# US 11,299,709 B2 · Pluripotent stem cell and T cell differentiated therefrom and application thereof

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- Number: US 11,299,709 B2
- Document ID: US11299709B2
- Status: Granted
- Kind: B2
- Application number: 16/312,794
- Filed: 2018-01-11
- Published: 2020-07-02
- Granted: 2022-04-12
- Earliest claimed: 2017-12-30
- Assignees: Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences
- Applicants: Guangzhou Institutes of Biomedicine and Health Chinese Academy of Sciences
- Inventors: Jinyong Wang; Rongqun Guo; Mengyun Zhang; Lijuan Liu; Xiaofei Liu; Cui Lv; Juan Du
- Web page: https://patentgrep.com/patent/US11299709B2

## CPC

- A61K 35/17: Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes (when activated by a specific antigen A61K39/00)
- C12N 5/0647: {Haematopoietic stem cells; Uncommitted or multipotent progenitors}
- C07K 14/47: from mammals
- C12N 5/0636: {T lymphocytes}
- C12N 15/85: for animal cells
- C12N 15/907: {in mammalian cells}
- C12N 2500/00: Specific components of cell culture medium
- C12N 2500/24: Iron; Fe chelators; Transferrin
- C12N 2501/115: Basic fibroblast growth factor (bFGF, FGF-2)
- C12N 2501/125: Stem cell factor [SCF], c-kit ligand [KL]
- C12N 2501/145: Thrombopoietin [TPO]
- C12N 2501/155: Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
- C12N 2501/16: Activin; Inhibin; Mullerian inhibiting substance
- C12N 2501/165: Vascular endothelial growth factor [VEGF]
- C12N 2501/2303: Interleukin-3 (IL-3)
- C12N 2501/2306: Interleukin-6 (IL-6)
- C12N 2501/26: Flt-3 ligand (CD135L, flk-2 ligand)
- C12N 2501/999: Small molecules not provided for elsewhere
- C12N 2502/1394: Bone marrow stromal cells; whole marrow
- C12N 2506/02: from embryonic cells
- C12N 2506/11: from blood or immune system cells
- C12N 2506/45: from artificially induced pluripotent stem cells
- C12N 2510/00: Genetically modified cells
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

The present invention provides a pluripotent stem cell comprising a co-expression vector in which Runx1 and Hoxa9 are of in tandem, and a T cell differentiated therefrom and application thereof. In the present invention, Pluripotent stem cells inducibly co-expressing exogenous Runx1 and Hoxa9 are successfully established by introducing an exogenous vector co-expressing Runx1 and Hoxa9 into pluripotent stem cells. The pluripotent stem cells are directionally differentiated into T-lineage progenitor cells and will be developed into T cells. The pluripotent stem cell-derived T cells obtained by the method of the present invention are not only functionally normal but also have no tumorigenic risk.

## Claims

### Claim 1

A method for directed differentiation of T cells using pluripotent stem cell, comprising the steps of:
(1) integrating an expression vector wherein a nucleic acid sequence encoding Runx1 and a nucleic acid sequence encoding Hoxa9 are linked in tandem into pluripotent stem cells at a Rosa26 site by gene recombination and performing resistance screening with Hygromycin B;
(2) culturing the pluripotent stem cells of step (1) with D0 medium, D2.5 medium, D3 medium, D4 medium, D5 medium, D6 medium and D7 medium sequentially, and directionally differentiating the same into hematopoietic stem cell precursors on day 11;
(3) co-culturing the hematopoietic stem cell precursors of step (2) with OP9-DL1 cells and inducing with Doxycycline for at least 10 days to induce expression of Runx1 and Hoxa9 to obtain T-lineage progenitor cells; and
(4) inducing the T-lineage progenitor cells of step (3) to differentiate into T cells, which are TCR β cells and/or TCR γ/δ cells,
wherein:
the D0 medium is a basic differentiation medium containing 3-8 ng/mL bone morphogenetic protein 4;
the D2.5 medium is a basic differentiation medium containing 3-8 ng/mL activin A and 3-8 ng/mL basic fibroblast growth factor;
the D3 medium is a basic differentiation medium containing 3-8 ng/mL Activin A, 3-8 ng/mL bone morphogenetic protein 4 and 3-8 ng/mL vascular endothelial growth factor;
the D4 medium is a basic differentiation medium containing 3-8 ng/mL bone morphogenetic protein 4 and 3-8 ng/mL vascular endothelial growth factor;
the D5 medium is a basic differentiation medium containing 3-8 ng/mL bone morphogenetic protein 4, 3-8 ng/mL vascular endothelial growth factor, 10-30 ng/mL recombinant mouse interleukin 3, 10-30 ng/mL recombinant mouse interleukin 6, 10-30 ng/mL recombinant mouse stem cell factor, 10-30 ng/mL recombinant human thrombopoietin and 10-30 ng/mL human Fms-associated tyrosine kinase 3 ligand;
the D6 medium is a basic differentiation medium containing 3-8 ng/mL bone morphogenetic protein 4, 3-8 ng/mL vascular endothelial growth factor, 10-30 ng/mL recombinant mouse interleukin 3, 10-30 ng/mL recombinant mouse interleukin 6, 10-30 ng/mL recombinant mouse stem cell factor, 10-30 ng/mL recombinant human thrombopoietin, and 10-30 ng/mL human Fms-associated tyrosine kinase 3 ligand and 1-2 μg/mL Doxycycline;
the D7 medium is a basic differentiation medium containing 10-30 ng/mL recombinant mouse interleukin 3, 10-30 ng/mL recombinant mouse interleukin 6, 10-30 ng/mL recombinant mouse stem cell factor, 10-30 ng/mL recombinant human thrombopoietin, and 10-30 ng/mL human Fms-associated tyrosine kinase 3 ligand and 1-2 μg/mL Doxycycline; and
the basic differentiation medium is IMDM medium comprising 10-20% fetal calf serum, 180-220 μg/mL iron-saturated transferrin, 4.5×10−4 M thioglycerol, 2 mM L-alanyl-L-glutamine dipeptide, and 0.5 mM ascorbic acid.

### Claim 2 (depends on 1)

The method of claim 1, wherein the D0 medium is a basic differentiation medium containing 5 ng/mL bone morphogenetic protein 4.

### Claim 3 (depends on 1)

The method of claim 1, wherein the D2.5 medium is a basic differentiation medium containing 5 ng/mL activin A and 5 ng/mL basic fibroblast growth factor.

### Claim 4 (depends on 1)

The method of claim 1, wherein the D3 medium is a basic differentiation medium containing 5 ng/mL Activin A, 5 ng/mL bone morphogenetic protein 4 and 5 ng/mL vascular endothelial growth factor.

### Claim 5 (depends on 1)

The method of claim 1, wherein the D4 medium is a basic differentiation medium containing 5 ng/mL bone morphogenetic protein 4 and 5 ng/mL vascular endothelial growth factor.

### Claim 6 (depends on 1)

The method of claim 1, wherein the D5 medium is a basic differentiation medium containing 5 ng/mL bone morphogenetic protein 4, 5 ng/mL vascular endothelial growth factor, 20 ng/mL recombinant mouse interleukin 3, 20 ng/mL recombinant mouse interleukin 6, 20 ng/mL recombinant mouse stem cell factor, 20 ng/mL recombinant human thrombopoietin and 20 ng/mL human Fms-associated tyrosine kinase 3 ligand.

### Claim 7 (depends on 1)

The method of claim 1, wherein the D6 medium is a basic differentiation medium containing 5 ng/mL bone morphogenetic protein 4, 5 ng/mL vascular endothelial growth factor, 20 ng/mL recombinant mouse interleukin 3, 20 ng/mL recombinant mouse interleukin 6, 20 ng/mL recombinant mouse stem cell factor, 20 ng/mL recombinant human thrombopoietin, and 20 ng/mL human Fms-associated tyrosine kinase 3 ligand and 1 μg/mL Doxycycline.

### Claim 8 (depends on 1)

The method of claim 1, wherein the D7 medium is a basic differentiation medium containing 20 ng/mL recombinant mouse interleukin 3, 20 ng/mL recombinant mouse interleukin 6, 20 ng/mL recombinant mouse stem cell factor, 20 ng/mL recombinant human thrombopoietin, 20 ng/mL human Fms-associated tyrosine kinase 3 ligand and 1 μg/mL Doxycycline.

### Claim 9 (depends on 1)

The method of claim 1, wherein the basic differentiation medium is IMDM medium comprising 15% fetal calf serum, 200 μg/mL iron-saturated transferrin, 4.5×10−4 M thioglycerol, 2 mM L-alanyl-L-glutamine dipeptide, and 0.5 mM ascorbic acid.

### Claim 10 (depends on 1)

The method of claim 1, wherein the pluripotent stem cells in step (1) are genetically-edited inducible pluripotent stem cells or embryonic pluripotent stem cell lines.