprise immobilized Notch ligand, such as DLL4, promotes differentiation of the cells to progenitor and precursor T cells that have the capacity to further differentiate to mature T cells under suitable culture conditions either in the hydrogel or outside of the hydrogel. The invention is also includes the spheroids formed during cell culture while the cells are still within the hydrogel. The cells and spheroids generated by the methods of the present invention using the hydrogels may be harvested from the hydrogels, through enzymatic reactions and / or hydrolysis depending on the second polymer. In the case of natural polymers such as gelatin, enzymes can be used, while in the case of PEG, it can be removed through hydrolysis. The harvested cells can then as described herein be used for a variety of applications, including as source of donor derived: T cells and progenitor and precursor T cells.IV. Uses of the invention

[0147] The inventors have demonstrated that the hydrogels of the invention can be used in the development of mature and functional T cells from both cord blood-derived and induced hematopoietic stem cells. The encapsulation of stem cells within the hydrogel prior to differentiation may also be suitable as an injectable material for in vivo T cell delivery and in situ T cell generation.

[0148] The data described herein establishes proof of concept for use of this hydrogel platform to produce mature, functional T cells from a patient’s own stem cells. Further data has been generated supporting use of the hydrogel as an injectable material for in situ T cell production.

[0149] In some embodiments of the invention, the functionalized hydrogels of the present invention could be formulated into a composition that can be administered or transplanted to a subject in need. In some aspects the hydrogels comprise HSPC cells, that can differentiate and expand in vivo and enhance a subjects ability to produce T cells and progenitor and precursor T cells and / or spheroids of any of the foregoing. In some other aspects the progenitor and precursor T cells and T cells and spheroids comprising same produced can be harvested and used in immunotherapy, T cell transfer therapies and CAR-T therapy. In other aspects the HSPC cells in the hydrogels are derived from the subjects own cells. In other aspects, the hydrogels comprising T cells and progenitor and precursor T cells could be administered or transplanted into a subject. In other aspects, the cells and spheroids produced by the methods described herein may be used in research and therapy / drug development and for other uses described and contemplated herein. In further aspects, the hydrogels of the present invention could be used in 3D printing applications. The below expands on some of the utility of the present invention.

[0150] In contrast to existing differentiation platforms using 2D substrates, 3D hydrogel-based systems can be used to provide a platform to study 3D ECM effects on T cell development and can replicate crucial elements of the thymic microenvironment required for T cell development, namely the thymus’s 3D architecture and complex ECM composition which determine stiffness and intricate cell-cell and cellmatrix interactions.

[0151] In contrast to existing platforms, the hydrogel composition of the invention provides a feeder- free and serum-free non-xenogeneic platform that allows for clinically useful T cell development from CB-derived and PSC-derived blood progenitor cells (HSPCs). The hydrogel composition of the invention can therefore potentially be used as both a T cell development and delivery platform for various T cell therapies by injecting the biomaterials (e.g., T cells and / or their progenitors and / or precursors encapsulated in the hydrogel) into a patient.

[0152] Furthermore, the hydrogel composition of the invention comprises biocompatible polymers, allowing the hydrogel composition to be used for T cell production in vivo. In an embodiment, the hydrogel composition is an artificial 3D thymic niche that may be used as a foundation for a platform to direct T cell production from a patient's own blood progenitor cells when injected in vivo. A similar approach was previously explored using alginate cryogels functionalized with Notch ligand (Shah et al., 2019). In contrast to the previously reported approach, the hydrogel of the present invention allows for direct encapsulation of cells and does not rely on attracting cells to migrate into the hydrogel. Moreover, while the previously reported alginate cryogel generated lymphoid progenitors, it did not demonstrate capability to produce mature T cells, unlike the hydrogel composition of the present invention which is capable of producing mature T cells.

[0153] The invention has substantial commercial potential. Mature functional T cells and / or their progenitors derived from the methods of the present invention can be used in various therapies, including cell replacement and immunotherapies. They can also be a source of genetically modified T cells. T cells derived from a patient’s own blood and engineered to recognize a target present on tumour cells are currently being used as potent treatments for hematological cancers. Despite their efficacy, these personalized or autologous treatments are extremely expensive and this has drastically limited their widespread adoption. PSCs have the capacity for unlimited growth and thus provide a renewable and inexpensive source of starting material for producing blood progenitor (HSPC)-derived T cells and / or their progenitors. Producing progenitor T cells and T cells from PSCs using the clinically compatible methods of the present invention could lower the cost of T cell therapy and make them more accessible. Cell therapy, including T cell immunotherapy represents a rapidly growing, multibillion dollar market with applications not only for hematological malignancy but for treating solid tumours, immunodeficiency and autoimmunity. The cells produced by or obtainable from the methods of the present invention can also be genetically modified and / or used for gene therapy. For example, using the biocompatible thymic niche-like hydrogel composition as a delivery reservoir in CAR HSPC transplantation may accelerate stem cell differentiation, allowing for more potent CAR T cells in cancer therapy (Agarwalla et al., 2022; Gschweng et al., 2014).

[0154] [ANY OTHER USES / APPLICATIONS. THE INVIVO APPLICATION MAY NOT BE SUPPORTED. ]EXAMPLES

[0155] The following examples are provided by way of illustration, not limitation.Methods and MaterialsModification of Protein G (ProG) with methyltetrazine

[0156] 10 mg of protein G (ProG) (ThermoFisher Scientific, Source: E. coli, MW -21.6 kDa, 98-100% purity) was dissolved in 1 mL phosphate-buffered saline (PBS) at room temperature. 4 mg of methyltetrazine-sulfo-NHS ester (Click Chemistry) was added to the ProG solution and stirred overnight at 4°C. Unreacted crosslinker molecule was removed by purifying the product of the reaction using 7 kDa spin desalting column (ThermoFisher Scientific) to obtain methyltetrazine-functionalized protein G (ProG-Tz). Purified ProG-Tz was characterized by using electrospray ionization mass spectrometry (ESI-MS).Synthesis of HA Hydrogel and Immobilization of DLL4 and VCAM1

[0157] HA was modified with reactive norbornene groups as described previously in Delplace et al. Nonswelling, ultralow content inverse electron-demand Diels-Alder hyaluronan hydrogels with tunable gelation time: Synthesis and in vitro evaluation. Adv Funct Materials 2020;30(14):1903978; doi: 10.1002 / adfm.201903978. 1 g of sodium hyaluronate (NaHA; Lifecore Biomedical, Source: bacterial fermentation, MW 151-300 kDa) was dissolved in 0.1 M 2-(N-morpholino) ethanesulfonic acid (MES) at pH 5.5 and reacted with 4-(4,6-dimethoxy-1 ,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM) (3 eq) for 30 minutes, followed by addition of 5-Norbornene-2-methylamine (1.5 eq). After 15 hours, the product of the reaction was purified by extensive dialysis (100 kDa Molecular Weight Cut-Off (MWCO) membrane) against 0.1 M NaCI (2 times per day, 1 day) and then deionized (DI) water (2 times per day, 4 days). The solution was lyophilized and stored at -20°C until further use. The degree of substitution was determined by1H NMR (500 MHz, D2O, 8).

[0158] HA-norbornene was reacted with purified ProG-Tz (5 eq) overnight at 4°C. After the reaction was completed, unreacted ProG-Tz was quenched with 5-Norbornene-2-carboxylic acid for 2 hours, followed by dialysis (100 kDa MWCO membrane) against 1x PBS buffer (2 times per day, 1 day) and then DI water (2 times per day, 4 days). The solution was lyophilized and stored at -20°C until further use. The amount of ProG immobilized on HA (HA-ProG) was quantified by amino acid analysis usingthe Waters Pico-Tag System and high-performance liquid chromatography (HPLC) (Waters Corporation, Milford MA).

[0159] HA samples containing ProG were hydrolyzed to individual amino acids using 6N HCI at 110°C for 24 hours. Excessive HCI was eliminated through evaporation under reduced pressure, and the hydrolyzed amino acids underwent derivatization using phenyl isothiocyanate (PITC). Subsequently, the amino acids labeled with phenyl groups were isolated, and samples were quantified by employing Pierce amino acid standard H. Separation was carried out using an ACQUITY UPLC BEH C18 column (2.1 mm X 10 cm) at 48°C. Detection of the signal occurred at 254 nm with an ACQUITY TUV detector, and the outcomes were managed using Waters Empower 2 chromatography software. For analysis purposes, glycine values were utilized, and each condition involved three gel replicates.

[0160] To conjugate DLL4-Fc (Sino Biological) or VCAM1-Fc (RnDSystems), HA-ProG was first dissolved at 25 mg / mL in StemSpan SFEM II media with cytokines (prepared as described elsewhere in the description), followed by addition and conjugation of DLL4-Fc or VCAM1-Fc overnight at 4°C.Synthesis of qelatin-methyltetrazine and PEG-methyltetrazine

[0161] To synthesize gelatin-methyltetrazine, 1 g of gelatin (Sigma-Aldrich, Source: cold water fish skin, MW 60 kDa) was first dissolved in 50 mL of 10x PBS. Then, 48.5 mg of methyltetrazine-sulfo-NHS ester was added to the solution and stirred overnight at 4°C. The product of the reaction was purified by extensive dialysis (12-14 kDa MWCO membrane) against DI water (2 times per day, 4 days). The solution was lyophilized and stored at -20°C until further use. The degree of substitution was determined by ultraviolet-visible (UV-Vis) spectroscopy.

[0162] The PEG crosslinker was modified with reactive methyltetrazine groups as previously described in Delplace et al., 2020. 0.29 g of methylphenyltetrazine acid (Click Chemistry Tools) was dissolved in 10 mL dichloromethane (Millipore Sigma) and activated with 0.16 g of N,N’-diisopropylcarbodiimide. 1 g of 4-arm PEG amine HCI salt (JenKem, MW 5.240 kDa) was added and stirred with 0.25 g of N,N- diisopropylethylamine (Sigma-Aldrich). After 72 hours, the solution was concentrated under vacuum and mixed with 20 mL of 1 :1 dimethylformamide (Sigma-Aldrich) and distilled water solution, followed by extensive dialysis (2 kDa MWCO membrane). The final product was lyophilized and stored at -20°C until further use.Mechanical properties

[0163] The compressive moduli of the hydrogels were measured using a Mach-1 micromechanical system (Biomomentum) connected to a Universal Motion Controller (Newport). Hydrogel samples of100 pL (0.32 cm2) were prepared in 16-well chamber slides at 37°C and equilibrated in PBS overnight before being removed from the chamber slides. Cylindrical samples were placed between two impermeable flat platens and maintained in 50 pL of PBS until they were tested to avoid evaporation (less than 5 minutes per sample) using a single-axis load cell (150 g, ATI Industrial Automation). The samples’ height (distance between the two platens) was measured using an initial force of 0.01 N. Uniaxial unconfined compression at 10% strain based on the gel height was applied to account for any surface defects. Sample compressive modulus was measured by applying a further 10% strain in 5 sequential 2% strain steps. The slope of resultant stress-strain curves for each sample was averaged to calculate the compressive modulus.T cell progenitor differentiation in hydrogel: transwell system

[0164] Standard 24-well plates were coated with DLL4-Fc and VCAM1-Fc solution at concentrations 15 pg / mL and 2.5 pg / mL, respectively, in 300 pL of PBS per well, resulting in a coating concentration of approximately 24 ng / mm2of DLL4-Fc and 4 ng / mm2of VCAM1-Fc. The coated plates were incubated either overnight at 4°C or for 3 hours at 37°C. Hydrogels with encapsulated CB-derived HSCs were placed on transwells (Corning) with a pore size of 8 pm, and the transwells were inserted into the well plates coated with DLL4-Fc and VCAM1-Fc or into uncoated well plates. 1 mL of StemSpan SFEM II media (STEMCELL Technologies) with 10% StemSpan lymphoid progenitor expansion supplement (STEMCELL Technologies) containing SCF, IL7, TPO and Flt3L was added to each well.Human cord blood (CB)-derived HSC sorting

[0165] Human CB was collected from consenting donors at hospitals according to institutional research ethics board policies. Density gradient centrifugation in Lymphoprep density gradient medium (STEMCELL Technologies) was used to isolate mononuclear cells. CD34(+) cells were isolated from CB using either EasySep Human CD34(+) Positive Selection Kit (STEMCELL Technologies) or MACS columns (Miltenyi Biotec) to >90% purity. Isolated CD34(+) HSCs were cryopreserved in fetal bovine serum (FBS) with 10% dimethyl sulfoxide (DMSO) and stored at -150°C. Prior to use, cells were thawed in a 37°C water bath, and 10x volume of 37°C Iscove’s Modified Dulbecco’s Medium + Bovine Insulin Transferrin (IMDM+BIT) was added dropwise. Cells were then centrifuged for 7 minutes at 300 g and washed with IMDM+BIT. This was followed by resuspending cells at the desired cell density in appropriate media.Generation of CD34(+) HSPCs from iPSCs

[0166] CD34(+) HSPCs were generated as described previously in Michaels et al., 2022. The human iPSC cell line iPS11 (ALSTEM Cell Advancements, derived from human foreskin fibroblasts) wascultured and maintained in serum-free mTeSR plus complete medium (STEMCELL Technologies) on Geltrex basement membrane extract (Life Technologies, A1413302). The growth medium was changed daily, and cells were maintained at 37°C, 5% CO2. The cells were grown to 90% confluency and dissociated into single cells with cell dissociation enzyme TrypLE Express for 3 minutes at 37°C. Dissociated cells were collected, counted and pelleted at 200 g for 5 minutes. Cells were resuspended in 2 mL of TO medium per well and deposited into AggreWell 400 six-well plates (STEMCELL Technologies, 34425) prepared with AggreWell Rinsing Solution (STEMCELL Technologies, 07010) according to the manufacturer’s instructions. Cells were seeded at a density of 1.26 x 106cells per well of a six-well plate and aggregated by centrifugation at 200 g for 5 minutes. For the duration of the CD34(+) induction, cells were cultured at 37°C in a hypoxic incubator at 5% CO2, 5% O2. CD34(+) induction was performed for 8 days. Preferred induction medium formulations are listed in Table 1.Table 1 : Media formulation used to promote induction of hemogenic endothelium from PSCs in AggreWells

[0167] 192 hours after initiating the CD34(+) induction, aggregates were collected and pelleted by centrifugation at 200 g for 5 minutes. Aggregates were dissociated in 3 mL of TrypLE supplementedwith deoxyribonuclease I (Millipore Sigma, 260913-1 OMU) for 10 to 15 minutes. Cells were pipetted to a single-cell suspension, washed and counted. CD34(+) cells were enriched using the CD34(+) positive selection kit (Miltenyi Biotec, 130-046-702) according to the manufacturer’s instructions. CD34(+)- enriched cells were cryopreserved using CryoStor CS10 (STEMCELL Technologies, 07930) for use in downstream culture.Endothelial to hematopoietic transition (EHT) culture

[0168] The CD34(+) cells generated above were used as input for EHT culture. The coating solution was prepared using sterile PBS combined with DLL4-Fc (15 pg / mL; Sino Biological, 10171-H02H) and VCAM1-Fc (2.5 pg / mL; R&D Systems, 643- VM). Tissue culture-treated 96-well plates (Fisher Scientific, 12-556-008) were precoated with 50 pL of coating solution overnight at 4°C. The coating solution was aspirated, and the plates were washed with PBS. CD34(+) enriched cells were resuspended in EHT medium (Table 2) at a concentration of 1 x 105cells / mL unless otherwise indicated. Cells (10,000 / 100 pL) were seeded onto each well of the 96-well plate unless otherwise indicated. Cultures were incubated at 37°C, 5% CO2 for 7 days, and nonadherent cells were harvested by gentle pipetting. Harvested cells were encapsulated into hydrogels for T cell differentiation.Table 2: Media formulation used to promote endothelial to hematopoietic transitionSorted CD34(+) HSPC encapsulation and in vitro culture

[0169] CD34(+) HSPCs were encapsulated in hydrogels at a cell density of 0.5 million cells per 1 mL of hydrogel. All polymers used for cell encapsulation were dissolved in StemSpan SFEM II media (STEMCELL Technologies) with StemSpan lymphoid progenitor expansion supplement (STEMCELL Technologies) containing SCF, IL7, TPO and Flt3L. Polymer solutions of 15 to 30 pL containing 0.75% of HA-ProG and 100 pg DLL4-Fc per 1 mL hydrogel and / or HA-DLL4-VCAM1 (at concentrations described in the operating examples) and different concentrations of gelatin-methyltetrazine were poured into cylindrical polydimethylsiloxane (PDMS) molds. After 15 minutes, hydrogels formed via methyltetrazine-norbornene crosslinking were placed in standard 48- or 96-well flat-bottom plates. Media was changed every 3 to 4 days according to Figure 12A for CB-derived HSPCs and according to Figure 15A for iPSC-derived HSPCs. In some embodiments, the lymphoid progenitor expansion supplement is replaced with StemSpan T cell progenitor maturation supplement (STEMCELL Technologies) containing IL7 and Flt3L but not SCF or TPO (Tabatabaei-Zavareh et al., 2017). Thehydrogels were dissolved at indicated time points with enzyme solution. The cells were subsequently recovered, washed with HF and prepared for further analysis.Verification of CB- and PSC-derived HSPCs to develop into T cell progenitors using 2D DLL4+VCAM1 system

[0170] Due to batch-to- batch variability in their ability to produce T cells, verification of cells’ potential to differentiate into T cells was performed (Figure 8: CB-derived; Figure 13c: PSC-derived). The DLL4- and VCAM1 -functionalized 2D system previously disclosed by the inventors in WO 2022 / 241558 A1 was used for this verification purpose. DLL4-Fc (Sino Biological) was diluted in PBS at 15 pg / mL along with VCAM1-Fc (R&D Systems) at 2.5 pg / mL. Standard flat-bottom 96-well plates were coated with 50 pL of prepared protein solution per well, resulting in a coating concentration of 24 ng / mm2of DLL4-Fc and 4 ng / mm2of VCAM1-Fc. Coated well plates were incubated either overnight at 4°C or for 3 hours at 37°C.

[0171] After incubation, each coated well was washed with 100 pL PBS twice and seeded with 10,000 CD34(+) CB- or PSC-derived HSPCs resuspended in 100 pL of StemSpan SFEM II media (STEMCELL Technologies) with 10% StemSpan lymphoid progenitor expansion supplement (STEMCELL Technologies) containing SCF, IL7, TPO and Flt3L. The media was exchanged every 3 to 4 days per the manufacturer’s recommendation. On day 14, the cells were collected and rinsed with PBS with Fc block and stained with Zombie UV viability dye (BioLegend) for 15 minutes at room temperature. Cells were stained with antibodies against Pro-T cell markers CD5 and CD7. The antibodies and dilutions used are listed in Table 3.Table 3: Antibodies used to verify ability of CB- and PSC-derived HSPCs to develop into T cell progenitorsCell imaging

[0172] For cell imaging with phalloidin / 4’,6-Diamidino-2-Phenylindole (DAPI), media was removed from cell culture, and hydrogels with encapsulated cells were washed with PBS three times with 5 minute intervals between washes. 4% paraformaldehyde in Dulbecco’s Phosphate-Buffered Saline (DPBS) wasadded to the hydrogels for 45 minutes followed by three washes with DPBS with 5 minute intervals between washes. This was followed by permeabilization of the cells inside the hydrogels with 0.1% Triton X-100 in DPBS for 20 minutes. Filamentous actin was stained with Alexa Fluor 488 conjugated to phalloidin for 45 minutes at 37°C and washed three times with DPBS. Actin staining was followed by staining of cell nuclei with DAPI (Invitrogen NucBlue) overnight at 4°C. After nuclei staining, hydrogels were washed three times with DPBS. Fluorescent images were obtained on a Nikon (Nikon AXR).Flow cytometry and antibodies

[0173] Surface marker staining for described experiments was performed with conjugated mouse antihuman antibodies (BD Biosciences). All samples were analyzed on a CytoFLEX LX cytometer (Beckman Coulter). On day 14 of culture, cells obtained from hydrogels were washed multiple times with Hanks’ Balanced Salt Solution (HBSS; Thermo Fisher Scientific) supplemented with 2% fetal bovine serum (FBS) (collectively, HBSS + FBS = HF buffer) and stained at 1 :300 dilution with antibodies against CD33, CD56, CD34, CD7, CD5 and CD1a for 20 minutes on ice. Cells were washed twice with HF, and dead cells were excluded using 7-AAD at 1 :1000 dilution. Alternatively, dead cells were excluded using Zombie LIV viability dye (BioLegend) at 1 :500 dilution in PBS for 15 minutes, followed by antibody staining. The antibodies and dilutions used are listed in Table 4.Table 4: Antibodies used to determine phenotype of cells differentiated from HSPCs in hydrogels

[0174] Cells that were assessed for their ability to generate mature T cells were collected on day 35 and rinsed with PBS with Fc block and stained with Zombie UV viability dye (BioLegend) for 15 minutes at room temperature. Cells were stained with antibodies against CD4, CD8, CD3 and TCRap. Cells were washed once and resuspended in HF buffer for analysis. The antibodies and dilutions used are listed in Table 5.Table 5: Antibodies used to assess phenotype and quantity of mature T cells generated from HSPCs in hydrogels

[0175] Flow data were analyzed and processed using CytExpert (Beckman Coulter) and FlowJo software and further analyzed in GraphPad Prism.Cytokine production

[0176] To assess IL-2, TNF-a and IFN-y production, T cells were harvested 7 days after initiation of expansion and were subjected to nonspecific stimulation. Cells were seeded at 50,000 to 100,000 cells / well of a 96-well plate in 200 pL of 37°C IMDM+BIT medium containing 25 ng / mL phorbol 12- myristate 13-acetate (PMA) and 1 pg / mL ionomycin (Sigma). After 1 hour, 3 pg / mL of brefeldin A was added, and cells were cultured for an additional 5 hours. Cells were fixed and stained for flow cytometry as described above.Statistical analysis

[0177] In all figure legends, n represents the number of independent biological replicates, and data are represented as mean ± standard deviation (SD) or mean ± standard error of the mean (SEM) as indicated. Statistical analysis was performed using GraphPad Prism software, and p-values were calculated by ordinary one-way or two-way ANOVA with Tukey's or Sidak’s multiple comparison tests to determine statistical significance. *p < 0.05; **p < 0.01 ; ***p < 0.001 and ****p < 0.0001 were considered significant.EXAMPLE 1 : Synthesis of hyaluronic acid-norbornene and polyethylene glycol-tetrazine hydrogel functionalized with protein G and Notch ligand for generating HSPC-derived Pro-T and Pre-T cells

[0178] Figure 1 is a schematic representation of a hydrogel composition used to mimic the thymic microenvironment for T cell production. The composition of the present invention comprises a serum-free feeder-free hyaluronic acid (HA)-based hydrogel functionalized with thymic components such as the DLL4 ligand to create a 3D thymic niche.

[0179] Methyltetrazine-norbornene click chemistry is used to control hydrogel crosslinking for cell encapsulation purposes due to its short reaction time, irreversibility, high specificity and the advantage of not necessitating a catalyst (Hansell et al., 2011). Furthermore, this reaction has previously been used for hydrogels with applications in 3D cell cultures, as the tetrazine-norbornene reaction demonstrates no effects on cell viability and is inert to the proteins (Delplace et al., 2020; Gopinathan & Noh, 2018; Koshy et al., 2016).

[0180] The hydrogel is functionalized with one or more Notch ligands. HSCs’ interaction with immobilized Notch ligand is essential for their commitment to the T cell lineage; therefore, DLL4, the most potent Notch ligand, is conjugated to HA hydrogels (Rutz et al., 2005). Previous studies have reported that immobilization of DLL4-Fc to HA functionalized with Protein G (ProG) facilitates cholangiocyte differentiation dependent on Notch signaling (Rizwan et al., 2022). Using ProG for Notch ligand immobilization makes the system more tunable and allows simultaneous immobilization of various signaling molecules. Thus, ProG is first conjugated to HA followed by immobilization of DLL4- Fc fusion protein. Additionally, this approach eliminates possible conformational changes of DLL4 during the synthesis.

[0181] First, HA is modified with 5-norbornene-2-methylamine, which may be characterized with1H NMR (Figure 2), followed by a reaction with protein G (ProG) modified with methyltetrazine using methyltetrazine-sulfo-NHS ester (Figure 3a-b). Modification of ProG with methyltetrazine [is I may be] verified using mass spectrometry prior to immobilization on HA-norbornene and may range from 1 to 4 tetrazine moieties per mole of ProG (Figure 4a). The amount of ProG immobilized per hydrogel may be measured using, for example, amino acid HPLC chromatography, and may comprise 24 pg ProG per 1 mg of HA. This ProG functionalized HA (HA-ProG) is modified with recombinant human DLL4-Fc via ProG affinity to Fc-containing proteins (Figure 3b). The hydrogel is crosslinked using gelatin modified with methyltetrazine-sulfo-NHS ester [and / or] 4-arm PEG amine modified with methyltetrazine acid (Figure 3c). The extent of modification with methyltetrazine may be confirmed using [UV-Vis spectroscopy or]1H NMR (Figures 4b, 5b).

[0182] When CB-derived CD34(+) HSCs are encapsulated at 500,000 cells / mL into either HA-ProG, HA-DLL4 or HA-ProG-DLL4 as single-cell suspensions, they form spheroids by day 7 of cell culture (Figure 6). All hydrogel formulations tested result in spheroid formation. The resulting encapsulated spheroids are not hollow, mimicking lobules in the thymus and resulting in maximized cell-cell interactions. The spheroids may lack interaction with the hydrogel.

[0183] Similar to T cell development in the native thymus and on DLL4- and VCAM1 -functionalized 2D substrates previously disclosed by the inventors in WO 2022 / 241558 A1 , HSCs encapsulated in the HA- ProG-DLL4 hydrogel differentiate to become CD7(+)CD5(+) T cell lineage-specific progenitor (Pro-T) cells and CD7(+)CD5(+)CD1a(+) T cell lineage-committed precursor (Pre-T) cells (Figure 7a-d). Figure 7b provides a schematic overview of the timeline and media used to promote differentiation of HSCs encapsulated in the hydrogels. Figure 7c shows a representative flow cytometry analysis of Pro-T and Pre-T cells generated from HSCs encapsulated in the HA-ProG-DLL4 hydrogel for 14 days with lymphoid progenitor expansion supplement containing SCF, IL7, TPO and Flt3L. Figure 7d shows that Pro-T cells are generated only in the HA-ProG-DLL4 hydrogel and not in HA-ProG or HA-DLL4 hydrogels.

[0184] Due to potential batch-to- batch variability in HSCs’ ability to produce T cells, verification of HSCs’ potential to differentiate into Pro-T cells may be performed using the DLL4- and VCAM1 -functionalized 2D substrates previously disclosed by the inventors in WO 2022 / 241558 A1 (Figure 8). Figure 8a shows a negative control wherein CB-derived HSCs are encapsulated in the HA-ProG hydrogel in the absence of DLL4 for 2 weeks. Figure 8b shows a positive control wherein CB-derived HSCs are cultured on a DLL4- and VCAM1 -functionalized 2D substrate for 2 weeks. Figure 8c represents CB-derived HSCs cultured in the HA-ProG-DLL4 hydrogel for 2 weeks.

[0185] Notably, as shown in Figure 8, the CB-derived HSCs cultured in the HA-ProG-DLL4 hydrogel (Figure 8c) generate greater proportions of CD7(+)CD5(+) Pro-T cells and CD7(+)CD5(+)CD1a(+) Pre- T cells than cells cultured on the DLL4- and VCAM1 -functionalized 2D substrate (Figure 8b), showing that the 3D hydrogel promotes enhanced Pro-T and Pre-T cell generation than the DLL4- and VCAM1- functionalized 2D substrate previously disclosed by the inventors in WO 2022 / 241558 A1. Furthermore, the CB-derived HSCs cultured in the HA-ProG-DLL4 hydrogel (Figure 8c) generate fewer CD33(+) myeloid cells than when cultured on the DLL4- and VCAM1 -functionalized 2D substrate (Figure 8b).

[0186] To determine a preferred amount of DLL4 to be added to HA-ProG for HSPC-derived T cell development, formulations with varying concentrations of DLL4-Fc may be assessed. Figure 7e shows that increasing the concentration of DLL4-Fc from 50 to 100 pg per mL of HA-ProG increases the output of Pro-T cells generated from CB-derived CD34(+) HSCs encapsulated in the hydrogels. However, further increasing the concentration of DLL4-Fc to 200 pg / mL does not further increase Pro-T cell output. Thus, in a preferred embodiment and in the operating examples disclosed herein, the hydrogel composition contains DLL4-Fc at a concentration of 100 pg / mLEXAMPLE 2: Gelatin enhances T cell commitment of CB-derived CD34(+) HSCs

[0187] Gelatin’s physico-chemical properties are similar to those of collagen; therefore, gelatin-based hydrogels may effectively imitate natural collagen-rich ECM. Accordingly, gelatin hydrogels have been shown to enhance cell viability and proliferation, including murine CD34(+) HSCs (Suraiya et al., 2020). Some studies have shown gelatin's ability to affect stem cell differentiation by providing ECM similar to the one where cells develop naturally (Yao et al., 2021 ; Y. Lee et al., 2017). In contrast to previous studies involving murine cells and / or non-hydrogel based systems (Lins, 2022), the inventors examined whether adding gelatin to the HA-ProG-DLL4 hydrogel increases in vitro human T cell differentiation by mimicking collagen-rich thymic ECM.

[0188] Gelatin is modified with methyltetrazine (Tz) groups and crosslinked with HA-norbornene by reacting gelatin with methyltetrazine-sulfo-NHS ester (Figure 9a). The degree of modification may be confirmed using UV-Vis spectroscopy with tetrazine absorbance at 524 nm. In a preferred embodiment, the degree of gelatin modification with Tz is 0.05 pmol of tetrazine per 1 mg of gelatin (Figure 4b).

[0189] To determine a preferred concentration of gelatin in the hydrogel composition for HSPC-derived T cell development, formulations of HA-ProG-DLL4 crosslinked to varying amounts of gelatin- methyltetrazine (G) may be synthesized and assessed. Figure 9b shows that increasing the gelatin content in the HA-ProG-DLL4-G hydrogels up to 5% significantly increases the output of Pro-T and / or Pre-T cells generated from CB-derived CD34(+) HSCs encapsulated in the hydrogels after 14 days of culture. Further increasing the gelatin content from 5 to 10% does not significantly affect differentiation but results in a more viscous solution with a shorter crosslinking time.

[0190] Previous studies have shown that Notch signaling can be influenced by ECM stiffness (Kretschmer et al., 2023; Safaee et al., 2017). Due to the role of Notch signaling in T cell development, the inventors determined whether the mechanical properties of the HA-ProG-DLL4-G hydrogel correlate with T cell differentiation by comparing the Young moduli of hydrogel formulations with varying levels of gelatin-methyltetrazine. Figure 9c shows that the Young modulus of hydrogels comprising 2% gelatin does not significantly differ from the Young modulus of hydrogels crosslinked with PEG (0% gelatin). Increasing the gelatin concentration above 2%, for example to 5 or 10%, generates stiffer hydrogels.

[0191] It is noteworthy that the hydrogel formulation comprising 5% gelatin, the optimal concentration for Pro-T and Pre-T cell differentiation from CB-derived HSCs encapsulated in the hydrogels, has a Young modulus of ~3.1 kPa (Figure 9c), which corresponds to a 3 kPa Young modulus of specific regions in the thymus (Asnaghi et al., 2021). A gelatin concentration of 5% may therefore improve T cell differentiation by increasing ECM stiffness to comparable in vivo thymic levels and / or increasing theamount of adhesion molecule interacting with the cells compared to formulations with lower concentrations of gelatin.

[0192] Thus, in a preferred embodiment and in the subsequent operating examples disclosed herein, the HA-ProG-DLL4-G hydrogel composition contains gelatin-methyltetrazine at a concentration of 5%.EXAMPLE 3: VCAM1 does not influence Pro-T and Pre-T cell yields generated from CB-derived CD34(+) HSCs in engineered hydrogels

[0193] VCAM1 synergizes with DLL4 to enhance Notch pathway activation and improve T cell differentiation in 2D differentiation platforms (Edgar et al., 2022; Michaels et al., 2022; Shukla et al., 2017). Figure 9d-e shows the proportions and yields of the cell types generated from CB-derived CD34(+) HSCs encapsulated for 14 days in the engineered hydrogels in the presence or absence of immobilized VCAM1-Fc with or without gelatin. The four hydrogel formulations include: HA-ProG-DLL4, HA-ProG-DLL4+VCAM1, HA-ProG-DLL4+G, and HA-ProG-DLL4+G+VCAM1. Flow cytometry may be used to quantify the cell populations generated after 14 days in the four hydrogel formulations.

[0194] Figure 9d shows that HSCs encapsulated in each of the four formulations differentiate into ETPs, Pro-T cells, Pre-T cells and myeloid cells. However, none of the formulations produce CD56(+) innate lymphoid cells (Figure 8c). The four formulations do not significantly vary in their ability to generate ETPs or CD33(+) myeloid cells, and most of the cells generated in each formulation are either Pro-T or Pre-T cells (collectively comprising at least 65% of cells generated by each formulation). Formulations containing gelatin significantly increase the proportion of generated cells that are Pre-T cells (Figure 9d) (p=0.0077 for HA-ProG-DLL4 vs. HA-ProG-DLL4+G, p=0.0133 for HA-ProG-DLL4 vs. HA-ProG- DLL4+G+VCAM1 , p=0.0146 for HA-ProG-DLL4+VCAM1 vs. HA-ProG-DLL4+G and p=0.0245 for HA- ProG-DLL4+VCAM1 vs. HA-ProG-DLL4+G+VCAM1) but do not significantly affect the combined yield of Pro-T and Pre-T cells within the hydrogels (Figure 9e), indicating that gelatin facilitates faster T cell commitment of encapsulated HSCs.

[0195] Conversely, despite the synergistic effects of DLL4 and VCAM1 on Notch activation and T cell differentiation in existing 2D differentiation platforms, adding VCAM1 to the hydrogel formulations does not significantly affect the proportion of generated cells that are Pre-T cells, nor the combined yield of Pro-T and Pre-T cells (Figure 9d-e). Thus, in a preferred embodiment and in the operating examples disclosed herein, VCAM1-Fc is not included in the HA-ProG-DLL4-G hydrogel composition when encapsulating CB-derived CD34(+) HSCs.EXAMPLE 4: Cells migrating out of the hydrogels develop into Pro-T cells in the presence of ligands that enhance Notch signaling

[0196] Cells derived from HSCs encapsulated in the engineered hydrogels begin to migrate out of the hydrogels on day 7 of culture. Figure 10a is a schematic representation of an experiment in which hydrogels with encapsulated HSCs are placed on transwells with a pore size of 8 pm that are inserted into well plates that are either uncoated or coated with DLL4 and VCAM1. This allows cells that migrate out of the hydrogel and pass through the transwell membrane to continue being cultured on 2D well plate surfaces in the presence or absence of DLL4 and VCAM1.

[0197] Figure 10b shows representative flow cytometry analysis of CD5 and CD7 expression on day 14 of culture for cells remaining inside the hydrogel, cells that migrate out of the hydrogel into wells coated with DLL4 and VCAM1 and cells that migrate into uncoated wells. Figure 10c shows the proportion of cells on day 14 of culture that are CD5(+)CD7(+) Pro-T cells, comparing cells that remain within the hydrogel, cells that migrate out of the hydrogel into wells coated with DLL4 and VCAM1 and cells that migrate out of the hydrogel into uncoated wells.

[0198] As shown in Figure 10c, the proportion of cells on day 14 of culture that are CD5(+)CD7(+) Pro- T cells is significantly lower among cells that migrate into uncoated wells compared to cells that remain in the hydrogel and compared to cells that migrate into wells coated with DLL4 and VCAM1. In contrast, cells that migrate into wells coated with DLL4 and VCAM1 generate Pro-T cells to the same extent as cells that remain in the hydrogel.

[0199] This experiment therefore confirms that encapsulated HSCs generate cells which have the potential to continue differentiating towards T cell lineage after migrating out of the hydrogel in the presence of ligands that enhance Notch signalling. This finding supports the in vivo and / or clinical application of the engineered hydrogel, for example, in therapies involving hydrogel-mediated delivery of T cells and / or their progenitors to target sites within a patient.EXAMPLE 5: HA-Gelatin hydrogels functionalized with thymic components support late stages of T cell development

[0200] The operating examples disclosed above show that encapsulating CB-derived HSCs in HA- ProG-DLL4-G hydrogels promotes and / or enhances differentiation into Pro-T and Pre-T cells after 2weeks of culture with a lymphoid progenitor expansion supplement containing SCF, IL7, TPO and Flt3L. To promote differentiation towards more mature stages of T cell development (Pre-T cells), encapsulated HSCs may be cultured for a longer duration whereby the lymphoid progenitor expansion supplement is replaced after 2 weeks with a T cell progenitor maturation supplement containing IL7 and Flt3L but not SCF or TPO.

[0201] Figure 11a shows the frequencies of Pro-T and Pre-T cells generated from HSCs encapsulated in HA-ProG-DLL4-G hydrogel over 4 weeks, wherein the lymphoid progenitor expansion supplement is replaced after 2 weeks with a T cell progenitor maturation supplement. Cell populations may be quantified using flow cytometry. After 4 weeks of culture in the HA-ProG-DLL4-G hydrogel, Pre-T cells comprise over 90% of the generated cells.

[0202] Figure 11c shows the frequencies of Pro-T and Pre-T cells generated from HSCs encapsulated in HA-ProG-DLL4-G hydrogel over 5 weeks, wherein the lymphoid progenitor expansion supplement is replaced after 3 weeks with a T cell progenitor maturation supplement. This 1-week delay in switching from expansion supplement to maturation supplement results in a corresponding 1-week delay in generating similar frequencies (-90%) of Pre-T cells. These findings indicate that Pro-T and Pre-T cells are generated at similar frequencies in HA-ProG-DLL4-G hydrogel until the lymphoid progenitor expansion supplement is replaced with a T cell progenitor maturation supplement, at which point the cells predominantly become Pre-T cells.

[0203] The previously described operating examples establish that VCAM1 does not affect T cell development during the first 2 weeks of culture in HA-ProG-DLL4-G hydrogel with the lymphoid progenitor expansion supplement. Figures 11 b and 11 d show that adding VCAM1-Fc to the HA-ProG- DLL4-G hydrogel similarly has no effect on the generation of Pre-T or Pro-T cells at later time points (after 4 and 5 weeks of culture, respectively) following the replacement of the expansion supplement with the maturation supplement (at 2 and 3 weeks of culture, respectively).

[0204] Thus, in a preferred embodiment, VCAM1-Fc is not included in the HA-ProG-DLL4-G hydrogel composition, and lymphoid progenitor expansion supplement is replaced with T cell progenitor maturation supplement after 2 weeks of culture (Figure 12a).EXAMPLE 6: Functional T cells develop from CB-derived CD34(+) HSCs in the functionalized HA- ProG-DLL4-G hydrogel

[0205] Figure 12a is a schematic representation of the timeline and culture conditions used for generating functional mature T cells from CB-derived HSCs encapsulated in the HA-ProG-DLL4-G hydrogel. In some embodiments using CB-derived HSCs, encapsulated cells are cultured with lymphoid progenitor expansion supplement containing SCF, IL7, TPO and Flt3L for 2 weeks, followed by T cell progenitor maturation supplement containing IL7 and Flt3L but not SCF or TPO for 3 weeks. In some embodiments, CD4(+)CD8(+) DP T cells are generated after 5 weeks of culture and then transferred to DLL4+VCAM1 coated plates and cultured for 2 weeks in media supplemented with a CD3 / CD28 activator and IL- 15 to generate functional mature CD3(+)TCRaP(+) and CD8(+) SP T cells.

[0206] Figure 12b shows the frequencies of ETPs, Pro-T cells, Pre-T cells and myeloid cells generated during the first 4 weeks of culture. By week 3, there is a decline in both myeloid (~5% of generated cells) and ETP (~4% of generated cells) populations. By week 4, the myeloid and ETP populations are entirely replaced by Pro-T and predominantly Pre-T cells (over 90% of generated cells). These results confirm that the HA-ProG-DLL4-G hydrogel supports late stages of T cell development.

[0207] As illustrated in Figure 7a, the latest stages of T cell development are characterized by initial expression of CD4 (ISP T cells), followed by co-expression of CD4 and CD8 (DP T cells) and finally single expression of CD4 or CD8 (SP T cells). DP and SP T cells also express CD3 and TCRap to varying extents. The mature SP T cells are capable of cytokine secretion.

[0208] Figure 12c shows a representative flow cytometry analysis of CD4, CD8b, CD3 and TCRap expression in cells generated from CB-derived HSCs encapsulated in the HA-ProG-DLL4-G hydrogel after 5 weeks of culture. Figure 12d shows the frequencies of ISP T cells, DP T cells and CD3(+)TCRaP(+) T cells generated after 5 weeks of culture. At week 5, the cells generated in the HA- ProG-DLL4-G hydrogel comprise ~23 to 43% ISP T cells, -29-52% DP T cells and -2.5 to 5.5% CD3(+)TCRaP(+) T cells.

[0209] At week 5, the majority of generated CD3(+)TCRaP(+) cells are DP T cells, which may be further differentiated to SP T cells. Previous studies have shown that generating mature SP T cells in a 3D platform requires long-term culture, for example using artificial thymic organoids to culture HSCs for 12 weeks (Seet et al., 2017). Due to the cell-cell interactions within the cell spheroids generated in the HA- ProG-DLL4-G hydrogel, the inventors hypothesized that a similar culture duration would promote selection and / or differentiation to CD8(+) SP T cells. Accordingly, Figure 12e-f shows representative flow cytometry analysis of CD4, CD8, CD3 and TCRap expression and frequencies of CD8(+) SP cellsand CD3(+)TCRaP(+) T cells generated from CB-derived HSCs after 12 weeks of culture in the HA- ProG-DLL4-G hydrogel. At week 12, the generated cells comprise -9 to 26% CD8(+) SP T cells and -3.75 to 8.75% CD3(+)TCRaP(+) T cells depending on donor. At week 12, generated cells do not express CD4.

[0210] Certain feeder-free systems utilize antibodies such as anti-CD3 and anti-CD28 to simulate positive selection and produce SP T cells. In contrast to previous studies requiring long-term culture (e.g., 12 weeks), the inventors demonstrate that differentiation to mature CD8(+) SP T cells may be expedited and / or enhanced by transferring DP T cells generated after 5 weeks in the HA-ProG-DLL4-G hydrogel to DLL4+VCAM1 coated plates and culturing for 1 or more weeks in media supplemented with a CD3 / CD28 activator and IL-15. At week 5, DP T cells may be extracted from the HA-ProG-DLL4-G hydrogel using enzyme solution containing 25 mg / mL hyaluronidase and 50 mg / mL collagenase in 1xPBS. The extracted DP T cells may then be transferred onto DLL4+VCAM1 coated plates and cultured in media supplemented with 12.5 pL / mL ImmunoCult CD3 / CD28 activator and 10 ng / mL IL-15 for an additional 2 weeks.

[0211] Figure 12g-h shows representative flow cytometry analysis of CD4, CD8, CD3 and TCRap expression and frequencies of CD4(+)CD8(+) DP, CD4(+) SP, CD8(+) SP and CD3(+)TCRaP(+) T cells generated from DP T cells extracted from the HA-ProG-DLL4-G hydrogel at week 5 and then cultured for 2 weeks on DLL4+VCAM1 coated plates in the presence of a CD3 / CD28 activator and IL- 15. These results confirm that this method successfully generates CD3(+)TCRaP(+) and CD8(+) SP T cells from CB-derived HSCs encapsulated in the HA-ProG-DLL4-G hydrogel.

[0212] To assess the functionality of mature T cells, T cells may be stimulated to produce cytokines with phorbol 12-myristate 13-acetate (PMA) and ionomycin. Brefeldin A may be used to accumulate the produced cytokines within the cells (preventing secretion), thus allowing flow cytometric analysis. Figure 12i-j shows representative flow cytometry analysis of cytokine production in cells that were cultured in the HA-ProG-DLL4-G hydrogel for 5 weeks, stimulated on DLL4+VCAM1 coated plates with CD3 / CD28 activator and IL-15 for 2 weeks and then stimulated with PMA and ionomycin for 6 hours, with brefeldin A added for the final 5 hours. As shown in Figure 12i-j, the stimulated cells derived from the method are capable of secreting IL-2, TNF-a and IFN-y, confirming the functionality of mature T cells generated in the HA-ProG-DLL4-G hydrogel.

[0213] These findings demonstrate that the functionalized HA-ProG-DLL4-G hydrogel enables serum- free, feeder-free production of functional mature T cells from CB-derived HSCs.EXAMPLE 7: Functional mature T cells develop from PSC-derived HSPCs in the functionalized HA-ProG-DLL4-PEG hydrogel

[0214] Herein, the inventors demonstrate a novel feeder-free, serum-free 3D platform for functional T cell generation from PSC-derived cells using a functionalized HA-ProG-DLL4-PEG hydrogel.

[0215] In a preferred embodiment, PSC-derived HSPCs are first generated in a blood induction step using the inventors’ previously reported protocol (Michaels et al., 2022). Briefly, human iPS11 cells undergo a hemogenic or blood induction step, wherein the cells are aggregated in AggreWell plates and cultured in staged CD34 induction conditions for 8 days (Figure13a, Table 1) at 37°C in a hypoxic incubator at 5% CO2 and 5% O2. Figure 13b shows representative flow cytometry analysis of cells generated in AggreWells after 8 days. The cells are then enzymatically and / or mechanically disaggregated into a single-cell suspension, and CD34(+) cells are enriched using, for example, magnetic cell isolation column. The CD34(+)-enriched cells which comprise hematopoietic endothelial cells (HECs) then undergo an endothelial-to-hematopoietic transition (EHT) step, wherein the HECs are transferred onto DLL4-Fc / VCAM1-Fc coated plates in EHT conditions (Table 2) for 7 days at 37°C with 5% CO2.

[0216] After EHT, budded HPSCs are collected and encapsulated into the functionalized hydrogel. Collected HSPCs may be validated for their ability to generate T cells using the inventors’ previously published 2D protocol using DLL4+VCAM1 coated plates (Figure 13c) (Michaels et al., 2022).

[0217] While the HA-ProG-DLL4-G hydrogel remains stable when culturing encapsulated CB-derived HSCs, this hydrogel formulation degrades within 7 days of culturing encapsulated PSC-derived HSPCs. Therefore, HA-ProG-DLL4-PEG hydrogel is used for culturing encapsulated PSC-derived HSPCs, since 4-arm PEG increases the crosslinking density of the hydrogel by increasing the methyltetrazine to norbornene ratio. Adding 2 L at 126 mg / mL of PEG crosslinker prevents hydrogel degradation.

[0218] Cells generated from PSC-derived HSPCs cultured in the HA-ProG-DLL4-PEG hydrogel for 2 weeks with lymphoid progenitor expansion media comprise -7.6% CD5(+)CD7(+) Pro-T cells (Figure 13 d, f). Thus, the frequency of Pro-T cells generated from PSC-derived HSPCs are lower than those from CB-derived HSCs in the engineered hydrogels (Figures 7c-e, 9d, 10c, 11a-b, 12b). This is consistent with existing 2D T cell differentiation systems which produce greater numbers of Pro-T cells using CB-derived HSCs (Edgar et al., 2022; Michaels et al., 2022). As demonstrated in Figure 11 , the media composition has a significant impact on the development of T cells from CB-derived HSCs encapsulated in the engineered hydrogels. Accordingly, replacing the lymphoid progenitor expansion supplement with T cell progenitor maturation supplement after 1 week of culturing PSC-derived HSPCsin the HA-ProG-DLL4-PEG hydrogel results in a substantial improvement in the generation of Pro-T cells, increasing from 7.6% to 29.71% (Figure 13d-f). Thus, in a preferred embodiment, lymphoid progenitor expansion supplement is replaced with T cell progenitor maturation supplement after 1 week of culture when encapsulating PSC-derived HSPCs in the HA-ProG-DLL4-PEG hydrogel (Figures 14a, 15a). This preferred media exchange results in differentiation kinetics within the hydrogels similar to those observed in the inventors’ previously reported 2D differentiation platform (Michaels et al., 2022).

[0219] In addition to media composition, DLL4 concentration and oxygen levels impact the development of T cells from PSC-derived HSPCs encapsulated within the DLL4-functionalized hydrogels. Figure 14b shows representative brightfield images of cells generated from PSC-derived HSPCs encapsulated for 2 weeks in DLL4-functionalized hydrogels with different oxygen conditions and concentrations of DLL4. Figures 14c-d show representative flow cytometry analysis and frequencies of generated CD5(+)CD7(+) Pro-T cells. As shown in Figure 14b, PSC-derived HSPCs generate fewer cells in hydrogels containing 50 pg DLL4 per mL of hydrogel compared to 100 pg / mL DLL4 after 2 weeks in both normoxic and hypoxic conditions. Under normoxic conditions, PSC-derived HSPCs also generate lower frequencies of Pro-T cells in hydrogels containing 50 pg / mL DLL4 compared to 100 pg / mL DLL4 (Figures 14c-d). However, under hypoxic conditions, the frequency of generated Pro-T cells does not differ between 50 pg / mL and 100 pg / mL DLL4, indicating that hypoxic conditions enhance the differentiation of PSC- derived HSPCs to Pro-T cells in DLL4-functionalized hydrogels.

[0220] Since PSC-derived HSPCs do not expand to the same extent as CB-derived HSCs, a greater hydrogel volume and number of encapsulated PSC-derived HSPCs are required to produce a sufficient number of cells for flow cytometric analysis. Surprisingly, increasing the hydrogel volume and number of encapsulated PSC-derived HSPCs (from 15 pL and 7,500 cells to 30 pL and 15,000 cells), while maintaining the same cell density used for CB-derived HSCs, not only generates a higher number of cells but also a higher frequency of generated Pro-T cells (Figure 15b). Thus, in a preferred embodiment, 15,000 PSC-derived HSPCs are encapsulated in 30 pL of HA-ProG-DLL4-PEG hydrogel.

[0221] Figures 15b-c show a representative flow cytometry analysis of generated CD5(+)CD7(+) Pro-T cells and the frequency of Pro-T cells generated from different batches of PSC-derived HSPCs after 2 weeks in HA-ProG-DLL4-PEG-G hydrogel in the presence or absence of VCAM1. Similar to CB-derived HSCs cultured in the HA-ProG-DLL4-G hydrogel (Figures 9d-e, 10), the addition of VCAM1 in the HA- ProG-DLL4-PEG-G hydrogel does not affect the formation of Pro-T cells from PSC-derived HSPCs. However, as shown in Figure 15c, the frequency of generated Pro-T cells varies significantly among different batches of PSC-derived HSPCs. Thus, in a preferred embodiment, VCAM1 is not added to the HA-ProG-DLL4-PEG hydrogel.

[0222] Figure 15a is a schematic representation of the timeline and culture conditions used for generating functional mature T cells from PSC-derived HSPCs encapsulated in the HA-ProG-DLL4- PEG hydrogel. In a preferred embodiment, PSC-derived HSPCs are first generated using the inventors’ previously reported protocol, whereby PSCs undergo 8 days of CD34 induction (days -15 to -7), CD34(+) cell enrichment and then 7 days of EHT (days -7 to 0) (Michaels et al., 2022). PSC-derived HSPCs are then encapsulated in HA-ProG-DLL4-PEG-G hydrogel with lymphoid progenitor expansion supplement containing SCF, IL7, TPO and Flt3L for 1 week, followed by T cell progenitor maturation supplement containing IL7 and Flt3L but not SCF or TPO for 2 weeks. CD4(+)CD8(+) DP T cells are generated after 3 weeks of culture in the hydrogel and then transferred to DLL4+VCAM1 coated plates and cultured for 2 or more weeks in maturation media supplemented with a CD3 / CD28 activator and IL-15 to generate functional mature CD3(+)TCRaP(+) and CD8(+) SP T cells.

[0223] Figures 15d-e show representative flow cytometry analysis of CD4, CD8b, CD3 and TCRap expression and the frequencies of CD4(+) ISP, CD8(+)CD4(+) DP, CD3(+)TCRaP(+) and CD8(+) SP T cells generated from PSC-derived HSPCs encapsulated in the HA-ProG-DLL4-PEG hydrogel after 3 weeks. At week 3, the cells generated in the HA-ProG-DLL4-PEG hydrogel comprise 29% DP T cells and 2% CD3(+)TCRaP(+) T cells.

[0224] Figure 15f shows a representative flow cytometry analysis of CD4, CD8b, CD3 and TCRap expression in cells generated from PSC-derived HSPCs encapsulated in the HA-ProG-DLL4-PEG hydrogel after 4 weeks. At week 4, the cells generated in the HA-ProG-DLL4-PEG hydrogel do not comprise CD4(+) ISP, CD8(+)CD4(+) DP or CD3(+)TCRaP(+) T cells. Notably 27% of the generated cells express CD8.

[0225] To obtain conventional CD8(+) SP T cells, DP T cells generated after 3 weeks of differentiation may be harvested, transferred to DLL4+VCAM1 coated plates and cultured for 2 weeks in maturation media supplemented with 12.5 pL / mL ImmunoCult CD3 / CD28 activator and 10 ng / mL IL-15. Figures 15g-h show representative flow cytometry analysis of CD4, CD8, CD3 and TCRap expression and frequencies of CD8(+) SP and CD3(+)TCRaP(+) T cells generated from DP T cells extracted from the HA-ProG-DLL4-PEG hydrogel at week 3 and then cultured for 2 weeks on DLL4+VCAM1 coated plates in the presence of a CD3 / CD28 activator and IL- 15. After 2 weeks of culture with the CD3 / CD28 activator, the generated cells comprise -28% mature CD8(+) SP T cells and -8% CD3(+)TCRaP(+) T cells, confirming that this method successfully generates mature T cells from PSC-derived HSPCs encapsulated in the HA-ProG-DLL4-PEG hydrogel.

[0226] To assess the functionality of mature T cells, T cells may be stimulated to produce cytokines using PMA and ionomycin, along with brefeldin A to accumulate the produced cytokines within the cells(preventing secretion), thus allowing flow cytometric analysis. Figure 16 shows representative flow cytometry analysis of cytokine production in PSC-derived HSPCs that are cultured in the HA-ProG- DLL4-PEG hydrogel for 3 weeks, stimulated on DLL4+VCAM1 coated plates with CD3 / CD28 activator and IL-15 for 3 weeks and then stimulated with PMA and ionomycin for 6 hours, with brefeldin A added for the final 5 hours. As shown in Figure 16, a large proportion of the stimulated mature T cells produce IFN-y, and a moderate proportion produce IL-2 and TNF-a.

[0227] Overall, these findings demonstrate that PSC-derived HSPCs encapsulated in the HA-ProG- DLL4-PEG hydrogel are capable of efficient differentiation not only into Pro-T cells but also into mature and functional CD8(+) T cells.EQUIVALENTS AND SCOPE

[0228] The contents of all cited references (including literature references, U.S. or foreign patents or patent applications, and websites) that are cited throughout this application are hereby expressly incorporated by reference as if written herein in their entireties for any purpose, as are the references cited therein. Where any inconsistencies arise, material literally disclosed herein controls.

[0229] It is to be appreciated the Summary and Abstract sections and Examples may set forth one or more but not all exemplary embodiments of the present disclosure as contemplated by the inventor(s), and thus, are not intended to limit the present disclosure. The above specification is not restrictive. Many variations and equivalents to the specific embodiments of the invention described herein will become apparent to those skilled in the art upon review of this specification. For example, certain aspects of the hydrogel composition and other culture conditions or selection of cells may be altered without departing from the inventive concepts described herein. In particular, it is contemplated by the inventorthat various substitutions, alterations, variations and modifications may be made to various aspects of the hydrogel, methods and uses without departing from the spirit and scope of the invention as defined by the claims. As such, the scope of the present invention is not intended to include such variations and equivalents.REFERENCES

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Claims

CLAIMSWhat is claimed is1. A three dimensional (3D) hydrogel comprising: a. a first biocompatible polymer that is a polysaccharide polymer; b. at least one second biocompatible polymer; and c. at least one signalling molecule immobilized directly or indirectly to the first and / or second polymer, d. wherein the first and second biocompatible polymers are crosslinked.

2. The hydrogel of claim 1 wherein the signalling molecule is one that promotes hematopoietic stem and progenitor cell (HSPC), hematopoietic stem cell (HSC) and / or hematopoietic progenitor cell expansion and differentiation to progenitor and precursor T cells and / or mature T cells.

3. The hydrogel of claim 2 wherein the signalling molecule is a Notch ligand and / or a cell adhesion or integrin receptor ligand.

4. The hydrogel of claim 3, wherein the Notch ligand is selected from one or more of: DLL1 , DLL3, DLL4, JAG1 , and JAG2 OR from DLL1 or DLL4 or a recombinant human form of any of the foregoing.

5. The hydrogel of claim 4, wherein the Notch ligand is DLL4 or a recombinant human form thereof.

6. The hydrogel of any one of claims 1 to 5, wherein the Notch ligand is immobilized directly or indirectly on the first biocompatible polymer.

7. The hydrogel of any one of claims 1 - 6 wherein the signalling molecule is present in a range of about 50 to about 200, or from about 50 to about 100 micrograms per mL of hydrogel.

8. The hydrogel of any one of claims 1 - 8 wherein the first biocompatible polymer is selected from the group consisting of: alginate, chitosan, cellulose, pectin, hyaluronic acid (HA), agarose, chondroitin sulfate, carrageenan, inulin, starch, salts or esters of any of the foregoing and any combinations of the foregoing.

9. The hydrogel of claim 8 wherein the first biocompatible polymer is selected from the group consisting of alginate, chitosan, cellulose, pectin, hyaluronic acid (HA), salts or esters of any of the foregoing and any combinations of the foregoing.

10. The hydrogel of claim 9 wherein the first biocompatible polymer is hyaluronic acid or salts or esters thereof, such as sodium hyaluronate, potassium hyaluronate, magnesium hyaluronate and calcium hyaluronate.

11. They hydrogel of claim 10, wherein the first polymer is from about 150 - 300 kDa.

12. The hydrogel of any one of claims 1 - 8, wherein one of the at least one second biocompatible polymer is selected from one or more of a natural polymer and / or a synthetic polymer, such as a synthetic polymer that is inert to degradation from cellular enzymes.

13. The hydrogel of claim 12, wherein one of the at least one second biocompatible polymer acts as (i) a crosslinker and (ii) one or more of a hydrogel stabilizer, a stiffening agent, a cell adhesion molecule, a RGD-based polymer that promotes cell adhesion and / or migration, and a polymer that has an integrin receptor binding cite, such as RGD.

14. The hydrogel of claim 12, wherein the at least one second biocompatible polymer is a synthetic polyvinyl alcohol, polyethylene glycol (PEG), sodium polyacrylate, acrylate polymers and copolymers, salts or esters thereof, or an about 5 to about 6 kDA synthetic polymer of any of the foregoing.

15. The 3D hydrogel of any one of claims 1 - 14, wherein the at least one second biocompatible polymer is a PEG, a gelatin or a combination thereof.

16. The hydrogel of claim 15, wherein the at least one second biocompatible polymer is a gelatin, such as 60 kDa gelatin.

17. The 3D hydrogel of any one of claims 1 - 16, wherein the 3D hydrogel is in a liquid state or a gel state; and the 3D hydrogel forms a crosslinked 3D matrix, such as upon gelling.

18. The 3D hydrogel of anyone of claims 1 - 17 seeded with hematopoietic stem and progenitor cells (HSPC), hematopoietic stem cells (HSC) and / or hematopoietic progenitor cells (HPC) or combinations thereof, encapsulated within the 3D hydrogel.

19. The 3D hydrogel of claim 18, wherein the cells are derived from any source of HSPC, HSC or HPC or from derived from cord blood or induced pluripotent stem cell (iPSC) derived cells or derived from any human source, human cord blood or human induced pluripotent stem cell.

20. The 3D hydrogel of claim 19 wherein the at least one second biocompatible polymer is both a crosslinker and a hydrogel stabilizer and / or a cell adhesion molecule, and / or is an RGD-based polymer that promotes cell adhesion and / or migration, and / or a polymer that has an integrin receptor binding cite, such as RGD, such as gelatin.

21. The 3D hydrogel of claim 20, wherein the at least one second biocompatible polymer further comprises at least one second biocompatible polymer that is a synthetic polymer, such as PEG or other polymer that is inert to cellular enzyme degradation.

22. The 3D hydrogel of any one of claims 1 - 21 wherein the first biocompatible polymer, the second biocompatible polymer and the signalling molecule are connected using click chemistry.

23. The 3D hydrogel of any one of claims 1 - 22 wherein the signalling molecule is an Fc-fusion protein signalling molecule, and bound to an Fc fusion protein binding agent, such as Protein A (ProA) or Protein G (ProG) or ProG.

24. The 3D hydrogel of claim 22 or 23 wherein the first and second biocompatible polymer and Fc fusion protein binding agent are modified to enable such conjugation through click chemistry.

25. A 3D hydrogel comprising: a. a first biocompatible polymer that is a polysaccharide polymer; b. at least one second biocompatible polymer; and c. at least one signalling molecule immobilized directly or indirectly to the first and / or second polymers, wherein the first biocompatible polymer is crosslinked to the at least one second biocompatible polymer through click chemistry, and wherein the first and second biocompatible polymers are modified to enable crosslinking through click chemistry; and wherein optionally the 3D hydrogel is seeded with at least one cell, and wherein optionally the at least one cell is selected from the group consisting of hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPCs), or any combinations thereof (i.e., HSPCs), which is encapsulated within the 3D hydrogel.

26. The 3D hydrogel of claim 25, wherein the signaling molecule is a Fc-fusion protein, the 3D hydrogel further comprising: a. a Fc fusion protein binding agent, b. wherein the first biocompatible polymer is conjugated to the Fc fusion protein binding agent through click chemistry, and wherein the first biocompatible polymer and Fc fusion protein binding agent are modified to enable such conjugation through click chemistry.

27. The 3D hydrogel of claim 25 or 26 for use in expansion and differentiation of the at least one cell to progenitor and precursor T cells and mature T cells.

28. The 3D hydrogel of any one of claims 1 - 27 wherein the 3D hydrogel components are selected to mimic the 3D structure of the extracellular matrix (ECM) of the thymus.

29. The 3D hydrogel of any one of claims 1 - 28 wherein a ratio of the components of the 3D hydrogel can be selected to mimic a microenvironment of a thymus.

30. The 3D hydrogel of any one of claims 18 - 29 wherein the at least one cell is a cord blood (CB)- derived HSPC or an induced pluripotent stem cell (iPSC)-derived HSPC.

31. The 3D hydrogel of any one of claims 18 - 30, wherein the 3D hydrogel is used for promoting T cell lineage development from the at least one seeded cell.

32. The 3D hydrogel of any one of claims 25 - 31 , wherein the signalling molecule is a Notch ligand, such as is DLL4 or DLL1 , or an integrin ligand, such as VCAM1.

33. The 3D hydrogel of claim 32, wherein the Notch ligand is DLL4.

34. The 3D hydrogel of any one claims 22 - 33, wherein the click chemistry is a norbornene-tetrazine conjugation chemistry.

35. The 3D hydrogel of claim 34 wherein the tetrazine is methyltetrazine.

36. The 3D hydrogel of claim 34 or 35, wherein the first polymer is modified with at least two norbornenes and the second polymer and the Fc fusion protein binding agent are each modified with at least one methyltetrazine.

37. The 3D hydrogel of any one of claims 25 - 36, wherein the first biocompatible polymer is a polysaccharide polymer selected from the group consisting of synthetic or natural polysaccharides such as alginate, chitosan, cellulose or hyaluronic acid (HA) or salts or esters of any of the foregoing.

38. The 3D hydrogel of claim 37, wherein the polysaccharide polymer is hyaluronic acid or a salt or ester thereof.

39. The 3D hydrogel of claim 38, wherein the hyaluronic acid or salt thereof is from 150 - 300 kDa.

40. The 3D hydrogel of any one of claims 26 - 39, wherein the Fc fusion protein binding agent is Protein A (ProA) or Protein G (ProG).

41. The 3D hydrogel of claim 40, wherein the Fc fusion protein is Protein G (ProG).

42. They 3D hydrogel of any one of claims 1 - 41, wherein the at least one second biocompatible polymer is selected from one or more of a natural polymer and / or a synthetic polymer, such as a synthetic polymer that is inert to degradation from cellular enzymes.

43. The hydrogel of claim 42, wherein the at least one second biocompatible polymer is a crosslinker and one or more of a hydrogel stabilizer, a stiffening agent, a cell adhesion molecule, a RGD- based polymer that promotes cell adhesion and / or migration and a polymer that has an integrin receptor binding cite, such as RGD.

44. The hydrogel of claim 42, wherein the at least one second biocompatible polymer is a synthetic polyvinyl alcohol, polyethylene glycol (PEG), sodium polyacrylate, acrylate polymers and copolymers, salts or esters thereof, an about 5 to about 6 kDA synthetic polymer, or a combination of any of the foregoing.

45. The 3D hydrogel of any one of claims 1 - 44, wherein the at least one second biocompatible polymer is a gelatin, a PEG or a combination thereof.

46. The hydrogel of claim 45, wherein the at least one second biocompatible polymer is a gelatin.

47. The 3D hydrogel of claim 46, wherein the gelatin is 40 - 70 kDa.

48. The 3D hydrogel of any one of claims 1 - 47, wherein in a gelled state, the hydrogel stiffness corresponds to a stiffness of a region of a human thymus.

49. The 3D hydrogel of any one of claims 1 - 48, wherein the 3D hydrogel stiffness has a Young modulus of 2.5 to 3.5 kPa.

50. The 3D hydrogel of any one of claims 1 - 49, for use in promoting expansion and differentiation of the at least one seeded cell to a T cell lineage, wherein the 3D hydrogel with the at least one seeded cell is cultured in media that promotes such expansion and differentiation.

51. The 3D hydrogel for use of claim 50, wherein: a. the media comprises a basal media supplemented with at least one cytokine, b. the basal media is selected from the group consisting of IMDM, RPMI, aMEM, SFEM II, StemPro34 and any suitable media designed and optimized for culturing human stem cells; and c. the at least one cytokine is selected from the group consisting of IL-7, Flt3L, SCF, TPO, CXCL12, TNF-a, IL3, other small molecules including apoptosis inhibitors, metabolites, vitamins, bovine serum albumin and human serum albumin and combinations thereof52. The 3D hydrogel for use of claims 50 or 51 , wherein cell spheroids are formed from the at least one seeded cell.

53. The 3D hydrogel for use of any one of claims 50 - 52, wherein cells of the T cell lineage are harvested and recovered from the 3D hydrogel using enzymes and or hydrolysis to dissolve the 3D hydrogel.

54. The 3D hydrogel for use of claim 53, wherein the cells of the T cell lineage are early T cell progenitor (ETP) cells, Pro-T cells, Pre-T cells, initial single positive (ISP) T cells, double positive (DP) T cells, mature single positive (SP) T cells, or a combination thereof.

55. The 3D hydrogel for use of claim 53, wherein the recovered cells can be further differentiated within the 3D hydrogel under suitable media or outside of the hydrogel on 2D substrates.

56. A method for producing the 3D hydrogel of any one of claims 1 - 55, comprising: a. a crosslinking step, wherein a first biocompatible polymer is crosslinked with at least one second biocompatible polymer to form a 3D hydrogel matrix, and wherein the crosslinking may be achieved using chemical means, such as click chemistry, or physical means, such as photo-crosslinking; and b. a signalling molecule immobilization step, wherein at least one signalling molecule is immobilized within the 3D hydrogel matrix by directly or indirectly conjugating the at least one signalling molecule to the first biocompatible polymer and / or the at least one second biocompatible polymer.

57. The method of claim 56, wherein the first biocompatible polymer and the at least one second biocompatible polymer are modified to enable the crosslinking step, to enable the signalling molecule immobilization step, or to enable both the crosslinking step and the signalling molecule immobilization step.

58. The method of claim 56 or 57, wherein the signalling molecule is modified to enable the signalling molecule immobilization step.

59. The method of any one of claims 56 - 58, wherein: a. a binding agent is used to immobilize the signalling molecule within the 3D hydrogel matrix; and b. the binding agent is modified to enable conjugation with the first biocompatible polymer and / or the at least one second biocompatible polymer.

60. The method of claim 59, wherein: a. the signalling molecule is a human Fc-fusion protein; and b. the binding agent is a protein that binds with high affinity to the Fc-fusion protein, such as Protein A or Protein G.

61. The method of any one of claims 56 - 60, wherein the first biocompatible polymer and the at least one second biocompatible polymer are modified with click chemistry reactive groups.

62. The method of any one of claims 56 - 61 , wherein the first biocompatible polymer is modified with a norbornene group and the at least one second biocompatible polymer is modified with a methyltetrazine group.

63. The method of any one of claims 59 - 62, wherein the binding agent is modified with a click chemistry reactive group, such as a norbornene group or a methyltetrazine group.

64. The method of any one of claims 56 - 63, wherein: a. the first biocompatible polymer, the at least one second biocompatible polymer and the signalling molecule are mixed in a solution to form the 3D hydrogel in a liquid state; b. at least one cell may be seeded in the liquid state 3D hydrogel, the at least on cell selected from the group consisting of hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPCs) and a combination thereof (i.e. , HSPCs); and then c. the 3D hydrogel becomes a gelled state, thereby encapsulating the at least one seeded cell.

65. A method for in vitro human T cell development, comprising: a. providing the functionalized hydrogel of any one of claims 1 to 32; b. seeding the 3D hydrogel with cells selected from the group of HSCs, HPCs or a combination thereof (HSPCs); and c. culturing the cells within the 3D hydrogel under conditions that promote lymphoid specification (lymphopoiesis), differentiation into progenitor and precursor T cells and T cell maturation, generating CD7(+) early T cell progenitors (ETPs), CD7(+)CD5(+) lineage-specific Pro-T cells, CD7(+)CD5(+)CD1a(+) lineage-committed Pre-T cells, initial single positive (ISP) CD4(+) cells, double positive (DP) CD4(+)CD8(+) cells, mature single positive (SP) CD4(+) or CD8(+) T cells, CD3(+)TCRaP(+) T cells capable of cytokine secretion or combinations thereof.

66. The method of claim 65, wherein the 3D hydrogel in liquid state is poured into a mold to form a gelled state.

67. The method of claim 65 or 66, wherein the cells are seeded in the 3D hydrogel at a cell density of about 300,000 - about 2,000,000 per 1 mL of 3D hydrogel.

68. The method of any one of claims 65 - 67, wherein the cells are seeded in the 3D hydrogel when the hydrogel is in a liquid state.

69. The method of any one of claims 65 - 68, wherein: a. the conditions are non-xenogeneic, feeder-free and serum-free; and b. the conditions comprise a defined culture medium.

70. The method of any one of claims 65 - 69, wherein: a. the conditions comprise a basal media supplemented with at least one cytokine; b. the basal media is selected from the group consisting of IMDM, RPMI, aMEM, SFEM II, StemPro34 and any suitable media designed and optimized for culturing human stem cells; and c. the at least one cytokine is selected from the group consisting of IL-7, Flt3L, SCF, TPO, CXCL12, TNF-a, IL3, other small molecules including apoptosis inhibitors, metabolites, vitamins, bovine serum albumin and human serum albumin and combinations thereof.

71. The method of any one of claims 65 - 70, wherein the cells are human umbilical cord blood (CB)-derived cells.

72. The method of claim 71 , wherein culturing the CB-derived cells within the 3D hydrogel comprises: a. a first culture stage, wherein the conditions comprise a basal media supplemented with a lymphoid progenitor expansion supplement comprising SCF, IL7, TPO and Flt3L for 2 to 3 weeks; and b. a second culture stage, wherein the conditions comprise a basal media supplemented with a maturation supplement comprising IL7 and Flt3L, but not SCF and TPO, for an additional 3 to 10 weeks.

73. The method of claim 72, further comprising: a. dissolving the 3D hydrogel after the second culture stage using an enzyme solution or hydrolysis; andb. isolating CD4(+)CD8(+) DP T cells, CD8(+) SP T cells, CD3(+)TCRaP(+) T cells or combinations thereof generated after the second culture stage.

74. The method of claim 73, wherein CD4(+)CD8(+) DP T cells are isolated after the second culture stage, further comprising: a. transferring the CD4(+)CD8(+) DP T cells to a two-dimensional (2D) substrate coated with DLL4 and VCAM1 ; and b. culturing the CD4(+)CD8(+) DP T cells in a basal media supplemented with IL7, Flt3L, a CD3 / CD28 activator and IL- 15 for 2 weeks to generate mature CD8(+) SP T cells and CD3(+)TCRaP(+) T cells.

75. The method of any one of claims 65 - 69, wherein the cells are human induced pluripotent stem cell (iPSC)-derived cells.

76. The method of claim 75, wherein culturing the iPSC-derived cells within the 3D hydrogel comprises: a. a first culture stage, wherein the conditions comprise a basal media supplemented with SCF, IL7, TPO and Flt3L for 1 to 2 weeks; and b. a second culture stage, wherein the conditions comprise a basal media supplemented with IL7 and Flt3L for an additional 2 to 10 weeks.

77. The method of claim 76, further comprising: a. dissolving the 3D hydrogel after the second culture stage using an enzyme solution or hydrolysis; and b. isolating CD4(+)CD8(+) DP T cells, CD8(+) SP T cells, CD3(+)TCRaP(+) T cells or combinations thereof generated after the second culture stage.

78. The method of claim 77, wherein CD4(+)CD8(+) DP T cells are isolated after the second culture stage, further comprising: a. transferring the CD4(+)CD8(+) DP T cells to a 2D substrate coated with DLL4 and VCAM1 ; and b. culturing the CD4(+)CD8(+) DP T cells in a basal media supplemented with IL7, Flt3L, a CD3 / CD28 activator and IL- 15 for 2 or more weeks to generate mature CD8(+) SP T cells and CD3(+)TCRaP(+) T cells.

79. The use of the hydrogels of any one claims 1 - 55 and the methods of claims 56 - 78 to produce mature T cells.

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