GENERATION OF INDUCED HUMAN THYMIC EPITHELIAL CELLS AND ITS APPLICATION IN CELL-BASED IMMUNOTHERAPIES

Abstract

Methods of differentiating pluripotent stem cells into thymic epithelial progenitor cells are provided.

Claims

1. A method of differentiating a Definitive Endoderm (DE) cell or Anterior Foregut (AFG) cell or an Anterior Foregut (AFG) plus cell to a HOXA3+, TBX1+/high, bipotent PPEIII cell that has a committed fate to PPEIII and has the potency to further differentiate into thymus and parathyroid epithelial lineages, the method comprising culturing the DE cell or AFG cell or AFG plus cell in a medium comprising at least: a Transforming Growth Factor (TGF-) signaling pathway inhibitor; a Bone Morphogenic Protein (BMP) signaling pathway inhibitor; a Retinoic Acid (RA) signaling pathway activator; a PI3K/AKT signaling pathway inhibitor, wherein the culturing of the DE or AFG or AFG-plus cell promotes differentiation to a bipotent PPEIII cell specified for PPEIII and primed for thymus or parathyroid fate.

2. The method of claim 1, wherein the medium further comprises a Fibroblast Growth Factor (FGF) signaling pathway activator.

3. The method of claim 1, wherein the medium further comprises a canonical WNT signaling pathway inhibitor.

4. The method of claim 1, further comprising differentiating the HOXA3+, TBX1+/high bipotent PPEIII cell into a human TBX1dim, PAX1+, PAX9+, FOXG1+ ventral PPEIII cell by: culturing the bipotent PPEIII cell in a second medium comprising: a WNT signaling pathway activator; a BMP signaling pathway activator, wherein the culturing promotes differentiation of the bipotent PPEIII cell to a ventral PPEIII cell.

5. The method of claim 4, wherein the second medium further comprises one, some or all of the following: an FGF/ERK/MAPK signaling pathway inhibitor, an IGF signaling pathway activator, and a Sonic Hedgehog (SHH) signaling pathway inhibitor.

6-8. (canceled)

9. The method of claim 4, further comprising differentiating the ventral PPEIII cell to a FOXN1+, PSMB11+, EPCAM+ TEPC cell, wherein the differentiation protocol comprises culturing the ventral PPEIII cell in a third medium comprising: an IFN type I or II signaling pathway activator; a FGF signaling pathway activator; and an IGF signaling pathway activator.

10. The method of claim 9, wherein the third medium further comprises one, both or all three of the following: a Sonic Hedgehog (SHH) signaling pathway inhibitor, a canonical WNT signaling pathway inhibitor and a RANK ligand.

11. The method of claim 1, wherein the DE or AFG cell is a primary DE or AFG cell, respectively.

12. The method of claim 1, wherein the method comprises obtaining the DE cell or AFG cell from a human.

13. The method of claim 1, wherein the method comprises introducing the bipotent PPEIII cell, ventral PPEIII cell, or TEPC cell into a human.

14. The method of claim 13, wherein the bipotent PPEIII cell, ventral PPEIII cell, or TEPC cell is autologous to the human individual.

15. The method of claim 13, wherein the bipotent PPEIII cell, ventral PPEIII cell, or TEPC cell is allogenic to the human individual.

16. The method of claim 13, wherein the human has congenital thymic aplasia, thymic injury, reduced thymic function (e.g., due to HSCT status, aGVHD, infection, tumor, irradiation, medications or iatrogenic surgical removal due to cardiac surgeries), or age-related decline in thymic function.

17. A method of differentiating a HOXA3+, TBX1+/high, bipotent PPEIII cell to a human a TBX1dim, PAX1+, PAX9+, FOXG1+ ventral PPEIII cell that can further differentiate into thymic epithelial progenitor cells, the method comprising culturing the bipotent PPEIII cell in a medium comprising at least: a WNT signaling pathway activator; a BMP signaling pathway activator, wherein the culturing promotes differentiation of the bipotent PPEIII cell to a ventral PPEIII cell.

18. The method of claim 17, wherein the medium further comprises one, multiple or all of the following: an FGF/ERK/MAPK signaling pathway inhibitor, an IGF signaling pathway activator, and a Sonic Hedgehog (SHH) signaling pathway inhibitor.

19. The method of claim 18, wherein the medium comprises an FGF/ERK/MAPK signaling pathway inhibitor.

20. The method of claim 18, wherein the medium comprises an IGF signaling pathway activator.

21. The method of claim 18, wherein the medium comprises a Sonic Hedgehog (SHH) signaling pathway inhibitor.

22. The method of claim 21, wherein the method comprises introducing the bipotent PPEIII cell, ventral PPEIII cell, or TEPC cell into a human.

23-24. (canceled)

25. A method of differentiating a TBX1dim, PAX1+, PAX9+, FOXG1+ ventral PPEIII cell to a FOXN1+, PSMB11+, EPCAM+ TEPC cell, the method comprising culturing the ventral PPEIII cell in a medium comprising at least: an IFN type I or II signaling pathway activator; a FGF signaling pathway activator; and an IGF signaling pathway activator.

26-38. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0044] FIG. 1 Overview of the 5 step TEPC differentiation protocol using human pluripotent stem cells (PSC). Activating and inhibiting signals are detailed.

[0045] FIG. 2 (A-B) shows the induction of DE and AFG. Homogenous expression of SOX17 in DE with <1% SOX17 negative cells. (B) Expression of Stage-specific markers in DE and AFG respectively are confirmed by Taqman qRT-PCR.

[0046] FIG. 3 illustrates the changes in expression levels of key developmental markers across the 6 stages of thymic epithelial development from pluripotency, DE. AFG, bipotent PPEIII, ventral PPEIII to TEPC fate. mRNA expression levels of individual markers are represented relative to the mRNA expression level of the housekeeping gene GAPDH.

[0047] FIG. 4A-B illustrates the differentiation of AFG to bipotent PPEIII under the combined induction of multiple signals. (4A) dynamic temporal changes of TBX1 mRNA expression 1) with TGF inhibition and BMP4 inhibition alone, 2) with TGF inhibition and BMP4 inhibition in conjunction with retinoic acid (RA), 3) with TGF inhibition and BMP4 inhibition in conjunction with RA, FGF, P13K inhibition and Canonical WNT inhibition. (4B) dynamic temporal changes of HOXA3 mRNA expression 1) with TGF inhibition and BMP4 inhibition alone, 2) with TGF inhibition and BMP4 inhibition in conjunction with retinoic acid (RA), 3) with TGF inhibition and BMP4 inhibition in conjunction with RA. FGF, PI3K inhibition and Canonical WNT inhibition. mRNA expression levels of individual markers are represented relative to the mRNA expression level of the housekeeping gene GAPDH.

[0048] FIG. 5 details the expression levels of key markers of the bipotent PPEIII stage (TBX1, HOXA3, FOXG1, PAX9, GATA3, GATA6 and SOX2) in response to different combinations of inductive signals which in their collectiveness (#5) constitute the methods claimed herein to differentiate a suitable precursor population (DE cell, AFG cells, AFG-plus cells) into bipotent PPEIII cells.

[0049] FIG. 6A-B: 6A illustrates the capacity of bipotent PPEIII cells to give rise to both, ventral PPEIII cells and subsequently TEPCs, as well as dorsal PPEIII cells and subsequently parathyroid epithelial precursor cells (PEPCs) when directed with the appropriate signals. (6B) Expression levels of the TEC/TEPC maker FOXN1 across different primary and iPSC-derived tissues relative to the housekeeping gene GAPDH. Expression levels of the parathyroid and PEPC maker GCM2 across different iPSC-derived tissues relative to the housekeeping gene GAPDH.

DETAILED DESCRIPTION OF THE INVENTION

[0050] The inventors have discovered an in vitro differentiation platform for the efficient generation of TEPCs from human pluripotent stem cells (PSC) by recapitulating the sequential stages of human thymic ontogeny: Definitive Endoderm (DE), Anterior Foregut (AFG), Bipotent Third Pharyngeal Pouch Endoderm (bipotent PPEIII), Ventral Third Pharyngeal Pouch Endoderm (ventral PPEIII) and Thymic Epithelial Progenitor Cells (TEPC). These TEPCs can further mature into functional cTECs and mTECs either in vivo after transplantation or in vitro through further signal induction. Accordingly, methods of differentiating cells into any of these intermediate stages or into bipotent PPEIII cells, ventral PPEIII cells or TEPCs are provided, starting from any cell type that can be differentiated into DE cells or AFG cells.

[0051] The process of differentiating pluripotent stem cells into functional TEPCs in vitro may be viewed as progressing through five consecutive stages, as is shown in the exemplary protocol depicted in FIG. 1 In this stepwise progression, Stage 1 refers to the first step in the differentiation process, the differentiation of PSC into cells expressing markers characteristic of DE cells. Stage 2 refers to the second step, the differentiation of cells expressing markers characteristic of DE cells into cells expressing markers characteristic of AFG cells. Stage 3 refers to the third step, the differentiation of cells expressing markers characteristic of AFG cells into cells expressing markers characteristic of bipotent PPEIII cells. Bipotent as used herein refers to the ability of Stage 3 cells to differentiate into thymus or parathyroid. Stage 4 refers to the fourth step, the differentiation of cells expressing markers characteristic of bipotent PPEIII cells into cells expressing markers of ventral PPEIII cells. Stage 5 refers to the fifth step, the differentiation of cells expressing markers characteristic of ventral PPIII cells into cells expressing markers characteristic of TEPCs. Not all cells in a particular population progress through these stages at the same rate, i.e., some cells may have progressed less, or more, down the differentiation pathway than the majority of cells present in the population.

[0052] In some embodiments, the methods begin with DE cells. Optionally DE cells can be generated from any cell that can be differentiated into a DE cell. Any human pluripotent cells can be used including but not limited to human embryonic stem cells (hESCs), human induced pluripotent stem cells (hiPSCs) or human embryonic germ cells (hEGCs). In some cases, human adult stem cells or progenitors may be used to induce TEPCs with the methods and medium composition disclosed herein.

[0053] Methods of generating DE are known and include but are not limited to Loh et al (Loh, Ang et al., 2014 Cell Stem Cell). The method of Loh et al can be used or adapted to generate DE cells from human PSCs. Other methods to generate DE cells, are also suitable.

[0054] In some embodiments, the methods begin with AFG cells. Optionally, AFG cells can be generated from any cell that can be differentiated into a AFG cell. Methods of making AFG cells are known and include but are not limited to Green et al. (Green, Chen et al. 2011 Nature Biotechnology). The method of Green et al can be used or adapted to generate AFG cells from DE cells. Other methods to generate AFG cells, are also suitable.

[0055] In some embodiments, the methods to differentiate bipotent PPEIII cells can start with AFG cells that have additionally been treated with dual SMAD inhibition and all-trans retinoic acid (ATRA) at concentrations from 5 nM to 5 uM or another retinoic acid agonist, or a PI3K signaling pathway inhibitor, or an FGF signaling pathway activator or a canonical WNT signaling pathway inhibitor for more than 24 hours. Cell derived in such a fashion are herein referred to as AFG-plus cells.

[0056] In some embodiments, either DE cells or AFG cells or AFG-plus cell can be used as starting material to generate bipotent PPEIII cells.

[0057] Basal medium can be, without limitation, chemically defined medium (CDM2), RPMI1640 or DMEM/F12. Basal medium can be supplemented with, but not limit to, Knockout serum replacement (KOSR), B27 or N2 supplements. This medium composition applies to all the stages of the TEPC differentiation methods. Other basal media with different bases as defined above can be used.

[0058] As demonstrated in the Examples and FIG. 1. DE or AFG or AFG-plus cells can be used as starting material for the methods of making bipotent PPEIII cells. The additional treatment of DE cell with dual SMAD inhibition or of AFG cells with any additional factors is not required as a starting material to induce the bipotent PPEIII stage. Bipotent PPEIII cells are biologically defined by their dual capacity to give rise to thymus and parathyroid. These cells are HOXA3+/high, TBX1+/high, wherein high is defined as respective expression levels of HOXA3 and TBX1 peaking at the bipotent PPEIII stage after which HOXA3 and TBX1 expression will decline as detailed above. The resulting cells have a committed fate to PPEIII and have the potency to further differentiate into thymus/parathyroid epithelial lineages. To achieve these bipotent PPEIII cells, DE cells or AFG cells or AFG-plus cells were cultured in basal medium, e.g., as described above, that further comprises a Transforming Growth Factor (TGF) signaling pathway inhibitor (e.g., A 83-01 at 1 nM-10 uM); a Bone Morphogenic Protein (BMP) signaling pathway inhibitor (e.g., LDN193189 at 1 nM-10 uM); a retinoic acid (RA) signaling pathway activator (e.g., ATRA at 5 nM-5 uM); and a PI3K/AKT signaling pathway inhibitor (e.g., PI 828 at 0.05 uM-50 uM). The inventors have found that the induction of the bipotent PPEIII stage is further enhanced by addition of one or more of a Fibroblast Growth Factor (FGF) signaling pathway activators (e.g., FGF7 and/or FGF10, at least 1 ng/ml) or a canonical WNT signaling pathway inhibitor (e.g., IWR-1 at 5 nM to 5 uM) or both to the medium to further improve conversion to bipotent PPEIII cells.

[0059] Exemplary TGF signaling pathway inhibitors include, without limitation, an ALK5 inhibitor including A 83-01, LY364947, LY580276, SB505124, SB431524, SB525334, SM16, SD208. D4476, GW6604, GW788388, TP0427736, BIBF-0775, CAS 446859-33-2, or an anti-TGF antibody including Vactoscrtib.

[0060] Exemplary BMP signaling pathway inhibitors include, without limitation, an ALK2 inhibitor or ALK3 inhibitor e.g., LDN193189, Noggin, DMH2, K02288, ML374, LDN214117, DMH-1, LDN212854, or Dorsomorphin dihydrochloride.

[0061] Exemplary retinoic acid (RA) signaling pathway activators include reagents which activates any retinoic acid receptor (RAR // and RXR), and wherein the RAR activator include, without limitation, all-trans retinoic acid (ATRA), and analogs of ATRA including TTNPB, Tamibarotene, BMS-189453, CD1530, Adapalene, Tazarotenic acid, CD437, BMS753. CD2314, BMS961, CH55, AM580, AM80, AC55649, AC261066, EC19, BMS493, DC271, or EC23. RXR activators include, without limitation, SRI1237 and 9-cis-Retinoic Acid.

[0062] Exemplary PI3K/AKT signaling pathway inhibitors include reagents that can inhibit the signal at any level of this pathway including IGF receptors, PI3K(///), AKT (AKT1/2/3) and their substrates. Some examples include, without limitation, PI 828 (0.05 uM-50 uM), LY294002, PI 103, GSK1059615. ETP45658, API-1. API-2. GSK690693, GSK1904529A, GDC-0941, PIK90, TGX-221, IC-87114, BKM120, BEZ235, and GS-1101.

[0063] Exemplary FGF signaling pathway activators include, without limitation. FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, and FGF22.

[0064] Exemplary canonical WNT signal inhibitors include, without limitation, endo-IWR 1 (5 nM-5 uM), IWP2, XAV939, JW55, SM04690, LF3, WIK14, G007-LK and G244-LM.

[0065] The duration of bipotent PPEIII induction under the aforementioned regimes can take, for example, 2 to 14 days, depending on the concentration of each individual reagent used, to reach the inductive dosage for PPEIII differentiation.

[0066] Accordingly, in some embodiments, a culture of cells is provided that is enriched for HOXA3+, TBX1+/high, bipotent PPEIII cells. For example, in some embodiments, the culture comprises at least 50, 60, 70, 80, 90, 95, or 99% HOXA3+, TBX1+/high, bipotent PPEIII cells. The cell cultures can be made for example using the methods described herein.

[0067] The present inventors have also discovered signals that work synergistically to specify bipotent PPEIII cells towards thymic fate. For example, one can culture a bipotent PPEIII cell into a ventral PPEIII cell, i.e., a TBX1dim, PAX1+, PAX9+, FOXG1+ cell that can further differentiate into a TEPC. For example, bipotent PPEIII cells can be cultured in a medium that activates WNT and BMP signals together, optionally with one or some or all of the following: FGF/ERK/MAPK signaling pathway inhibitor, an IGF signaling pathway activator, and a Sonic Hedgehog (SHH) signaling pathway inhibitor.

[0068] In some embodiments, the culture medium comprises a WNT activator and a BMP activator. In some embodiments, the culture medium comprises a WNT activator and a BMP activator and a SHH inhibitor. In some embodiments, the culture medium comprises a WNT activator and a BMP activator and an FGF/ERK/MAPK inhibitor. In some embodiments, the culture medium comprises a WNT activator and a BMP activator and an IGF/PI3K/AKT activator. In some embodiments, the culture medium comprises a WNT activator and a BMP activator and an FGF/ERK/MAPK inhibitor and an IGF/PI3K/AKT activator. In some embodiments, the culture medium comprises a WNT activator and a BMP activator and an FGF/ERK/MAPK inhibitor and a SHH inhibitor. In some embodiments, the culture medium comprises a WNT activator and a BMP activator and an FGF/ERK/MAPK inhibitor and an IGF/PI3K/AKT activator and a SHH inhibitor.

[0069] WNT signal activation can be achieved, for example, by WNT proteins and/or small molecule agonists targeting the WNT pathway. CHIR99021 (1 nM-10 uM) is in some embodiments used as a reagent in this regime for WNT activation, which works as a potent canonical WNT agonist by targeting GSK3b. Canonical WNT signal family members such as WNT3a (at least 1 ng/ml) works as alternative to activate WNT signaling. Noncanonical WNT proteins can be used in combination with canonical WNT proteins. In addition, R-spondin (RSOP) reagents can be used in combination with WNT proteins to enhance WNT signal activation. Canonical and noncanonical WNT proteins include, without limitation, WNT1, WNT2, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16. Small molecule WNT activators include, without limitation, CHIR99021. LiCL, WAY-316606, ABC99, IQ1. QS 11. SB-216763, BIO(6-bromoindirubin-3-oxime), LY2090314, DCA, 2-amino-4-13,4-(methylenedioxy) benzyl-amino]-6-(3-methoxyphenyl) pyrimidine.

[0070] BMP signal activation is achieved by BMP4 (at least 1 ng/ml). Other BMP family members can be added in the regime together with BMP4 or replace BMP4. These BMP family members include, without limitation, BMP1, BMP2, BMP3, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10, BMP11, BMP12, BMP13.

[0071] Exemplary FGF/ERK/MAPK signaling pathway inhibitors can include inhibitors at the various levels of the pathway. e.g., a FGF receptor antagonist including, without limitation, SU 5402 (1 nM-10 uM), SU6668, PD161570, PD166285, PD173073: a MAPK inhibitor including, without limitation, U0126 (5 nM-50 uM), PD98059; an ERK inhibitor including, without limitation, SCH772984, FR180204 and TCS ERK11e.

[0072] Exemplary IGF/PI3K/AKT signaling pathway activators include, without limitation, IGF-1 (at least 0.1 ng/ml), IGF-2 (at least 1 ng/ml) and insulin.

[0073] Exemplary SHH signaling pathway inhibitors include, without limitation, Vismodegib (0.1 nM-1 uM), XL-139 (0.1 nM-1 uM), GANT61, MK-4101, HPI-4, Jervine, cyclopamine, PF-5274857, JK184, LY2940680 or Sonidegib. SHH agonizing and antagonizing signals can drive cell fate in such bipotent PPEIII cells further towards thymus and parathyroid fate respectively. Exemplary SHH signaling pathway activators driving further differentiation towards parathyroid include, without limitation. SAG (0.1 nM-1 uM) or SAG 21k (0.1 nM-1 uM).

[0074] The duration of induction of bipotent PPEIII cells into TBX1dim, PAX1+, PAX9+, FOXG1+ ventral PPEIII cells under the aforementioned regime can be for example 3-5 days. In some cases, it can take 1 to 10 days, depending on the concentration of each individual reagent used and the responsiveness of different samples.

[0075] Accordingly, in some embodiments, a culture of cells is provided that is enriched forTBX1dim, PAX1+, PAX9+, FOXG1+ ventral PPEIII cells. For example, in some embodiments, the culture comprises at least 50, 60, 70, 80, 90, 95, or 99% TBX1dim, PAX1+, PAX9+, FOXG1+ ventral PPEIII cells. The cell cultures can be made for example using the methods described herein.

[0076] The present inventors have also discovered one can culture and differentiate a TBX1dim, PAX1+, PAX9+, FOXG1+ ventral PPEIII cell to a TEPC (e.g., FOXN1+, PSMB11+, EPCAM+). A TEPC can further mature into functional cTECs and mTECs either in vivo after transplantation or in vitro through further signal induction. cTECs are mature TECs and can further differentiate to AIRE+ mTEC lineage cells. To generate TEPCs, ventral PPEIII cells can be cultured in a medium that contains one, some or all of the followings: IFN type I and/or II signaling activators, FGF signaling pathway activators, and IGF signaling pathway activators, optionally with inhibition of SHH signaling pathways and/or inhibition of the canonical WNT signaling pathway

[0077] In some embodiments, the culture medium comprises an IFN type I and/or II signal activator and a FGF signal activator and an IGF signal activator. In some embodiments, the culture medium comprises an IFN type I and/or II signal activator and a FGF signal activator and an IGF signal activator and a SHH signal inhibitor. In some embodiments, the culture medium comprises an IFN type I and/or II signal activator and a FGF signal activator and an IGF signal activator and a canonical WNT signal inhibitor. In some embodiments, the culture medium comprises an IFN type I and/or II signal activator and a FGF signal activator and an IGF signal activator and a SHH signal inhibitor and a canonical WNT signal inhibitor.

[0078] IGF signaling pathway activators, SHH signaling pathway inhibitors, FGF signaling pathway activators, and canonical WNT signaling pathway inhibitors can be selected as described above.

[0079] IFN type I and/or II signal activation can be achieved with, without limitation, IFN-alpha (at least 1 U/ml), IFN-beta (at least 1 pg/ml), IFN-gamma (at least 0.05 ng/ml).

[0080] Accordingly, in some embodiments, a culture of cells is provided that is enriched for FOXN1+, PSMB11+, EPCAM+ TEPCs. For example, in some embodiments, the culture comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99% FOXN1+, PSMB11+, EPCAM+ TEPC cells. The cell cultures can be made for example using the methods described herein.

[0081] In some embodiments, one can combine the above methods, for example to start with DE or AFG or AFG-plus cells, differentiate them into bipotent PPEIII cells, which in turn can optionally be differentiated into ventral PPEIII cells and optionally subsequently into TEPC cells.

[0082] Any of the cells made as described herein, e.g., HOXA3+, TBX1+/high, bipotent PPEIII cells: TBX1 dim, PAX1+, PAX9+, FOXG1+ ventral PPEIII cells: or FOXN1+, PSMB11+, EPCAM+ TEPC cells can be administered to a human, for example to treat a disorder. In some embodiments the cells are derived from the individual human, differentiated by one or more of the methods described herein and introduced to the same individual. In this case the cells are termed autologous. In other embodiments, cells from one or more individuals are differentiated by one or more of the methods described herein and introduced into a different individual. In this case, the cells are termed allogeneic. For allogenic transplantation, in some embodiments, the cells will be partly or fully HLA-matched to the individual.

[0083] Optionally, the cells can be genetically modified before introduction into an individual. For example, in embodiments in which the recipient has a defective gene, the cells can be modified to have a wildtype copy of the relevant gene, and then can be introduced into the individual. Any method available can be used to modify the cells. For example, in some embodiments, a targeted nuclease introduces a double-stranded break at a target site in a chromosome of the cells and homology directed repair (HDR) is used in the presence of a polynucleotide that is introduced into the double-stranded break. In some embodiments, for example, the targeted nuclease is selected from the group consisting of an RNA-guided nuclease domain, a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN) and a megaTAL. In some embodiments, CRISPR/Cas9 or other CRISPR methods are used to target and introduce the double-stranded break and introduce the polynucleotide into the cell.

[0084] The cells described herein can be used to ameliorate, for example, any diseases or disorders involving defective thymic epithelial cells. Exemplary individuals receiving the cells can include, for example, individuals born with congenital thymic aplasia, due to 22q11 Deletion Syndrome, or mutations in TBX1, FOXN1 or CHD7, for example who can present with T-cell immunodeficiency. In other embodiments, the cells described herein can be administered to human patients before or during or after hematopoietic stem cell transplantation (HSCT), for example to mitigate the injury caused to thymic cells by acute graft-versus-host disease (aGVHD). In other embodiments, the cells described herein can be administered to individuals having compromised thymic function, for example but not limited to, caused by infection, cancer, irradiation, medication or iatrogenic surgical removal due to cardiac surgeries. In other embodiments, the cells described herein can be administered to elderly individuals, e.g., over 50, 55, 60, 65, 70, 75, or 80 years old, for example individuals experiencing reduced thymic function due to age (immune senescence).

[0085] In some embodiments, the cells as described herein can be used to induce tolerance to transplanted solid organs (for example but not limited to kidney, heart, lung, islet cells or liver). In some embodiments, autologous or HLA-matched allogeneic cells as described herein are used to induce stable donor-specific immune tolerance during allogeneic solid organ transplant. This can reduce the dependence of patients on immunosuppressive drugs and address the current shortage of HLA-matched donor organs.

[0086] In some embodiments, the cells as described herein can be administered to a recipient of pancreatic islet (-cells) to treat type I diabetes mellitus (DM). Thymic dysfunction contributes to the development of type I DM through impaired presentation of tissue-restricted antigens and/or negative selection of autoreactive T cells, thus is permissive of the production of self-reactive T cells that target and destroy islet cells. The thymic transplant can eliminate these self-reactive T cells through negative selection (clonal deletion) of autoreactive T cells on AIRE-positive thymic epithelial cells that express tissue restricted antigens (including pro-insulin) and through the generation of regulatory T cells to further protect islet function through induction of central tolerance.

[0087] In some embodiments, the cells as described herein can be cultured in a bioreactor. Optionally, the bioreactor further comprises T-cells, which can be amplified. This approach, for example, can be used to enable highly efficient in vitro T cell production for any T cell editing/engineering purpose and solves the limitations in T cell generation for any type of T cell therapies.

[0088] In some embodiments, the cells as described herein can be formed into organoids or synthetic organs. In some embodiments, 3D thymic organoids are formed by coculturing in vitro derived TEPCs with mesenchymal cells in a synthetic matrix. Exemplary matrices include but are not limited to hydrogels containing elastin-like protein (ELP) and hyaluronic acid. These 3D thymic organoids can be used in any of the aforementioned applications in place of TEPCs generated in 2D monolayer cultures. Mesenchymal cells can be primary cells derived from human thymic tissues or differentiated in vitro from pluripotent stem cells.

EXAMPLES

[0089] The outline of the stepwise differentiation protocol to thymic epithelial progenitor cells using human pluripotent stem cells is illustrated in FIG. 1. The entire course of in vitro differentiation can be divided into 5 stages based on the phenotype of the differentiated cells achieved at each stage under corresponding inductive signals: [0090] Stage 1: generation of DE cells from human pluripotent stem cells [0091] Stage 2: generation of AFG or AFG-plus cells from DE cells [0092] Stage 3: generation of bipotent PPEIII cells from AFG cells [0093] Stage 4: generation of ventral PPEIII cells from bipotent PPEIII cells [0094] Stage 5: generation of thymic epithelial progenitor cells (TEPCs) from ventral PPEIII cells

[0095] In vitro differentiation stage 1: generation of DE cells from human pluripotent stem cells.

[0096] The method to generate DE cells is adapted from the work of Loh (Loh, Ang et al, 2014, Cell Stem Cell). As shown in FIG. 2, such DE cells express endodermal markers such as SOX17, CXCR4, and FOXA2 in nearly 100% of the population (FIG. 2A).

[0097] In vitro differentiation stage 2: generation of AFG or AFG-plus cells from DE cells

[0098] The method of AFG generation provided in this present patent is based on inhibition of both TGF and BMP signaling pathways (a.k.a, dual SMAD inhibition). Dual SMAD inhibition was first reported by Green et al (Green, Chen et al. 2011 Nature Biotechnology) to induce AFG cells.

[0099] In the disclosed method, DE cells were cultured in basal medium containing dual SMAD inhibition reagents to induce AFG cells with or without low concentrations of ATRA for 2 days. For example, TGF signal inhibitor A 83-01 (1 uM) combined with BMP signal inhibitor LDN193189 (250 nM), can specify DE cells into AFG cells that express SOX2, FOXA2 and OTX2 (FIG. 2B). Optional addition of ATRA (5 nM-5 uM) can induce AFG-plus cells

[0100] In vitro differentiation stage 3: generation of bipotent PPEIII cells from AFG or AFG-plus cells

[0101] The present invention provided a novel approach to advance AFG or AFG-plus cells to bipotent PPEIII cells. As shown in FIGS. 4 and 5, continued dual SMAD inhibition together with PI3K/AKT pathway inhibition (e.g., PI 828 at 5 uM) plus canonical WNT signal inhibition (e.g., endo-1WR 1 at 250 nM) plus FGF/ERK/MAPK pathway activation (e.g., FGF 7 at 20 ng/ml and FGF10 at 20 ng/ml) and optimal ATRA signal activation (e.g., ATRA at 50-100 nM) for 3-7 days drives AFG or AFG-plus cells into bipotent PPEIII cell stage. Compared to AFG or AFG-plus cells, the expression of TBX1, HOXA3 and PAX9 in bipotent PPEIII cells is significantly increased while GATA4 and GATA6 expression is significantly decreased which inhibits the differentiation bifurcating to liver and lung development respectively. The highly controlled modulation of RA in combination of the aforementioned signals is necessary to achieve the synchronized expression of the bipotent PPEIII markers (HOXA3, PAX9, FOXG1 and TBX1), which are detected in >95% of cells in our bipotent PPEIII protocol. Notably, the RA treatment dosage in the experiments in FIG. 5 throughout conditions #3 through #5 was lower than what was used in FIGS. 3 and 4 which accounts for the overall lower absolute expression levels for TBX1 and HOXA3 in this set of experiments.

[0102] In vitro differentiation stage 4: generation of ventral PPEIII cells from bipotent PPEIII cells

[0103] Bipotent PPEIII cells are biologically defined by their dual capacity to give rise to thymus and parathyroid. The present inventors discovered critical signals that work synergistically to specify bipotent PPEIII cells towards thymic fate through ventral PPEIII stage. As shown in FIG. 3, TBX1dim, PAX1+, PAX9+, FOXG1+ ventral PPEIII cells were generated through combined activation of WNT signal (e.g., CHIR99021 at 2 uM). BMP signal (e.g., BMP4 at 10 ng/ml) and IGF signal (e.g., IGF2 at 10 ng/ml) together with FGF/ERK/MAPK signal inhibition (e.g., U0126 at 10 uM), and SHH signal inhibition (e.g., Vismodegib at 200 nM) for 5 days.

[0104] Dynamic downregulation of TBX1 was observed in ventral PPEIII cells under the aforementioned differentiation regime, while upregulation in GCM2 and persistent TBX1 expression remained in cells committed to parathyroid fate under SHH signal activation (FIG. 6).

[0105] In vitro differentiation stage 5: generation of TEPCs from ventral PPEIII cells

[0106] The present inventors discovered signals further driving the maturation of ventral PPEIII cells into TEPCs, which include at least an IFN type I or II signaling pathway activator: an FGF signaling pathway activator: and an IGF signaling pathway activator. Under the induction of IFN-alpha (1 U/ml), IFN-gamma (0.05 ng/ml), FGF7 and FGF10 (10 ng/ml), and IGF2 (20 ng/ml) for 5 days, the results in FIG. 6 showed the level of FOXN1 expression in the TEPCs differentiated under the aforementioned regime reached approximately 20% of the level found in purified human fetal thymic epithelial cells (EPCAM+ selected by MACS sorting).

[0107] The examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.