Materials and methods for the production of pluripotent stem cells

Abstract

Provided herein are methods of generating induced pluripotent stem cells (iPSCs) and isolated populations of the generated iPSCs. Also provided herein are methods of treating a subject in need thereof using the generated iPSCs or pharmaceutical compositions comprising the generated iPSCs.

Description

[Technical Field]

[0001] (Cross-reference to related applications) This application claims the interests of U.S. Patent Application No. 63 / 040,373, U.S. Patent Application No. 63 / 040,374, U.S. Patent Application No. 63 / 040,392, U.S. Patent Application No. 63 / 040,397, and U.S. Patent Application No. 63 / 040,398, filed on June 17, 2020, each of which is incorporated herein by reference in whole.

[0002] (Reference to electronically submitted sequence listings) This application includes a sequence listing submitted electronically via EFS-Web in ASCII format, filed as "14620-526-228_SEQ_LISTING", created on May 30, 2021, and having a size of 21,884 bytes. The sequence listing submitted via EFS-Web is part of this specification and is incorporated herein by reference in its entirety. 1. Field of Invention

[0003] This specification provides, in particular, methods for producing induced pluripotent stem cells (iPSCs), isolated populations of the produced induced pluripotent stem cells (iPSCs), and their uses. [Background technology]

[0004] 2.Background technology Pluripotent stem cells, such as embryonic stem (ES) cells and induced pluripotent stem cells (iPSCs), possess the ability to differentiate in the body and generate multiple cell types, exhibiting proliferative and developmental capabilities. Therefore, the therapeutic and scientific potential of these cells is exceptional, albeit uncertain, particularly because research has revealed that they can alter the gene expression profiles in somatic cells and epigenetically reprogram them into pluripotent stem cells (see, for example, Takahashi, K., & Yamanaka, S, Nat. Rev. Mol. Cell Biol., 2016, 17(3):183-93).

[0005] Embryonic stem cells can be derived from the inner cell mass of mammalian blastocysts; see, for example, Human Genes and Genomes: Science, Health, Society (Rosenberg, LE & Rosenberg, DD, 1st ed. 2012). In addition, somatic cell nuclear transfer (SCNT)-mediated reprogramming has also been used to generate pluripotent ES cells, and in some cases, cloned animals have been used (Wilmut, I., et al., Nature, 1997, 385:810-813, Wakayama, T., et al., Nature, 1998, 394:369-374). Nevertheless, SCNT has been plagued by various technical (e.g., epigenetic) barriers because the destruction of embryos and the introduction of mammalian genetic information into unfertilized eggs are controversial (Matoba, S. & Zhang, Y., supra; Kastenberg, ZJ & Odorico, JS, Transplant Rev., 2008, 22(3):215-22).

[0006] Alternative technologies for reprogramming somatic cells into pluripotent stem cells remain a subject of interest. Induced pluripotent stem cell (iPSC) technology emerged as one such option when Yamanaka et al. reported that the transcription factors Oct3 / 4, Sox2, Klf4, and c-Myc can confer pluripotency to adult somatic cells and to the ability to generate iPSCs (Takahashi, K., & Yamanaka, S, Cell, 2006, 126(4):663-76; Wernig, M., et al., Nature, 2007, 448:318-324; Maherali, N., et al., Cell Stem Cell, 2007, 1(1):55-70). [Overview of the Initiative]

[0007] 3. Outline of the Invention Given this background, improved materials and methods for reprogramming somatic cells into a pluripotent state are still needed. In one embodiment, a method for producing induced pluripotent stem cells (iPSCs) is provided herein, comprising: (a) contacting an isolated cell population with an activated culture, wherein the activated culture contains IL-15 and zoledronic acid; (b) culturing the isolated cell population in the activated culture to enrich and / or activate γδT cells in the isolated cell population; (c) transducing the γδT cells using a viral vector encoding one or more reprogramming factors; and (d) culturing the transduced γδT cells under conditions suitable for reprogramming mammalian somatic cells into a pluripotent state.

[0008] In certain embodiments, the activated culture further comprises IL-2.

[0009] In a particular embodiment, the viral vector is a Sendai virus (SeV) vector.

[0010] In certain embodiments, the method further includes obtaining an isolated population of cells from the subject.

[0011] In certain embodiments, the isolated cell population consists of peripheral blood mononuclear cells (PBMCs). In certain embodiments, the isolated cell population consists of terminally differentiated cells. In certain embodiments, the isolated cell population consists of mammalian cells. In certain embodiments, the isolated cell population consists of human cells.

[0012] In certain embodiments, the isolated cell population is cultured in an activated culture for 1 to 20 days. In certain embodiments, the isolated cell population is cultured in an activated culture for 1 to 17 days. In certain embodiments, the isolated cell population is cultured in an activated culture for 1 to 15 days. In certain embodiments, the isolated cell population is cultured in an activated culture for 1 to 13 days. In certain embodiments, the isolated cell population is cultured in an activated culture for 1 to 11 days. In certain embodiments, the isolated cell population is cultured in an activated culture for 1 to 9 days. In certain embodiments, the isolated cell population is cultured in an activated culture for 1 to 7 days. In certain embodiments, the isolated cell population is cultured in an activated culture for 1 to 5 days. In certain embodiments, the isolated cell population is cultured in an activated culture for 1 to 3 days. In certain embodiments, the isolated cell population is cultured in an activated culture for 12 to 72 hours. In certain embodiments, the isolated cell population is cultured in an activated culture for 12 to 60 hours. In certain embodiments, the isolated cell population is cultured in an activated culture for 12–48 hours. In certain embodiments, the isolated cell population is cultured in an activated culture for 12–36 hours. In certain embodiments, the isolated cell population is cultured in an activated culture for 12–24 hours. In certain embodiments, the isolated cell population is cultured in an activated culture for 8–16 hours. In certain embodiments, the isolated cell population is cultured in an activated culture for 4–8 hours. In certain embodiments, the isolated cell population is cultured in an activated culture for 2–4 hours.

[0013] In a particular embodiment, the isolated cell population is cultured in an activated culture for up to 13 days, up to 10 days, up to 9 days, up to 8 days, up to 7 days, up to 6 days, up to 5 days, up to 4 days, up to 3 days, up to 2 days, or up to 1 day.

[0014] In certain embodiments, the isolated cell population is cultured in the activation culture for up to 3 days. In certain embodiments, the isolated cell population is cultured in the activation culture for about 3 days. In certain embodiments, the isolated cell population is cultured in the activation culture for 50 to 80 hours. In certain embodiments, the isolated cell population is cultured in the activation culture for 55 to 75 hours. In certain embodiments, the isolated cell population is cultured in the activation culture for 60 to 75 hours. In certain embodiments, the isolated cell population is cultured in the activation culture for 70 to 75 hours.

[0015] In certain embodiments, the cell population isolated after being cultured in an activated culture contains 5% to 100% γδT cells. In certain embodiments, the cell population isolated after being cultured in an activated culture contains 5% to 95% γδT cells. In certain embodiments, the cell population isolated after being cultured in an activated culture contains 5% to 90% γδT cells. In certain embodiments, the cell population isolated after being cultured in an activated culture contains 5% to 85% γδT cells. In certain embodiments, the cell population isolated after being cultured in an activated culture contains 5% to 80% γδT cells. In certain embodiments, the cell population isolated after being cultured in an activated culture contains 5% to 75% γδT cells. In certain embodiments, the cell population isolated after being cultured in an activated culture contains 5% to 70% γδT cells. In certain embodiments, the cell population isolated after being cultured in an activated culture contains 5% to 65% γδT cells. In certain embodiments, the cell population isolated after being cultured in an activated culture contains 5% to 60% γδT cells. In certain embodiments, the cell population isolated after being cultured in an activated culture contains 5% to 55% γδT cells. In certain embodiments, the cell population isolated after being cultured in an activated culture contains 5% to 50% γδT cells. In certain embodiments, the cell population isolated after being cultured in an activated culture contains 5% to 45% γδT cells. In certain embodiments, the cell population isolated after being cultured in an activated culture contains 5% to 40% γδT cells. In certain embodiments, the cell population isolated after being cultured in an activated culture contains 5% to 35% γδT cells. In certain embodiments, the cell population isolated after being cultured in an activated culture contains 15% to 35% γδT cells. In certain embodiments, the cell population isolated after being cultured in an activated culture contains 25% to 35% γδT cells. In certain embodiments, the isolated cell population after being cultured in an activated culture contains 30%–35% γδT cells. In certain embodiments, the isolated cell population after being cultured in an activated culture contains 5%–30% γδT cells.In certain embodiments, after culturing in an activation culture, the isolated cell population comprises 5% to 25% γδ T cells. In certain embodiments, after culturing in an activation culture, the isolated cell population comprises 5% to 20% γδ T cells. In certain embodiments, after culturing in an activation culture, the isolated cell population comprises 5% to 15% γδ T cells.

[0016] In certain embodiments, after culturing in an activation culture, the isolated cell population comprises less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 45%, less than 40%, less than 35%, or less than 30% γδ T cells. In certain embodiments, after culturing in an activation culture, the isolated cell population comprises less than 35% γδ T cells.

[0017] In certain embodiments, the method further comprises enriching γδ T cells in the isolated cell population.

[0018] In certain embodiments, the γδ T cells are enriched by cell - aggregate concentration. In certain embodiments, at least a portion of the γδ T cells are activated to Vγ9 + γδ T cells in step (b).

[0019] In certain embodiments, at least a portion of the γδ T cells are activated to Vγ9δ2 + γδ T cells in step (b).

[0020] In certain embodiments, one or more reprogramming factors are selected from the group consisting of OCT3 / 4, SOX2, KLF4, LIN28, and c - Myc.

[0021] In certain embodiments, in step (d), the transduced γδT cells are cultured in the presence of one or more supporting cell layers. In certain embodiments, in step (d), the transduced γδT cells are cultured in the presence of a single layer of supporting cells. In certain embodiments, the supporting cell layer comprises mouse embryonic fibroblasts (MEFs).

[0022] In certain embodiments, the method further comprises isolating and / or purifying the prepared iPSCs. In certain embodiments, the method further comprises administering the isolated iPSCs to the target.

[0023] In certain embodiments, the method further includes differentiating iPSCs in vitro into cells of a desired cell type.

[0024] In certain embodiments, the method further comprises administering differentiated cells to a target. In certain embodiments, the target is a mammal. In certain embodiments, the target is a human. In certain embodiments, the target has hyperproliferative disorders or hematopoietic cancer.

[0025] In certain embodiments, the generated iPSCs are negative for the Sendai virus (SeV) vector. In certain embodiments, the generated iPSCs are derived from γδT cells. In certain embodiments, the generated iPSCs have rearrangement genes at the TRG and TRD loci. In certain embodiments, the generated iPSCs are not derived from αβT cells.

[0026] In certain embodiments, the generated iPSCs do not produce polymerase chain reaction (PCR) products from the TCRA and TCRB loci. In some embodiments, the generated iPSCs have Vγ9 and Vδ2 gene configurations. In certain embodiments, the generated iPSCs are genomically stable without chromosome loss. In certain embodiments, the genomic stability of the generated iPSCs is determined by karyotype analysis. In certain embodiments, the generated iPSCs can be grown in a culture medium without supporting cells after adoption.

[0027] In another embodiment, induced pluripotent stem cells (iPSCs) prepared according to a method provided herein are provided herein.

[0028] In another embodiment, a pharmaceutical composition comprising an iPSC provided herein and a pharmaceutically acceptable excipient is provided herein.

[0029] In another embodiment, differentiated cells prepared according to a method provided herein are provided herein.

[0030] In another embodiment, a pharmaceutical composition comprising differentiated cells provided herein and a pharmaceutically acceptable excipient is provided herein.

[0031] In yet another embodiment, a method for treating a subject in need of treatment is provided herein, comprising: (i) obtaining a cell population comprising peripheral blood mononuclear cells (PBMCs) from the subject; (ii) reprogramming γδT cells in the cell population to produce iPSCs according to a method for producing iPSCs provided herein; and (iii) optionally differentiating the iPSCs into one or more desired cell types, and then administering the produced iPSCs or a pharmaceutical composition containing the produced iPSCs to the subject. In certain embodiments, the subject is human. In certain embodiments, the subject has hyperproliferative disorder or hematopoietic cancer.

[0032] In yet another embodiment, an isolated population of induced pluripotent stem cells (iPSCs) is provided herein, wherein the isolated population of iPSCs comprises pluripotent cells, the pluripotent cells expressing one or more reprogramming factors and / or the pluripotent cells contain nucleotide sequences encoding rearrangements of the TRG and TRD genes.

[0033] In yet another embodiment, a method for producing induced pluripotent stem cells (iPSCs) is provided herein, comprising the steps of (a) enriching and / or activating γδT cells in an isolated cell population, and (b) reprogramming the γδT cells into a pluripotent state. In yet another embodiment, induced pluripotent stem cells (iPSCs) produced according to the method provided herein are provided herein.

[0034] In yet another embodiment, an isolated population of induced pluripotent stem cells (iPSCs) comprising pluripotent cells, wherein the pluripotent cells include means for expressing one or more reprogramming factors, and / or the pluripotent cells include means for encoding the rearrangement of TRG and TRD genes, is provided herein. [Brief explanation of the drawing]

[0035] [Figure 1] This shows the abundance of TCRVγ9+γδT cells in concentrated intercellular aggregates of PBMC cultures stimulated with Zol+IL-2+IL-15 at various days. The numbers in the representative FACS plots indicate the frequency of TCRγδ and αβT cells (top row) and TCRVγ9+ cells (bottom row) in all PBMCs, as well as in γδT cells, at day 3 (left column), day 8 (center column), and day 13 (right column) of PBMCs stimulated with Zol+IL-2+IL-15. Arrows represent parental and progeny gates. [Figure 2A]This shows microscopic observations of iPSC colonies derived from PBMC cultures stimulated with Zol+IL-2+IL-15 for 3 days. Figure 2A shows a representative microscopic image of iPSCs on the MEF-supported cell layer, appearing as round colonies with tight, smooth boundaries and dense intracellular boundaries. The elongated cells in the background are mitomycin C-treated MEF-supported cell layers. [Figure 2B] This shows microscopic observations of iPSC colonies derived from PBMC cultures stimulated with Zol+IL-2+IL-15 for 3 days. Figure 2B shows representative microscopic images of iPSC colonies from three individual clones at various passages, namely passages 3, 4, 5, and 7. The iPSC colonies were located on the irradiated MEF-supported cell layer. [Figure 3] This paper demonstrates the evaluation of gene rearrangements at the TRG locus using the IdentiClone® TCRG gene rearrangement assay. Genomic DNA was isolated from all five colonies. Genomic PCR was performed using primers from the IdentiClone® TCRG gene rearrangement assay kit, following the manufacturer's protocol. Representative peaks show the amplicon size (in base pairs) from the genomic PCR of clones A, B, C, D, and E. [Figure 4] This study evaluates gene rearrangements at the TRG and TRD loci between iPSC colonies. Representative agarose gel images show amplification of genomic DNA from clones A, B, and C mediated by either Vγ9 or Vδ2 forward primers combined with various joining region primers. Genomic DNA from the 22Rv1 cell line was used as a negative control. [Figure 5-1] This shows a representative sequence alignment demonstrating the similarity between the sequence obtained from the amplicon (from clone B) and the human TCR Vγ9 and Vδ2 sequences. [Figure 5-2] This is a continuation of Figure 5-1. [Figure 6A-1]This study evaluates the sequence homology between genomic DNA amplified amplicons and the TRGV9 and TRDV2 genes for five iPSC clones. Genomic DNA was isolated from all five iPSC clones, and genomic PCR was performed using primers for TCRVγ9(FP), JP1 / JP2, JP or J1 / J2(RV), and TCRVδ2(FP), Jδ1(RP), or Jδ3(RP). The amplification products were gel-eluted, top-cloned, and sequenced. The sequences were subjected to BLAST against the whole human genome. Figure 6A shows clone A. Representative sequence alignments show the sequence homology between the amplicons and the TRGV9 and TRDV2 gene sequences. [Figure 6A-2] This is a continuation of Figure 6A-1. [Figure 6B] This study evaluates the sequence homology between genomic DNA amplified amplicons and TRGV9 and TRDV2 for five iPSC clones. Genomic DNA was isolated from all five iPSC clones, and genomic PCR was performed using primers for TCRVγ9(FP), JP1 / JP2, JP or J1 / J2(RV) and TCRVδ2(FP), Jδ1(RP) or Jδ3(RP). The amplification products were gel-eluted, top-cloned, and sequenced. The sequences were subjected to BLAST against the whole human genome. Figure 6B shows clone C. Representative sequence alignments show the sequence homology between the amplicons and the TRGV9 and TRDV2 gene sequences. [Figure 6C-1] This study evaluates the sequence homology between genomic DNA amplified amplicons and TRGV9 and TRDV2 for five iPSC clones. Genomic DNA was isolated from all five iPSC clones, and genomic PCR was performed using primers for TCRVγ9(FP), JP1 / JP2, JP or J1 / J2(RV) and TCRVδ2(FP), Jδ1(RP) or Jδ3(RP). The amplification products were gel-eluted, top-cloned, and sequenced. The sequences were subjected to BLAST against the whole human genome. Figure 6C shows clone D. Representative sequence alignments show the sequence homology between the amplicons and the TRGV9 and TRDV2 gene sequences. [Figure 6C-2]This is a continuation of Figure 6C-1. [Figure 6D-1] This study evaluates the sequence homology between genomic DNA amplified amplicons and TRGV9 and TRDV2 for five iPSC clones. Genomic DNA was isolated from all five iPSC clones, and genomic PCR was performed using primers for TCRVγ9(FP), JP1 / JP2, JP or J1 / J2(RV) and TCRVδ2(FP), Jδ1(RP) or Jδ3(RP). The amplification products were gel-eluted, top-cloned, and sequenced. The sequences were subjected to BLAST against the whole human genome. (Figure 6D shows clone E). Representative sequence alignments show the sequence homology between the amplicons and the TRGV9 and TRDV2 gene sequences. [Figure 6D-2] This is a continuation of Figure 6D-1. [Figure 7A] This section describes the characterization of iPSCs derived from γδ T cells. Figure 7A shows representative agarose gel images of RT-PCR amplicons for Oct3 / 4, Nanog, Sox2, Lin28, Sendai virus (SeV), GAPDH, TCRα, and β derived from iPSC clones A, B, and C. [Figure 7B] This shows the characterization of γδT cell-derived iPSCs. Figure 7B shows overlay immunohistochemistry (IHC) images visualizing cells positive for DAPI (blue only), markers (Nanog / Oct3 / 4 / Sox2, red only), or DAPI+ marker (pink) among iPSC clones. [Figure 7C] This section describes the characterization of γδT cell-derived iPSCs. Figure 7C shows a representative histogram of the frequency of cells positive for SSEA-4 and TRA1-60 surface expression, as well as Oct-3 and Sox2 intracellular expression, among iPSC clones A, B, C, D, and E. [Figure 8A] This shows the evaluation of the genomic stability of iPSC clones B, C, D, and E via karyotype analysis. Figure 8A is clone B. [Figure 8B]This shows the evaluation of the genomic stability of iPSC clones B, C, D, and E via karyotype analysis. Figure 8B is clone D. [Figure 8C] This shows the evaluation of the genomic stability of iPSC clones B, C, D, and E via karyotype analysis. Figure 8C is clone C. [Figure 8D] This shows the evaluation of the genomic stability of iPSC clones B, C, D, and E via karyotype analysis. Figure 8D shows clone E. [Figure 9] Representative bright-field microscopy images are shown of support cell-based iPSC colonies from all five clones (clones A-E) adopted under conditions without support cells, in the presence of different matrix and culture medium combinations. [Modes for carrying out the invention]

[0036] 5. Modes for Carrying Out the Invention This disclosure provides, in part, an improved method for generating iPSCs from T cells, particularly γδT cells.

[0037] γδ T cells are a subset of T lymphocytes that express a different TCR than that expressed by αβ T cells, which are the major subset of T lymphocytes in human peripheral blood (Kalyan, S. & Kabelitz, D., Cell Mol. Immunol., 2013, 10(1):21-29). Vγ9 Vδ2 T cells are a major subset of γδ T cells and exhibit significant effector function against tumor cells (Tyler, CJ, et al., Cellular Immunology, 2015, 296(1):10-21; Silva-Santos.B., Nat Rev Immunol., 2015, 15:683-91). Furthermore, unlike αβ T cells, the antigen recognition and antitumor efficiency of Vγ9 Vδ2 T cells are unrestricted by the major histocompatibility complex (MHC) (Kalyan, S. & Kabelitz, D., supra). For these reasons, Vγ9 Vδ2 T cells are considered an attractive option for cancer immunotherapy and have been clinically explored and utilized (Kakimi, K., et al., Transl Lung Cancer Res., 2014, 3(1):23-33).

[0038] 5.1. Definition As used herein, the terms “about” or “approximately” refer to a quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length that varies by 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. The range of a quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length may be ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% with respect to the quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length.

[0039] A "cell culture medium" (also referred to herein as "culture medium," "culture," or "culture") is a medium for culturing cells that contains nutrients that maintain cell viability and support proliferation. A cell culture medium may contain, in appropriate combinations, any or any of the following: salts, buffers, amino acids, glucose or other sugars, antibiotics, serum or serum substitutes, and other components such as peptide growth factors. Cell culture media commonly used for specific cell types are known to those skilled in the art. Several non-limiting examples are provided herein.

[0040] As used herein, “cell line” typically refers to a population of cells that are largely or substantially identical, derived from a single ancestral cell or from a defined population and / or substantially identical population of ancestral cells. A cell line may be maintained, or can be maintained, in a culture for a long period of time (e.g., months, years, or an unlimited period). It may undergo a spontaneous or induced process of transformation that confers an unlimited culture life to the cells. A cell line includes all cell lines recognized as such in the art. It will be understood that cells acquire mutations and, possibly epigenetic changes, over time, such that at least some characteristics of the individual cells in a cell line may differ from one another.

[0041] As used herein, terms such as “differentiate” and “differentiation” refer to the process by which unspecialized (or undetermined) or relatively unspecialized cells acquire the characteristics of specialized cells, such as blood cells or muscle cells. Differentiated or differentiated cells are cells that occupy a more specialized (or determined) position within a cell line. A cell is determined when, under normal circumstances, it continues to differentiate into a particular cell type or subset of cell types, and has progressed in its differentiation pathway to a point where, under normal circumstances, it cannot differentiate into a different cell type or revert to a relatively undifferentiated cell type.

[0042] As used herein, the term “coding” refers to the inherent properties of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, which has either a defined sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties arising therefrom, that serve as a template for the synthesis of other polymers and macromolecules in a biological process. Thus, in a cell or other biological system, if the transcription and translation of mRNA corresponding to a gene produces a protein, that gene codes for a protein. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is typically provided in a sequence listing, and the non-coding strand, which is used as a template for the transcription of a gene or cDNA, can be referred to as coding for a protein or other product of that gene or cDNA.

[0043] As used herein, the term “exogenous” is intended to mean that the reference molecule or activity is introduced into a host cell. This molecule can be introduced, for example, by introducing the coding nucleic acid into the host genetic material, such as by integration into the host chromosome, or as non-chromosomal genetic material such as a plasmid. Therefore, when used in relation to the expression of coding nucleic acids, this term refers to the introduction of the coding nucleic acid into a cell in an expressible form. The term “endogenous” refers to the reference molecule or activity present in the host cell. Similarly, when used in relation to the expression of coding nucleic acids, this term refers to the expression of a coding nucleic acid that is contained within the cell and is not exogenous.

[0044] As used herein, the term “expression” refers to cellular processes involved in the production of RNA and proteins, and, if applicable, the secretion of proteins, including, but not limited to, transcription, translation, folding, modification, and processing, where applicable. “Expression products” include RNA transcribed from genes and polypeptides obtained by the translation of mRNA transcribed from genes.

[0045] As used herein, the terms “induced pluripotent stem cells” or “iPSCs” refer to stem cells derived from differentiated adult cells that have been induced or modified (i.e., reprogrammed) into cells capable of differentiating into all three tissues of the embryo or cortex: mesoderm, endoderm, and ectoderm.

[0046] As used herein, terms such as “isolated” are intended to mean, when used in reference to cells, cells that substantially contain at least one component, as the cells referred to are found in nature. This term includes cells separated from some or all of the components as they are found in their natural environment. This term also includes cells separated from at least one, some or all of the components as the cells are found in an environment in which they do not naturally occur. Thus, isolated cells are partially or completely separated from other substances so that they grow, are stored or exist as they are found in nature or in an environment in which they do not naturally occur. Specific examples of isolated cells include partially pure cells, substantially pure cells, and cells cultured in a medium in which they do not naturally occur.

[0047] As used herein, terms such as “purify” refer to increasing purity. For example, purity can be increased to at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%.

[0048] As used herein, the term “pluripotency” refers to a cell’s ability to form all lineages of the body or cell body (i.e., the embryonic body). For example, embryonic stem cells are a type of pluripotent stem cell that can form cells from each of the three germ layers: the ectoderm, mesoderm, and endoderm. Pluripotency is a continuum of developmental potential, from incomplete or partial pluripotent cells (e.g., epiblast stem cells or EpiSCs) that cannot produce a complete organism to more primitive and more pluripotent cells (e.g., embryonic stem cells) that can produce a complete organism.

[0049] As used herein, the term “population” refers, when used in relation to T lymphocytes, to a group of cells containing two or more T lymphocytes. An isolated population of T lymphocytes may contain only one type of T lymphocyte or two or more types of T lymphocytes. An isolated population of T lymphocytes may be a homogeneous population of one type of T lymphocyte or a heterogeneous population of two or more types of T lymphocytes. An isolated population of T lymphocytes may also be a heterogeneous population containing T lymphocytes and at least other cells, such as B cells, macrophages, neutrophils, erythrocytes, hepatocytes, endothelial cells, epithelial cells, muscle cells, brain cells, etc. A heterogeneous population may contain 0.01% to about 100% T lymphocytes. Therefore, an isolated population of T lymphocytes may contain at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% T lymphocytes. An isolated population of T lymphocytes may contain only one type of T lymphocyte or a mixture of two or more types of T lymphocytes. An isolated population of T lymphocytes may contain one or more, or all, of the different types of T lymphocytes, including but not limited to those disclosed herein. An isolated population of T lymphocytes may contain all known types of T lymphocytes. In an isolated population of T lymphocytes containing two or more types of T lymphocytes, the proportion of each type of T lymphocyte may range from 0.01% to 99.99%. The isolated population may also be a clonal population of T lymphocytes, where all T lymphocytes in the population are clones of a single T lymphocyte.

[0050] A "recombinant" polynucleotide is a polynucleotide that is not in its natural state, for example, by containing a nucleotide sequence not found in nature, or by being in a situation other than that in nature, such as being separated from a nucleotide sequence that is normally adjacent in nature, or being adjacent (or consecutive) to a nucleotide sequence that is not normally adjacent. For example, the sequence in question can be cloned into a vector, or otherwise can be recombined with one or more further nucleic acids.

[0051] As used herein, “reprogramming” refers to the process of altering or reversing the differentiation state of somatic cells. Cells can be partially or terminally differentiated before reprogramming. Reprogramming includes the complete reversal of the differentiation state of somatic cells (e.g., T cells) to a pluripotent state. Reprogramming also includes the partial reversal of the differentiation state of somatic cells to a state that makes the cells more sensitive to complete reprogramming to a pluripotent state when subjected to further operations as described herein. Such contact may result in the expression of specific genes by the cells, and this expression contributes to reprogramming. In certain embodiments of the present invention, somatic cell reprogramming makes somatic cells pluripotent and ES-like. The resulting cells are referred herein to as reprogrammed pluripotent somatic cells or induced pluripotent stem cells (iPSCs). In some embodiments, reprogramming also includes the partial reversal of the differentiation state of somatic cells to a multipotent state.

[0052] Reprogramming is not simply maintaining the existing undifferentiated state of cells that are already pluripotent, nor is it maintaining the existing non-differentiated state of cells that are already pluripotent (e.g., hematopoietic stem cells). Reprogramming is also not simply promoting the self-regeneration or proliferation of cells that are already pluripotent or multipotent. In some embodiments, the methods described herein contribute to establishing a pluripotent state through reprogramming. In some embodiments, the methods described herein may be carried out not on cells that are already pluripotent or multipotent, but on fully differentiated cells and / or certain types of cells (e.g., γδT cells).

[0053] As used herein, “reprogramming factor” refers to a gene, RNA, or protein that promotes or contributes to cell reprogramming, (e.g., in vitro). Examples of target reprogramming factors for reprogramming somatic cells to be pluripotent in vitro include Oct3 / 4, Klf4, c-Myc, Nanog, Sox2, and Lin28, as well as any gene / protein that can replace one or more of these in a method of reprogramming somatic cells, for example, in vitro.

[0054] As used herein, the terms “T lymphocyte” and “T cell” are used interchangeably and refer to the primary type of leukocyte that completes maturation in the thymus and plays various roles in the immune system, including the identification of specific foreign antigens in the body and the activation and deactivation of other immune cells. T lymphocytes can be any T lymphocyte, such as cultured T lymphocytes, e.g., primary T lymphocytes, or T lymphocytes from cultured T cell lines, e.g., T lymphocytes from Jurkat, SupT1, etc., or T lymphocytes obtained from mammals. T lymphocytes can be CD3+ cells. T lymphocytes can be any type of T lymphocyte, including but not limited to any developmental stage, such as CD4+ / CD8+ double-positive T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor infiltrating lymphocytes (TILs), memory T cells, naive T cells, regulatory T cells, gamma delta (γδ) T cells, etc. T lymphocytes can be nTreg (native Tregs), iTreg (inducible Tregs), CD8+ T cells, etc. + T regulatory cells may include Treg, Tr1 regulatory cells, and Th3 cells. Further types of helper T cells include Th3(Treg), Th17, Th9, or Tfh cells. Further types of memory T cells include central memory T cells (T CMCells), effector memory T cells (T EM Cells and T EMRA Examples of cells include T lymphocytes. T lymphocytes can also refer to genetically modified T lymphocytes, such as T lymphocytes that have been modified to express the T cell receptor (TCR) or chimeric antigen receptor (CAR). Furthermore, T lymphocytes can also be differentiated from stem cells, final angioblasts, CD34+ cells, HSCs (hematopoietic stem cells and progenitor cells), hematopoietic pluripotent progenitor cells, or T cell progenitor cells.

[0055] As used herein, the term “γδT cell” refers to a T cell having T cell receptors containing γ and δ chains on its surface.

[0056] As used herein, the term “selection marker” refers to a gene, RNA, or protein that, when expressed, confers a selectable phenotype to a cell, such as resistance to cytotoxic agents or cell division inhibitors (e.g., antibiotic resistance), nutritional prototrophy, or the expression of a particular protein that can be used as a basis for distinguishing cells that express that protein from cells that do not. Proteins whose expression can be readily detected, such as fluorescent or luminescent proteins, or enzymes that act on a substrate to produce a colored, fluorescent, or luminescent substance (“detectable markers”), constitute a subset of selection markers. The presence of selection markers linked to native expression regulators for genes that are normally selectively or exclusively expressed in pluripotent cells makes it possible to identify and select somatic cells reprogrammed to a pluripotent state. Various selectable marker genes can be used, including the neomycin resistance gene (neo), puromycin resistance gene (puro), guanine phosphoribosyl transferase (gpt), dihydrofolate reductase (DHFR), adenosine deaminase (ada), puromycin-N-acetyltransferase (PAC), hygromycin resistance gene (hyg), multidrug resistance gene (mdr), thymidine kinase (TK), hypoxanthine-guanine phosphoribosyltransferase (HPRT), and the hisD gene. Detectable markers include green fluorescent protein (GFP), blue, sapphire, yellow, red, orange, and cyan fluorescent proteins, as well as any of these variants.Luciferases (e.g., firefly or sea urchin luciferase) are also useful. As will be apparent to those skilled in the art, as used herein, the term “selection marker” may refer to a gene or a gene expression product, e.g., an encoded protein.

[0057] In some embodiments, a selection marker confers a growth and / or survival benefit to cells expressing it compared to cells that do not express it or cells that express it at significantly lower levels. Such a growth and / or survival benefit typically occurs when cells are maintained under certain conditions, i.e., “selective conditions.” To ensure effective selection, the cell population can be maintained under conditions and for a sufficient period of time such that cells that do not express the marker do not grow and / or survive and are eliminated from the population, or their numbers are reduced to only a very small proportion of the population. The process of selecting cells that express a marker conferring a growth and / or survival benefit by maintaining the cell population under selective conditions to largely or completely eliminate cells that do not express the marker is referred to herein as “positive selection,” and the marker is said to be “useful for positive selection.” Negative selection and markers useful for negative selection are also of interest in certain methods described herein. The expression of such markers confers disadvantages to growth and / or survival to cells that express the markers compared to cells that do not express them or that express them at significantly lower levels (or, conversely, cells that do not express the markers have advantages in growth and / or survival compared to cells that express them). Therefore, cells that express the markers can be largely or completely excluded from a cell population when maintained under selective conditions for a sufficient period of time.

[0058] As used herein, “feeder cell” (or “feeder”) is a term referring to one cell type co-cultured with a second cell type, where the supporting cell provides stimuli, growth factors, and nutrients for supporting the second cell type, thereby providing an environment in which the second cell type can proliferate, expand, or differentiate. Supporting cells may, by choice, originate from a different species than the cells they support. For example, certain species of human cells, including stem cells, may be supported by primary cultures of mouse embryonic fibroblasts or immortalized mouse embryonic fibroblasts. In another example, peripheral blood-derived cells or transformed leukemia cells support the expansion and maturation of natural killer cells. Supporting cells may typically be inactivated by irradiation or treatment with antimitotic agents such as mitomycin to prevent them from proliferating beyond the supporting cells when co-cultured with other cells. Supporting cells may include endothelial cells, stromal cells (e.g., epithelial cells or fibroblasts), and leukemia cells. While not limited to the above, one specific supporting cell type could be human supporting cells, such as human skin fibroblasts. Another supporting cell type could be mouse embryonic fibroblasts (MEFs). In general, various supporting cells can be used partially to maintain pluripotency, direct differentiation towards a particular lineage, enhance proliferative capacity, and promote maturation into specialized cell types such as effector cells.

[0059] As used herein, “feeder-free” (FF) environment means an environment such as culture conditions, cell culture, or culture medium that is essentially free of supporting cells or stromal cells and / or has not been pre-conditioned by supporting cell culture. “Pre-conditioned” medium means a medium that has been collected after supporting cells have been cultured in the medium for a certain period of time, such as at least one day. Pre-conditioned medium contains many mediator substances, including growth factors and cytokines secreted by supporting cells cultured in the medium. In some embodiments, a feeder-free environment is free of both supporting cells and stromal cells and has not been pre-conditioned by supporting cell culture.

[0060] The term "pluripotency-related gene" refers to a gene whose expression occurs in pluripotent stem cells under normal conditions (e.g., in the absence of genetic engineering or other manipulations designed to alter gene expression), is typically limited to pluripotent stem cells, and is important for its own functional identity. It is understood that polypeptides encoded by genes functionally related to pluripotency may exist as maternal factors in oocytes. This gene may be expressed by at least some cells of the embryo, for example, for at least a portion of the preimplantation period and / or in the adult germ cell precursor.

[0061] The term "pluripotency factor" is used to refer to the expression products of pluripotency-related genes, such as polypeptides encoded by the genes. In some embodiments, pluripotency factors are not normally substantially expressed in somatic cell types (excluding germ cells or their precursors) that make up the body of an adult animal. For example, the average level of a pluripotency factor in ES cells may be at least 50 or 100 times higher than its average level in terminally differentiated cell types present in the body of an adult mammal. In some embodiments, pluripotency factors are essential for maintaining the viability or pluripotency of ES cells and / or ES cells induced using conventional methods in vivo. Therefore, if the gene encoding the factor is knocked out or inhibited (i.e., its expression is eliminated or substantially reduced), ES cells will not form, will die, or, in some embodiments, will differentiate. In some embodiments, inhibiting the expression of genes having functions related to pluripotency in ES cells (e.g., resulting in a reduction of at least 50%, 60%, 70%, 80%, 90%, 95%, or more from the mean steady-state levels of RNA transcripts and / or proteins encoded by the gene) results in viable but no longer pluripotent cells. In some embodiments, the gene is characterized by a decrease in its expression in ES cells when the cell differentiates into a terminal cell (e.g., resulting in a reduction of at least 50%, 60%, 70%, 80%, 90%, 95%, or more from the mean steady-state levels of RNA transcripts and / or proteins encoded by the gene).

[0062] As used herein, “pluripotency-inducing gene” refers to a gene whose expression contributes to the reprogramming of somatic cells into a pluripotent state. “Pluripotency-inducing factor” refers to the expression product of a pluripotency-inducing gene. A pluripotency-inducing factor may, but does not have to be, a pluripotency factor. The expression of an exogenously introduced pluripotency-inducing factor may be transient, that is, it may be required during at least a portion of the reprogramming process to induce pluripotency and / or establish a stable pluripotent state, but may not be required thereafter to maintain pluripotency. For example, the factor may induce the expression of endogenous genes that have functions related to pluripotency. These genes may then be able to maintain the reprogrammed cells in a pluripotent state.

[0063] The term "polynucleotide" is used herein interchangeably with "nucleic acid" and refers to a polymer of nucleosides. Typically, the polynucleotides of the present invention consist of nucleosides naturally found in DNA or RNA (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) linked by phosphodiester bonds. However, the term includes molecules containing nucleosides or nucleoside analogs that include chemically or biologically modified bases, modified backlines, etc., whether or not they are found in naturally occurring nucleic acids, and such molecules may be preferred for certain applications. When this application refers to polynucleotides, it is understood that both DNA and RNA, and both single-stranded and double-stranded forms in each case (and complements of each single-stranded molecule) are provided. As used herein, “polynucleotide sequence” may refer to the sequence information (i.e., a sequence of letters used as abbreviations for bases) that biochemically characterizes the substance polynucleotide and / or a particular nucleic acid. Unless otherwise indicated, the polynucleotide sequences presented herein are presented in the 5' to 3' direction.

[0064] A “polypeptide” refers to a polymer of amino acids. The terms “protein” and “polypeptide” are used interchangeably herein. Peptides are relatively short polypeptides, typically about 2 to 60 amino acids in length. Polypeptides as used herein typically contain amino acids, such as the 20 most commonly found L-amino acids in proteins. However, other amino acids and / or amino acid analogs known in the art may be used. One or more amino acids in a polypeptide may be modified by the addition of chemical components, such as carbohydrate groups, phosphate groups, fatty acid groups, linkers for conjugation and functionalization. A polypeptide is still considered a “polypeptide” even if a non-polypeptide moiety is covalently or non-covalently associated with it. Exemplary modifications include glycosylation and palmitoylation. Polypeptides can be purified from natural sources, produced using recombinant DNA technology, and synthesized through chemical means such as conventional solid-phase peptide synthesis. As used herein, the terms “polypeptide sequence” or “amino acid sequence” may refer to sequence information (i.e., a sequence of letters or three-letter codes used as abbreviations for amino acid names) that biochemically characterize the polypeptide material itself and / or the polypeptide. Unless otherwise indicated, polypeptide sequences presented herein are presented in the direction from the N-terminus to the C-terminus.

[0065] When applied to isolated cells, terms such as “process,” “to process,” and “process” include subjecting cells to any kind of process or conditions, or performing any kind of operation or procedure on cells. When applied to subjects, these terms refer to providing medical or surgical treatment, care, or management to an individual.

[0066] 5.2. Abbreviations A list of abbreviations used in this disclosure is provided in Table 1 below.

[0067] [Table 1]

[0068] 5.3. Method for producing induced pluripotent stem cells (iPSCs) In one embodiment, a method for reprogramming somatic cells (e.g., T cells) into a poorly differentiated state is provided herein. The resulting cells are referred herein to as reprogrammed somatic cells. The reprogrammed somatic cells may be reprogrammed somatic cells in various differentiated states. In some embodiments, the reprogrammed somatic cells are induced pluripotent stem cells. This disclosure is based in part on the surprising discovery that various combinations of factors, for example, a combination of zoledronic acid and interleukin-15 (IL-15), can activate γδT cells and thus improve the efficiency of pluripotency induction in non-pluripotent mammalian T cells transformed with transcription factors. Thus, in one embodiment, this disclosure relates to non-pluripotent mammalian γδT cells (e.g., Vγ9 + The present invention provides a method for inducing pluripotency in γδT cells, comprising contacting peripheral blood mononuclear cells (PBMCs) with an activated culture containing IL-15 and zoledronic acid.

[0069] In some embodiments, a method for reprogramming somatic cells is provided herein, comprising: (a) contacting an isolated cell population with an activated culture, the activated culture comprising IL-15 and zoledronic acid; (b) culturing the isolated cell population in the activated culture to enrich and / or activate γδT cells in the isolated cell population; (c) transducing the γδT cells using one or more viral vectors encoding one or more reprogramming factors; and (d) culturing the transduced γδT cells under conditions suitable for reprogramming mammalian somatic cells into a poorly differentiated state. In some embodiments, the poorly differentiated state is a pluripotent state. In some embodiments, the poorly differentiated state is a pluripotent state.

[0070] In some more specific embodiments, a method for producing induced pluripotent stem cells (iPSCs) is provided herein, comprising: (a) contacting an isolated cell population with an activated culture, wherein the activated culture comprises IL-15 and zoledronic acid; (b) culturing the isolated cell population in the activated culture to enrich and / or activate γδT cells in the isolated cell population; (c) transducing the γδT cells using one or more viral vectors encoding one or more reprogramming factors; and (d) culturing the transduced γδT cells under conditions suitable for reprogramming mammalian somatic cells into a pluripotent state.

[0071] In certain embodiments, the activated culture further comprises one or more additional agents or compounds, for example, to improve the efficiency of activation or induction. In one embodiment, the activated culture further comprises interleukin-2 (IL-2).

[0072] Accordingly, in some embodiments, a method for reprogramming somatic cells is provided herein, comprising: (a) contacting an isolated cell population with an activated culture, the activated culture comprising IL-15, zoledronic acid, and IL-2; (b) culturing the isolated cell population in the activated culture to enrich and / or activate γδT cells in the isolated cell population; (c) transducing the γδT cells using one or more viral vectors encoding one or more reprogramming factors; and (d) culturing the transduced γδT cells under conditions suitable for reprogramming mammalian somatic cells into a poorly differentiated state.

[0073] In some more specific embodiments, a method for producing induced pluripotent stem cells (iPSCs) is provided herein, comprising: (a) contacting an isolated cell population with an activated culture, the activated culture comprising IL-15, zoledronic acid, and IL-2; (b) culturing the isolated cell population in the activated culture to enrich and / or activate γδT cells in the isolated cell population; (c) transducing the γδT cells using one or more viral vectors encoding one or more reprogramming factors; and (d) culturing the transduced γδT cells under conditions suitable for reprogramming mammalian somatic cells into a pluripotent state.

[0074] In certain embodiments, the method further includes obtaining an isolated cell population from a subject. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human.

[0075] In certain embodiments, the isolated cell population is peripheral blood cells, umbilical cord blood cells, or bone marrow cells. In one embodiment, the isolated cell population is peripheral blood mononuclear cells (PBMCs).

[0076] Methods for identifying reprogrammed mammalian somatic cells having a poorly differentiated or pluripotent state are known in the art. For example, in some embodiments, reprogrammed somatic cells are identified by selecting cells that express appropriate selection markers. In some embodiments, reprogrammed somatic cells are further evaluated for pluripotency. The presence of pluripotency indicates that the somatic cell has been reprogrammed to a pluripotent state.

[0077] The differentiation states of cells are a continuous spectrum, with terminal differentiation at one end and dedifferentiation (pluripotency) at the other. Reprogramming, as used herein, refers to the process of changing or reversing the differentiation state of a somatic cell that can partially or terminally differentiate. Reprogramming includes complete reversal, as well as partial reversal, of the differentiation state of a somatic cell. In other words, as used herein, the term “reprogramming” includes any transition to a less differentiated state along the spectrum of cell differentiation states. For example, reprogramming includes reverting a pluripotent cell back to a pluripotent cell, or reverting a terminally differentiated cell back to either a pluripotent or pluripotent cell. In one embodiment, somatic cell reprogramming is returning a somatic cell to a pluripotent state. In another embodiment, somatic cell reprogramming is returning a somatic cell to a pluripotent state. Thus, as used herein, the term “less differentiated state” is a relative term and includes complete dedifferentiation and partial differentiation.

[0078] The term "pluripotency" refers to many characteristics associated with pluripotency, including, for example, the ability to differentiate into all types of cells, as well as expression patterns characteristic of pluripotent cells, such as the expression of pluripotency genes, the expression of other ES cell markers, and a distinctive expression profile known at an overall level as the "stem cell molecular sign" or "stem cell-like."

[0079] Therefore, to evaluate the pluripotency of reprogrammed somatic cells, such cells may be analyzed for various growth characteristics and ES cell-like morphologies. In some embodiments, cells may be subcutaneously injected into immunodeficient SCID mice to induce teratomas (standard assay for ES cells). ES-like cells can differentiate into embryoid bodies (another ES-specific characteristic). Furthermore, ES-like cells can be differentiated in vitro by adding certain growth factors known to drive differentiation into specific cell types. Self-renewal ability, marked by the induction of telomerase activity, is another pluripotency characteristic that can be monitored.

[0080] In some embodiments, a functional assay of reprogrammed somatic cells may be performed by introducing the reprogrammed somatic cells into a blastocyst to determine whether the cells are capable of giving rise to all cell types. If the reprogrammed cells are capable of forming some cell types of the body, they are pluripotent; if the reprogrammed cells are capable of forming all cell types of the body, including germ cells, they are pluripotent.

[0081] In other embodiments, the expression of individual pluripotency genes in reprogrammed somatic cells may be examined to evaluate their pluripotency characteristics.

[0082] In addition, the expression of other ES cell markers may be evaluated. Stage-specific embryo 1 antigens-1, -3, and -4 (SSEA-1, SSEA-3, SSEA-4) are glycoproteins specifically expressed during early embryonic development and are markers of ES cells (Solter and Knowles, 1978, Proc. Natl. Acad. Sci. USA 75:5565-5569; Kannagi et al., 1983, EMBO J 2:2355-2361).

[0083] High expression of the enzyme alkaline phosphatase (AP) is another marker associated with undifferentiated embryonic stem cells (Wobus et al., 1984, Exp. Cell 152:212-219; Pease et al., 1990, Dev. Biol. 141:322-352). Other stem / progenitor cell markers include intermediate nerve filament nestin (Lendahl et al., 1990, Cell 60:585-595; Dah-Istrand et al., 1992, J. Cell Sci. 103:589-597), membrane glycoprotein prominin / AC133 (Weigmann et al., 1997, Proc. Natl. Acad. USA 94:12425-12430; Corbeil et al., 1998, Blood 91:2625-22626), transcription factor Tcf-4 (Korinek et al., 1998, Nat. Genet. 19:379-383; Lee et al., 1999, J. Biol. Chem. 274. 1566-1572), and transcription factor Cdx1 (Duprey et al. Examples include al., 1988, Genes Dev. 2: 1647-1654; Subramania'n et al., 1998, Difference 64: 11-18).

[0084] In some embodiments, expression profiling of reprogrammed somatic cells may be used to assess their pluripotency. Pluripotent cells, such as embryonic stem cells, and multipotent cells, such as adult stem cells, are known to have characteristic patterns in their overall gene expression profiles. This characteristic pattern is called the "stem cell molecular sign" or "stem cell-like." See, for example, Ramalho-Santos et al., Science 298:597-600 (2002); Ivanova et al., Science 298:601-604.

[0085] Somatic cells can be reprogrammed to acquire any of the complete sets of pluripotency traits, and thus are pluripotent. Alternatively, somatic cells can be reprogrammed to acquire only a subset of pluripotency traits. In another option, somatic cells can be reprogrammed to be multipotent.

[0086] activation culture In certain embodiments, the isolated cell population is cultured in an activated culture for a first period. In certain embodiments, the first period is 1 to 20 days. In certain embodiments, the first period is 1 to 17 days. In certain embodiments, the first period is 1 to 15 days. In certain embodiments, the first period is 1 to 13 days. In certain embodiments, the first period is 1 to 11 days. In certain embodiments, the first period is 1 to 9 days. In certain embodiments, the first period is 1 to 7 days. In certain embodiments, the first period is 1 to 5 days. In certain embodiments, the first period is 1 to 3 days. In certain embodiments, the first period is 12 to 72 hours. In certain embodiments, the first period is 12 to 60 hours. In certain embodiments, the first period is 12 to 48 hours. In certain embodiments, the first period is 12 to 36 hours. In a particular embodiment, the first period is 12 to 24 hours. In a particular embodiment, the first period is 8 to 16 hours. In a particular embodiment, the first period is 4 to 8 hours. In a particular embodiment, the first period is 2 to 4 hours. In a particular embodiment, the first period is 4 to 8 hours. In a particular embodiment, the first period is 50 to 80 hours. In a particular embodiment, the first period is 4 to 8 hours. In a particular embodiment, the first period is 55 to 75 hours. In a particular embodiment, the first period is 4 to 8 hours. In a particular embodiment, the first period is 60 to 75 hours. In a particular embodiment, the first period is 4 to 8 hours. In a particular embodiment, the first period is 70 to 75 hours. In a particular embodiment, the first period is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days.

[0087] In certain embodiments, the isolated cell population is cultured in the activated culture for a certain period of time or less. For example, the isolated cell population is cultured in the activated culture for up to 13 days, up to 12 days, up to 11 days, up to 10 days, up to 9 days, up to 8 days, up to 7 days, up to 6 days, up to 5 days, up to 4 days, up to 3 days, up to 2 days, or up to 1 day. In certain embodiments, the isolated cell population is cultured in the activated culture for up to 5 days. In certain preferred embodiments, the isolated cell population is cultured in the activated culture for up to 3 days. In certain preferred embodiments, the isolated cell population is cultured in the activated culture for about 3 days.

[0088] In certain embodiments, the isolated cell population after being cultured in an activated culture for a first period contains 5% to 100% γδT cells. In certain embodiments, the isolated cell population after being cultured in an activated culture for a first period contains 5% to 95% γδT cells. In certain embodiments, the isolated cell population after being cultured in an activated culture for a first period contains 5% to 90% γδT cells. In certain embodiments, the isolated cell population after being cultured in an activated culture for a first period contains 5% to 85% γδT cells. In certain embodiments, the isolated cell population after being cultured in an activated culture for a first period contains 5% to 80% γδT cells. In certain embodiments, the isolated cell population after being cultured in an activated culture for a first period contains 5% to 75% γδT cells. In certain embodiments, the isolated cell population after being cultured in an activated culture for a first period contains 5% to 70% γδT cells. In certain embodiments, the isolated cell population after being cultured in an activated culture for a first period contains 5% to 65% γδT cells. In certain embodiments, the isolated cell population after being cultured in an activated culture for a first period contains 5% to 60% γδT cells. In certain embodiments, the isolated cell population after being cultured in an activated culture for a first period contains 5% to 55% γδT cells. In certain embodiments, the isolated cell population after being cultured in an activated culture for a first period contains 5% to 50% γδT cells. In certain embodiments, the isolated cell population after being cultured in an activated culture for a first period contains 5% to 45% γδT cells. In certain embodiments, the isolated cell population after being cultured in an activated culture for a first period contains 5% to 40% γδT cells. In certain embodiments, the isolated cell population after being cultured in an activated culture for a first period contains 5% to 35% γδT cells. In a particular embodiment, the isolated cell population, after being cultured in an activated culture for a first period, contains 15%–35% γδT cells.In certain embodiments, the isolated cell population after being cultured in an activated culture for a first period contains 25% to 35% γδT cells. In certain embodiments, the isolated cell population after being cultured in an activated culture for a first period contains 30% to 35% γδT cells. In certain embodiments, the isolated cell population after being cultured in an activated culture for a first period contains 5% to 30% γδT cells. In certain embodiments, the isolated cell population after being cultured in an activated culture for a first period contains 5% to 25% γδT cells. In certain embodiments, the isolated cell population after being cultured in an activated culture for a first period contains 5% to 20% γδT cells. In certain embodiments, the isolated cell population after being cultured in an activated culture for a first period contains 5% to 15% γδT cells.

[0089] In certain embodiments, the isolated cell population after being cultured in an activated culture for a first period contains about 100%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5% γδT cells. In certain embodiments, the isolated cell population after being cultured in an activated culture for a first period contains less than about 90%, about 80%, about 70%, about 60%, about 50%, about 45%, about 40%, about 35%, or less than 30% γδT cells.

[0090] In one embodiment, the isolated cell population after being cultured in an activated culture for a first period contains less than 60% γδT cells. In another embodiment, the isolated cell population after being cultured in an activated culture for a first period contains less than 55% γδT cells. In yet another embodiment, the isolated cell population after being cultured in an activated culture for a first period contains less than 50% γδT cells. In yet another embodiment, the isolated cell population after being cultured in an activated culture for a first period contains less than 45% γδT cells. In yet another embodiment, the isolated cell population after being cultured in an activated culture for a first period contains less than 40% γδT cells. In yet another embodiment, the isolated cell population after being cultured in an activated culture for a first period contains less than 35% γδT cells.

[0091] In certain embodiments, the γδ T cells in the isolated cell population after being cultured in an activated culture for a first period contain 5% to 100% TCRVγ9+ T cells. In certain embodiments, the γδ T cells in the isolated cell population after being cultured in an activated culture for a first period contain 5% to 95% TCRVγ9+ T cells. In certain embodiments, the γδ T cells in the isolated cell population after being cultured in an activated culture for a first period contain 5% to 90% TCRVγ9+ T cells. In certain embodiments, the γδ T cells in the isolated cell population after being cultured in an activated culture for a first period contain 5% to 85% TCRVγ9+ T cells. In certain embodiments, the γδ T cells in the isolated cell population after being cultured in an activated culture for a first period contain 5% to 80% TCRVγ9+ T cells. In certain embodiments, the γδ T cells in the isolated cell population after being cultured in an activated culture for a first period contain 5% to 75% TCRVγ9+ T cells. In certain embodiments, the γδ T cells in the isolated cell population after being cultured in an activated culture for a first period contain 5% to 70% TCRVγ9+ T cells. In certain embodiments, the γδ T cells in the isolated cell population after being cultured in an activated culture for a first period contain 5% to 65% TCRVγ9+ T cells. In certain embodiments, the γδ T cells in the isolated cell population after being cultured in an activated culture for a first period contain 5% to 60% TCRVγ9+ T cells. In certain embodiments, the γδ T cells in the isolated cell population after being cultured in an activated culture for a first period contain 5% to 55% TCRVγ9+ T cells. In certain embodiments, the γδ T cells in the isolated cell population after being cultured in an activated culture for a first period contain 5% to 50% TCRVγ9+ T cells. In certain embodiments, the γδ T cells in the isolated cell population after being cultured in an activated culture for a first period contain 5% to 45% TCRVγ9+ T cells.In certain embodiments, after culturing in an activation culture over a first period, the γδ T cells in the isolated cell population comprise 5% to 40% TCRVγ9+ T cells. In certain embodiments, after culturing in an activation culture over a first period, the γδ T cells in the isolated cell population comprise 5% to 35% TCRVγ9+ T cells. In certain embodiments, after culturing in an activation culture over a first period, the γδ T cells in the isolated cell population comprise 5% to 30% TCRVγ9+ T cells. In certain embodiments, after culturing in an activation culture over a first period, the γδ T cells in the isolated cell population comprise 5% to 25% TCRVγ9+ T cells. In certain embodiments, after culturing in an activation culture over a first period, the γδ T cells in the isolated cell population comprise 5% to 20% TCRVγ9+ T cells. In certain embodiments, after culturing in an activation culture over a first period, the γδ T cells in the isolated cell population comprise 5% to 15% TCRVγ9+ T cells.

[0092] In certain embodiments, after culturing in an activation culture over a first period, the γδ T cells in the isolated cell population are about 100%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5% TCRVγ9 + T cells (also known as Vγ9 + T cells). In certain embodiments, after culturing in an activation culture over a first period, the γδ T cells in the isolated cell population are less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, or less than about 30% TCRVγ9 + T cells.

[0093] In one embodiment, the γδ T cells in a cell population isolated after being cultured in an activated culture for a first period contain less than 60% TCRVγ9+ T cells. In another embodiment, the γδ T cells in a cell population isolated after being cultured in an activated culture for a first period contain less than 55% TCRVγ9+ T cells. In yet another embodiment, the γδ T cells in a cell population isolated after being cultured in an activated culture for a first period contain less than 50% TCRVγ9+ T cells. In yet another embodiment, the γδ T cells in a cell population isolated after being cultured in an activated culture for a first period contain less than 45% TCRVγ9+ T cells. In yet another embodiment, the γδ T cells in a cell population isolated after being cultured in an activated culture for a first period contain less than 40% TCRVγ9+ T cells. In yet another embodiment, the γδ T cells in a cell population isolated after being cultured in an activated culture for a first period contain less than 35% TCRVγ9+ T cells.

[0094] In certain embodiments, the method further comprises enriching γδT cells in an isolated cell population. In certain embodiments, γδT cells are enriched by intercellular aggregate enrichment.

[0095] In a particular embodiment, at least a portion of the activated γδT cells in step (b) are Vγ9 + These are γδT cells.

[0096] In a particular embodiment, at least a portion of the activated γδT cells in step (b) are Vγ9δ2 + These are γδT cells.

[0097] cell This disclosure is based on the discovery that isolated cell populations, such as isolated γδT cell populations, can be activated (e.g., in the presence of zoledronic acid and IL-15) and reprogrammed to be pluripotent by the introduction of transcription factors (e.g., by a Sendai virus vector).

[0098] The isolated cell populations of this disclosure include any T cells of the body that are not stem cells, germ cells, or iPSCs. Non-limited examples of non-iPSCs include T cells derived from any tissue of the body, including viscera, skin, bone, blood, nerve tissue, and connective tissue.

[0099] In certain embodiments, the isolated cell population is blood cells. In certain embodiments, the blood cells are preferably peripheral blood mononuclear cells (PMBCs) and may include all types of blood cells present in the entire differentiation process from hematopoietic stem cells to terminal differentiation into peripheral blood. In one embodiment, the blood cells include, for example, hematopoietic stem cells, lymphoid stem cells, lymphoid dendritic cell precursors, lymphoid dendritic cells, T lymphocyte precursors, T cells, B lymphocyte precursors, B cells, plasma cells, NK precursors, NK cells, monocytes, and macrophages.

[0100] In some embodiments, the isolated cell population may be peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor-infiltrating lymphocytes (TILs), or a combination thereof. In some embodiments, the isolated cell population is peripheral blood mononuclear cells (PBMCs).

[0101] In certain embodiments, the isolated cell population is T cells. In some embodiments, the isolated T cell population can be selected from the group consisting of CD4+ / CD8+ double-positive T cells, cytotoxic T cells, Th3 (Treg) cells, Th9 cells, Thαβ helper cells, Tfh cells, stem memory TSCM cells, central memory TCM cells, effector memory TEM cells, effector memory TEMRA cells, gamma delta T cells, and any combination thereof.

[0102] In some embodiments, the isolated cell populations are derived from easily accessible and minimally invasive cell types, such as fibroblasts, skin cells, umbilical cord blood cells, peripheral blood cells, and renal epithelial cells.

[0103] In certain embodiments, the isolated cell population consists of terminally differentiated cells. In certain embodiments, the isolated cell population consists of terminally differentiated T cells. In certain embodiments, the isolated cell population consists of terminally differentiated PBMC cells. In certain embodiments, the isolated cell population consists of terminally differentiated γδT cells.

[0104] The isolated cell populations of this disclosure may be derived from mammals, preferably humans, but include, but are not limited to, non-human primates, rodents (i.e., mice and rats), dogs, cats, horses, cattle, sheep, pigs, goats, and the like.

[0105] In a particular embodiment, the isolated cell population is mammalian cells.

[0106] In a particular embodiment, the isolated cell population is human cells.

[0107] In some embodiments, the isolated cell population is human PBMC cells.

[0108] Introduction of pluripotency-related genes This disclosure also relates to introducing endogenous loci, which are pluripotency-related genes, into an activated cell population. In some embodiments, such pluripotency-related genes can be introduced using an expression vector. In some embodiments, such pluripotency-related genes can be introduced using a CRISPR activation system having at least one sgRNA targeting a desired locus. In some embodiments, such pluripotency-related genes can be introduced by expression from a recombinant expression cassette introduced into target cells. In some embodiments, such pluripotency-related genes can be introduced by incubating cells in the presence of exogenous reprogramming transcription factor polypeptides.

[0109] In certain embodiments, the expression vector used to introduce pluripotency-related genes comprises modified viral polynucleotides derived from adenoviruses, Sendai viruses, herpesviruses, or retroviruses (such as lentiviral vectors). The expression vector is not limited to recombinant viruses and includes non-viral vectors such as DNA plasmids and in vitro transcribed mRNA. In one preferred embodiment, a Sendai virus vector is used.

[0110] To address safety issues arising from target cell genomes containing incorporated exogenous sequences, several modified gene protocols have been developed and can be used in the production methods described herein. These protocols generate potentially reduced-risk iPS cells and deliver reprogramming genes using non-integrated adenovirus (Stadtfeld, M., et al., Science, 2008, 322:945-949), transient transfection of reprogramming plasmids (Okita, K., et al., Science, 2008, 322:949-953), piggyBac transposition system (Woltjen, K., et al., Nature, 2009, 458:766-770; Yusa, et al., Nat. Methods, 2009, 6:363-369; Kaji, K., et al. (2009)), and Cre cleavable virus (Soldner, F., et al.). This includes al., Cell, 2009, 136:964-977, and oriP / EBNA1-based episome expression systems (Yu, J., et al., Science, 2009, 324(5928):797-801).

[0111] Non-limiting examples of pluripotency-related genes (genes encoding reprogramming transcription factors) include Oct3 / 4, Sox2, Nanog, Klf4, c-Myc, Nanog, Lin28, Nr5a2, Glis1, Cebpa, Esrrb, and Rex1. In some embodiments, the endogenous locus is Oct4 or Sox2.

[0112] In certain embodiments, the isolated cell population endogenously expresses at least one protein from the group consisting of Oct3 / 4 polypeptide, Klf4 polypeptide, c-Myc polypeptide, Sox2 polypeptide, Nanog polypeptide, Lin28 polypeptide, Nr5a2 polypeptide, Glis1 polypeptide, Cebpa polypeptide, Esrrb polypeptide, and Rex1 polypeptide. In certain embodiments, the isolated cell population does not endogenously express any reprogramming transcription factors.

[0113] In a particular embodiment, the reprogramming factors include Oct3 / 4, Sox2, Klf4, and c-Myc.

[0114] In a particular embodiment, the reprogramming factors are Oct3 / 4, Sox2, KLF4, c-Myc, and Lin28.

[0115] In a particular embodiment, the reprogramming factors are Oct3 / 4, Sox2, Klf4, and c-Myc.

[0116] Exogenous introduction of pluripotency genes can be carried out in several ways. In one embodiment, exogenously introduced pluripotency genes may be expressed from a chromosomal locus different from the endogenous locus of the pluripotency gene. Such chromosomal loci may be open chromatin loci and may contain one or more genes that are not essential for somatic cells. In other words, the desired chromosomal locus contains one or more genes whose disruption does not cause cell death. Exemplary chromosomal loci include, for example, the mouse ROSA26 locus and the type II collagen (Col2a1) locus (see Zambrowicz et al., 1997).

[0117] Exogenously introduced pluripotency genes can be expressed via inducible promoters so that their expression can be regulated as desired.

[0118] In alternative embodiments, exogenously introduced pluripotency genes may be transiently transfected into cells, either individually or as part of a cDNA expression library prepared from pluripotent cells. Such pluripotent cells may include embryonic stem cells, oocytes, blastomeres, inner cell mass cells, embryonic germ cells, embryoid body (embryonic) cells, morula-derived cells, teratoma (teratocarcinoma) cells, and pluripotent, partially differentiated embryonic stem cells isolated at later stages of the embryogenetic process.

[0119] cDNA libraries are prepared by conventional techniques. Briefly, mRNA is isolated from the organism of interest. RNA-dependent DNA polymerase is used for the first strand synthesis, using mRNA as a template. The second strand synthesis is performed using DNA-dependent DNA polymerase, which produces the cDNA product. Following conventional processing to facilitate cDNA cloning, the cDNA is inserted into an expression vector so that the cDNA is operably ligated to at least one regulatory sequence. The selection of expression vectors for use in relation to cDNA libraries is not limited to a specific vector. Any expression vector suitable for use in mouse cells is appropriate. In one embodiment, the promoter driving expression from the cDNA expression construct is an inductive promoter. The term regulatory sequence includes promoters, enhancers, and other expression regulatory elements. Exemplary regulatory sequences are described in Goeddel, Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, Calif. (1990). For example, any of the wide variety of regulatory sequences that control the expression of a DNA sequence when functionally ligated can be used in these vectors to express cDNA. Such useful regulatory sequences include, for example, the early and late promoters of SV40, the tet promoter, the earliest promoters of adenovirus or cytomegalovirus, the lac system, the trp system, the TAC or TRC system, the T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage λ, the regulatory regions of fd coat proteins, promoters of 3-phosphoglycerate kinase or other glycolytic enzymes, acid phosphatases such as the promoter of Pho5, the promoter of the yeast α-conjugation factor, the polyhedron promoter of the baculovirus system, and other sequences known to control the expression of genes in prokaryotic or eukaryotic cells or their viruses, as well as various combinations thereof.It should be understood that the design of expression vectors may depend on factors such as the selection of host cells to be transformed and / or the type of protein to be expressed. Furthermore, the copy number of the vector, its ability to control its copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered.

[0120] Exogenously introduced pluripotent genes can be expressed from inducible promoters. As used herein, the term “inducible promoter” refers to a promoter that, in the absence of an inducer (such as a chemical agent and / or biological agent), does not direct the expression of a functionally linked gene (including cDNA) to a low level, and whose ability to direct expression in response to an inducer is enhanced. Examples of inducible promoters include promoters that respond to heavy metals (CRC Boca Raton, Fla. (1991), 167-220; Brinster et al. Nature (1982), 296, 39-42), heat shock, and hormones (Lee et al. PNASUSA (1988), 85, 1204-1208; (1981), 294, 228-232; Klock et al. Nature (1987), 329, 734-736; Israel and Kaufman, Nucleic Acids Res. (1989), 17, 2589-2604), as well as promoters that respond to chemical agents such as glucose, lactose, galactose, or antibiotics.

[0121] Tetracycline-inducible promoters are an example of antibiotic-responsive inducible promoters. See Gossen et al., 2003. A tetracycline-inducible promoter comprises a minimal promoter operably ligated to one or more tetracycline operators. In the presence of tetracycline or one of its analogues, a transcription activator binds to the tetracycline operator sequence, which activates the minimal promoter and thus activates the transcription of the associated cDNA. Tetracycline analogues include any compound that exhibits structural homology to tetracycline and can activate a tetracycline-inducible promoter. Exemplary tetracycline analogues include, for example, doxycycline, chlorotetracycline, and anhydrotetracycline.

[0122] Accordingly, in one embodiment, the disclosure provides somatic cells possessing at least one pluripotency gene expressed as a transgene under an inducible promoter. Somatic cells possessing such inducible pluripotency transgenes may be more likely to be reprogrammed.

[0123] Any of the genetically engineered somatic cells of this disclosure may be used in the Method. In one embodiment, the somatic cells used in the Method contain only one endogenous pluripotency gene linked to a first selection marker, and the selection step is performed to select for the expression of the first selection marker. In an alternative embodiment, the somatic cells used in the Method contain any number of endogenous pluripotency genes, each linked to a different selection marker, and the selection step is performed to select for at least a subset of the selection markers. For example, the selection step may be performed to select for all selection markers linked to various endogenous pluripotency genes.

[0124] In alternative embodiments, the somatic cells used in this method include a selection marker linked to an endogenous pluripotency gene, and further pluripotency genes expressed as transgenes under an inducible promoter. For these cells, the reprogramming method may include inducing the expression of the pluripotency transgenes and selecting the expression of the selection marker.

[0125] In certain embodiments, in step (d) described above, the transduced γδT cells are cultured in the presence of one or more supporting cell layers. In certain embodiments, in step (d), the transduced γδT cells are cultured in the presence of a monolayer of supporting cell layers. In certain embodiments, the supporting cell layer comprises mouse embryonic fibroblasts (MEFs). In certain embodiments, in step (d), the transduced γδT cells are cultured in the presence of a monolayer of supporting cell layers. In certain embodiments, in step (d), the transduced γδT cells are cultured in the presence of mitotically inactivated mouse embryonic fibroblasts (MEFs). In certain embodiments, in step (d), the transduced γδT cells are cultured under conditions that do not include supporting cells. In certain embodiments, in step (d), the transduced γδT cells are cultured in an iMatrix-511 coated plate.

[0126] In a particular embodiment, following step (d), the method further comprises isolating and / or purifying the iPSCs prepared in step (e).

[0127] In a particular, more specific embodiment, a method for producing induced pluripotent stem cells (iPSCs) is provided herein, comprising: contacting an isolated cell population with an activated culture, wherein the activated culture comprises IL-15, zoledronic acid, and IL-2; culturing the isolated cell population in the activated culture to enrich and / or activate γδT cells in the isolated cell population; transducing the γδT cells using a Sendai virus (SeV) vector encoding one or more reprogramming factors; and culturing the transduced γδT cells under conditions suitable for reprogramming mammalian somatic cells into a pluripotent state.

[0128] In a particular, more specific embodiment, a method for producing induced pluripotent stem cells (iPSCs) is provided herein, comprising: obtaining a population of cells isolated from a subject (e.g., human) (e.g., terminally differentiated cells such as peripheral blood mononuclear cells (PBMCs)); contacting the isolated cell population with an activated culture, wherein the activated culture comprises IL-15, zoledronic acid, and IL-2; culturing the isolated cell population in the activated culture to enrich and / or activate γδT cells in the isolated cell population; transducing the γδT cells using a Sendai virus (SeV) vector encoding one or more reprogramming factors; and culturing the transduced γδT cells under conditions suitable for reprogramming mammalian somatic cells into a pluripotent state.

[0129] In other, more specific embodiments, a method for producing induced pluripotent stem cells (iPSCs) is provided herein, comprising: obtaining a population of cells isolated from a subject (e.g., human) (e.g., terminally differentiated cells such as peripheral blood mononuclear cells (PBMCs)); contacting the isolated cell population with an activated culture, wherein the activated culture contains IL-15, zoledronic acid, and IL-2; culturing the isolated cell population in the activated culture for about 3 days to enrich and / or activate the γδT cells in the isolated cell population; transducing the γδT cells using a Sendai virus (SeV) vector encoding one or more reprogramming factors; and culturing the transduced γδT cells under conditions suitable for reprogramming mammalian somatic cells into a pluripotent state.

[0130] In other, more specific embodiments, a method for producing induced pluripotent stem cells (iPSCs) is provided herein, comprising: obtaining a population of cells isolated from a subject (e.g., human) (e.g., terminally differentiated cells such as peripheral blood mononuclear cells (PBMCs)); contacting the isolated cell population with an activated culture, wherein the activated culture contains IL-15, zoledronic acid, and IL-2; culturing the isolated cell population in the activated culture for approximately 3 days, wherein after culturing in the activated culture the isolated cell population contains less than 35% γδT cells; transducing the γδT cells using a Sendai virus (SeV) vector encoding one or more reprogramming factors; and culturing the transduced γδT cells under conditions suitable for reprogramming mammalian somatic cells into a pluripotent state.

[0131] In other, more specific embodiments, a method for producing induced pluripotent stem cells (iPSCs) is provided herein, comprising: obtaining a cell population (e.g., terminally differentiated cells such as peripheral blood mononuclear cells (PBMCs)) isolated from a subject (e.g., human); contacting the isolated cell population with an activated culture, wherein the activated culture contains IL-15, zoledronic acid, and IL-2; culturing the isolated cell population in the activated culture for approximately 3 days, wherein after culturing in the activated culture the isolated cell population contains less than 35% γδT cells; further enriching the γδT cells by intercellular aggregate concentration; transducing the γδT cells using a Sendai virus (SeV) vector encoding one or more reprogramming factors; and culturing the transduced γδT cells under conditions suitable for reprogramming mammalian somatic cells into a pluripotent state.

[0132] In other, more specific embodiments, a method for producing induced pluripotent stem cells (iPSCs) is provided herein, comprising: obtaining a cell population (e.g., terminally differentiated cells such as peripheral blood mononuclear cells (PBMCs)) isolated from a subject (e.g., human); contacting the isolated cell population with an activated culture, wherein the activated culture contains IL-15, zoledronic acid, and IL-2; culturing the isolated cell population in the activated culture for approximately 3 days, wherein after culturing in the activated culture the isolated cell population contains less than 35% γδT cells; optionally further enriching the γδT cells by intercellular aggregate concentration; transducing the γδT cells using a Sendai virus (SeV) vector encoding one or more reprogramming factors selected from the group consisting of OCT3 / 4, SOX2, KLF4, LIN28, and c-Myc; and culturing the transduced γδT cells under conditions suitable for reprogramming mammalian somatic cells into a pluripotent state.

[0133] In another more specific embodiment, a method for producing induced pluripotent stem cells (iPSCs) is provided, comprising: obtaining a cell population (e.g., terminally differentiated cells such as peripheral blood mononuclear cells (PBMCs)) isolated from a subject (e.g., human); contacting the isolated cell population with an activated culture, wherein the activated culture contains IL-15, zoledronic acid, and IL-2; and culturing the isolated cell population in the activated culture for approximately 3 days, wherein after culturing in the activated culture, the isolated cell population contains less than 35% γδT cells. A method is provided herein that comprises culturing sea urchins, optionally further enriching γδT cells by intercellular aggregate concentration, transducing γδT cells with a Sendai virus (SeV) vector encoding one or more reprogramming factors selected from the group consisting of OCT3 / 4, SOX2, KLF4, LIN28, and c-Myc, and culturing the transduced γδT cells under conditions suitable for reprogramming mammalian somatic cells into a pluripotent state in the presence of one or more layers of supporting cells.

[0134] In another more specific embodiment, a method for producing induced pluripotent stem cells (iPSCs) is provided, comprising: obtaining a cell population (e.g., terminally differentiated cells such as peripheral blood mononuclear cells (PBMCs)) isolated from a subject (e.g., human); contacting the isolated cell population with an activated culture, wherein the activated culture contains IL-15, zoledronic acid, and IL-2; and culturing the isolated cell population in the activated culture for approximately 3 days, wherein after culturing in the activated culture, the isolated cell population contains less than 35% γδT cells. A method is provided herein that comprises: further enriching γδT cells by intercellular aggregate concentration, optionally; transducing γδT cells using a Sendai virus (SeV) vector encoding one or more reprogramming factors selected from the group consisting of OCT3 / 4, SOX2, KLF4, LIN28, and c-Myc; and culturing the transduced γδT cells under conditions suitable for reprogramming mammalian somatic cells into a pluripotent state in the presence of a monolayer of supporting cells including mouse embryonic fibroblasts (MEFs).

[0135] iPSCs derived from γδT cells In certain embodiments, the generated iPSCs are derived from γδT cells. In certain embodiments, the generated iPSCs have rearranged genes at the TRG and TRD loci. In certain embodiments, the generated iPSCs do not produce polymerase chain reaction (PCR) products from the TCRG and TCRD loci.

[0136] In certain embodiments, the generated iPSCs do not originate from αβT cells.

[0137] In a particular embodiment, the produced iPSCs are negative for the Sendai virus (SeV) vector.

[0138] In certain embodiments, the generated iPSCs are genomically stable without chromosome loss. In one embodiment, the genomic stability of the generated iPSCs is determined by karyotype analysis.

[0139] In certain embodiments, the produced iPSCs can be grown and maintained in a culture medium that does not contain supporting cells after adoption.

[0140] In certain embodiments, the method further includes differentiating the produced iPSCs into a desired cell type in vitro or in vitro. In certain embodiments, the method further includes differentiating the produced iPSCs into a desired cell type in vitro. In certain embodiments, the method further includes differentiating the produced iPSCs into a desired cell type in vitro.

[0141] In certain embodiments, the method further includes administering the generated iPSCs.

[0142] In certain embodiments, the method further comprises administering differentiated cells derived from iPSCs prepared herein.

[0143] 5.4. T cell-derived induced pluripotent stem cells (iPSCs) Furthermore, isolated populations of induced pluripotent stem cells (iPSCs) with novel properties are also provided herein. In some embodiments, the isolated population of iPSCs comprises pluripotent cells expressing one or more reprogramming factors and containing nucleotide sequences encoding rearrangements of the TRG and TRD genes.

[0144] In certain embodiments, isolated populations of iPSCs are prepared according to the methods described herein (for example, in Section 5.3).

[0145] In a particular embodiment, the reprogramming factor is selected from the group consisting of Oct3 / 4, Sox2, Klf4, c-Myc, and Lin28.

[0146] In a particular embodiment, the reprogramming factors include Oct3 / 4, Sox2, Klf4, and c-Myc.

[0147] In a particular embodiment, the reprogramming factors are Oct3 / 4, Sox2, KLF4, c-Myc, and Lin28.

[0148] In a particular embodiment, the reprogramming factors are Oct3 / 4, Sox2, Klf4, and c-Myc.

[0149] In certain embodiments, the isolated population of iPSCs is derived from γδT cells. In certain embodiments, the isolated population of iPSCs has rearranged genes at the TRG and TRD loci. In certain embodiments, the isolated population of iPSCs does not produce PCR products from the TCRG and TCRD loci.

[0150] In certain embodiments, the isolated population of iPSCs does not originate from αβT cells. In certain embodiments, the isolated population of iPSCs does not have rearrangement genes at the TRA and TRB loci. In certain embodiments, the isolated population of iPSCs does not produce PCR products from the TCRA and TCRB loci.

[0151] In certain embodiments, isolated populations of iPSCs are negative for Sendai virus (SeV) vectors.

[0152] In certain embodiments, isolated populations of iPSCs are genomically stable without chromosome loss. In one embodiment, the genomic stability of isolated populations of iPSCs is determined by karyotype analysis.

[0153] In certain embodiments, an isolated population of iPSCs can be grown and maintained in a culture medium that does not contain supporting cells after recruitment.

[0154] In some embodiments, an isolated population of induced pluripotent stem cells (iPSCs) comprising pluripotent cells expressing one or more reprogramming factors, wherein (i) the pluripotent cells contain nucleotide sequences encoding rearrangements of the TRG and TRD genes, or have rearrangement genes for the TRG and TRD loci, (ii) the reprogramming factors are selected from the group consisting of Oct3 / 4, Sox2, Klf4, c-Myc, and Lin28, and (iii) the isolated population of iPSCs is negative for the Sendai virus (SeV) vector. (iv) The isolated population of iPSCs is derived from γδT cells but not from αβT cells; (v) The isolated population of iPSCs does not produce PCR products from the TCRA and TCRB loci; (vi) The isolated population of iPSCs is genomically stable without chromosomal loss, as determined, for example, by karyotype analysis; and / or (vii) The isolated population of iPSCs can be grown and maintained in a culture medium without supporting cells after adoption.

[0155] In a specific embodiment, the following is provided herein: an isolated population of induced pluripotent stem cells (iPSCs) comprising pluripotent cells expressing one or more reprogramming factors, wherein the pluripotent cells comprise nucleotide sequences encoding rearrangements of the TRG and TRD genes, and the reprogramming factors are selected from the group consisting of Oct3 / 4, Sox2, Klf4, c-Myc, and Lin28.

[0156] In another specific embodiment, an isolated population of induced pluripotent stem cells (iPSCs) is provided herein, comprising pluripotent cells expressing one or more reprogramming factors, wherein the pluripotent cells comprise nucleotide sequences encoding rearrangements of the TRG and TRD genes, and the reprogramming factors are Oct3 / 4, Sox2, Klf4, c-Myc, and Lin28.

[0157] In yet another specific embodiment, an isolated population of induced pluripotent stem cells (iPSCs) is provided herein, comprising pluripotent cells expressing one or more reprogramming factors, wherein the pluripotent cells comprise nucleotide sequences encoding rearrangements of the TRG and TRD genes, and the reprogramming factors are Oct3 / 4, Sox2, Klf4, and c-Myc.

[0158] In yet another specific embodiment, an isolated population of induced pluripotent stem cells (iPSCs) is provided herein, comprising pluripotent cells expressing one or more reprogramming factors, wherein the pluripotent cells comprise nucleotide sequences encoding rearrangements of the TRG and TRD genes, and the isolated population of iPSCs is negative for the Sendai virus (SeV) vector.

[0159] In yet another specific embodiment, an isolated population of induced pluripotent stem cells (iPSCs) is provided herein, comprising pluripotent cells expressing one or more reprogramming factors, wherein the pluripotent cells comprise nucleotide sequences encoding rearrangements of the TRG and TRD genes, the reprogramming factors are selected from the group consisting of Oct3 / 4, Sox2, Klf4, c-Myc, and Lin28, and the isolated population of iPSCs is negative for the Sendai virus (SeV) vector.

[0160] In yet another specific embodiment, an isolated population of induced pluripotent stem cells (iPSCs) comprising pluripotent cells expressing one or more reprogramming factors, wherein the pluripotent cells comprise nucleotide sequences encoding rearrangements of the TRG and TRD genes, and the isolated population of iPSCs is derived from γδT cells but not from αβT cells.

[0161] In yet another specific embodiment, an isolated population of induced pluripotent stem cells (iPSCs) comprising pluripotent cells expressing one or more reprogramming factors, wherein the pluripotent cells comprise nucleotide sequences encoding rearrangements of the TRG and TRD genes, and the isolated population of iPSCs does not produce PCR products from the TCRA and TCRB loci.

[0162] In yet another specific embodiment, an isolated population of induced pluripotent stem cells (iPSCs) is provided herein, comprising pluripotent cells expressing one or more reprogramming factors, wherein the pluripotent cells contain nucleotide sequences encoding rearrangements of the TRG and TRD genes, or have rearrangement genes at the TRG and TRD loci, and the isolated population of iPSCs is genomically stable without chromosomal loss, as determined, for example, by karyotype analysis.

[0163] In yet another specific embodiment, an isolated population of induced pluripotent stem cells (iPSCs) comprising pluripotent cells expressing one or more reprogramming factors, wherein the pluripotent cells contain nucleotide sequences encoding rearrangements of the TRG and TRD genes, or have rearrangement genes at the TRG and TRD loci, and the isolated population of iPSCs can be grown and maintained in a culture medium without supporting cells after adoption.

[0164] In yet another specific embodiment, an isolated population of induced pluripotent stem cells (iPSCs) is provided herein, comprising pluripotent cells expressing one or more reprogramming factors, wherein the pluripotent cells contain nucleotide sequences encoding rearrangements of the TRG and TRD genes, or have rearrangement genes at the TRG and TRD loci, the reprogramming factors are selected from the group consisting of Oct3 / 4, Sox2, Klf4, c-Myc, and Lin28, the isolated population of iPSCs is negative for Sendai virus (SeV) vectors, the isolated population of iPSCs is derived from γδT cells but not from αβT cells, the isolated population of iPSCs does not produce PCR products from the TCRA and TCRB loci, the isolated population of iPSCs is genomically stable without chromosomal loss, as determined, for example, by karyotype analysis, and the isolated population of iPSCs can be grown and maintained in a culture medium without supporting cells after adoption.

[0165] In yet another specific embodiment, an isolated population of induced pluripotent stem cells (iPSCs) is provided herein, comprising pluripotent cells expressing one or more reprogramming factors, wherein the pluripotent cells contain nucleotide sequences encoding rearrangements of the TRG and TRD genes, or have rearrangement genes for the TRG and TRD loci, the reprogramming factors are selected from the group consisting of Oct3 / 4, Sox2, Klf4, c-Myc, and Lin28, the isolated population of iPSCs is negative for Sendai virus (SeV) vectors, the isolated population of iPSCs does not produce PCR products from the TCRA and TCRB loci, the isolated population of iPSCs is genomically stable without chromosome loss, as determined, for example, by karyotype analysis, and the isolated population of iPSCs can be grown and maintained in a culture medium without supporting cells after adoption.

[0166] In yet another specific embodiment, an isolated population of induced pluripotent stem cells (iPSCs) is provided herein, wherein the pluripotent cells contain nucleotide sequences encoding rearrangements of the TRG and TRD genes, or have rearrangement genes at the TRG and TRD loci, the isolated population of iPSCs is negative for Sendai virus (SeV) vectors, the isolated population of iPSCs is derived from γδT cells but not from αβT cells, the isolated population of iPSCs does not produce PCR products from the TCRA and TCRB loci, the isolated population of iPSCs is genomically stable without chromosome loss, as determined, for example, by karyotype analysis, and the isolated population of iPSCs can be grown and maintained in a culture medium without supporting cells after adoption.

[0167] In yet another specific embodiment, an isolated population of induced pluripotent stem cells (iPSCs) is provided herein, wherein the pluripotent cells contain nucleotide sequences encoding rearrangements of the TRG and TRD genes, or have rearrangement genes at the TRG and TRD loci, the isolated population of iPSCs is negative for Sendai virus (SeV) vectors, the isolated population of iPSCs does not produce PCR products from the TCRA and TCRB loci, the isolated population of iPSCs is genomically stable without chromosome loss, as determined, for example, by karyotype analysis, and the isolated population of iPSCs can be grown and maintained in a culture medium without supporting cells after adoption.

[0168] In yet another specific embodiment, an isolated population of induced pluripotent stem cells (iPSCs) is provided herein, wherein the pluripotent cells contain nucleotide sequences encoding rearrangements of the TRG and TRD genes, or have rearrangement genes at the TRG and TRD loci, the isolated population of iPSCs does not produce PCR products from the TCRA and TCRB loci, the isolated population of iPSCs is genomically stable without chromosome loss, as determined, for example, by karyotype analysis, and the isolated population of iPSCs can be grown and maintained in a culture medium without supporting cells after adoption.

[0169] In yet another specific embodiment, an isolated population of induced pluripotent stem cells (iPSCs) is provided herein, wherein the pluripotent cells contain nucleotide sequences encoding rearrangements of the TRG and TRD genes, or have rearrangement genes at the TRG and TRD loci, the isolated population of iPSCs does not produce PCR products from the TCRA and TCRB loci, and the isolated population of iPSCs is genomically stable without chromosomal loss, as determined, for example, by karyotype analysis.

[0170] 5.5. Pharmaceutical Compositions Also provided herein are “pharmaceutical compositions” comprising iPSCs or differentiated cells therefrom prepared according to the methods described herein, and one or more pharmaceutically acceptable carriers. In certain embodiments, the prepared iPSCs or differentiated cells therefrom are present in therapeutically effective amounts. In certain embodiments, the prepared iPSCs or differentiated cells therefrom are present in prophylactically effective amounts. The pharmaceutical compositions can be used according to the methods and uses provided herein. For example, the pharmaceutical compositions can be administered to a subject to carry out the therapeutic or prophylactic methods and uses provided herein. The pharmaceutical compositions provided herein can be formulated to suit the intended method or route of administration, exemplary routes of administration are described herein.

[0171] A pharmaceutical composition typically comprises at least one of a therapeutically effective amount of prepared iPSCs or differentiated cells therefrom, and a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include, but are not limited to, antioxidants (e.g., ascorbic acid), preservatives (e.g., benzyl alcohol, methylparaben, p-hydroxybenzoate), emulsifiers, suspending agents, dispersants, solvents, buffers, lubricants, fillers, and / or diluents. For example, a suitable vehicle may be physiological saline. Typical buffers that can be used include, but are not limited to, pharmaceutically acceptable weak acids, weak bases, or mixtures thereof. The buffer component may also include water-soluble reagents such as phosphoric acid, tartaric acid, succinic acid, citric acid, acetic acid, and their salts.

[0172] The vehicle may contain other pharmaceutically acceptable excipients for modifying or maintaining the pH, molar osmotic pressure, viscosity, or stability of the pharmaceutical composition. In specific embodiments, the vehicle is an aqueous buffer. In specific embodiments, the vehicle contains, for example, sodium chloride.

[0173] The pharmaceutical compositions provided herein may contain further pharmaceutically acceptable formulations for modifying or maintaining the administration rate of the prepared iPSCs or differentiated cells therefrom as described herein. Such formulations include, for example, substances known to those skilled in the art when preparing sustained-release or controlled-release formulations. For pharmaceutically acceptable formulations, see, for example, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042), pp. 1435–1712, and The Merck Index, 12th Ed. (1996, Merck Publishing Group, Whitehouse, NJ).

[0174] In specific embodiments, the pharmaceutical composition is provided in a single-use container (e.g., a single-use vial, ampoule, syringe, or auto-injector). In specific embodiments, the pharmaceutical composition is provided in a multi-use container (e.g., a multi-use vial or cartridge). The iPSC or pharmaceutical composition described herein may be delivered using any drug delivery device, including intravenous infusion.

[0175] Pharmaceutical compositions can be formulated to conform to their intended route of administration, as described herein.

[0176] Pharmaceutical compositions may also include carriers to protect the composition from decomposition or elimination from the body. Various antimicrobial and antifungal agents, such as parabens, chlorobutanol, ascorbic acid, and thimerosal, may be included in the pharmaceutical composition.

[0177] 5.6. Treatment methods and use Furthermore, reprogrammed somatic cells, including reprogrammed pluripotent somatic cells such as iPSCs, produced by the methods disclosed herein are also provided herein. These methods, useful for generating cells of a desired cell type, have a wide range of applications. For example, these methods have medical applications in the treatment or prevention of conditions.

[0178] Accordingly, in one embodiment, a method for treating or preventing a disease or disorder in a mammal is provided herein. In one embodiment, the method begins by obtaining somatic cells from an organism and reprogramming the somatic cells thus obtained by the method of the present invention to obtain iPSCs. The iPSCs are then cultured under conditions suitable for the development of iPSCs into cells of a desired cell type. The developed cells of the desired cell type are collected and introduced into the organism to treat the disease or disorder. In an alternative embodiment, the method begins by obtaining somatic cells from an organism and reprogramming the somatic cells according to the method. The iPSCs are then cultured under conditions suitable for the development of iPSCs into organs of a desired type, collected, and introduced into the organism to treat the disease or disorder.

[0179] In some embodiments, the reprogrammed somatic cells of the present invention are ES-like cells and can therefore be induced to differentiate to obtain a desired cell type according to known methods for differentiating ES cells. For example, iPSCs can be induced to differentiate into hematopoietic stem cells, muscle cells, cardiomyocytes, hepatocytes, chondrocytes, epithelial cells, urinary tract cells, etc., by culturing such cells in a differentiation medium under conditions that result in cell differentiation. Appropriate culture conditions, media and methods that result in the differentiation of embryonic stem cells are known in the art.

[0180] In some specific embodiments, iPSCs are induced to differentiate into hematopoietic stem cells, as described, for example, in Palacios et al., Proc. Natl. Acad. Sci., USA, 92:7530-37 (1995), which teaches the production of hematopoietic stem cells from embryonic cell lines by subjecting stem cells to a certain induction procedure, which includes first culturing aggregates of such cells in a suspension culture medium lacking retinoic acid, then culturing them in the same medium containing retinoic acid, and then transplanting the cell aggregates onto a substrate that provides cell adhesion.

[0181] In other specific embodiments, iPSCs are induced to differentiate according to a method such as that described in Pedersen, J. Reprod. Fertil. Dev., 6:543-52 (1994), which refers to numerous papers disclosing methods for in vitro differentiation of embryonic stem cells to produce various differentiated cell types, including hematopoietic cells, muscle cells, cardiac muscle cells, and nerve cells.

[0182] In other specific embodiments, iPSCs are induced to differentiate according to Bain et al., Dev. Biol., 168:342-357 (1995), which teaches in vitro differentiation of embryonic stem cells to produce nerve cells with neuronal properties.

[0183] These references describe reported exemplary methods for obtaining differentiated cells from embryonic cells or stem-like cells. These references, and in particular the disclosures therein regarding methods for differentiating embryonic stem cells, are incorporated herein by reference in their entirety.

[0184] Therefore, using known methods and culture media, those skilled in the art can culture target embryonic cells or stem-like cells to obtain desired differentiated cell types, such as nerve cells, muscle cells, hematopoietic cells, etc. In addition, the use of inducible Bcl-2 or Bcl-x1 may be useful to enhance the in vitro development of specific cell lineages. In vivo, Bcl-2 prevents many, but not all, forms of apoptotic cell death that occur during lymphoid and neurogenesis. A thorough discussion of how Bcl-2 expression may be used to inhibit apoptosis of relevant cell lineages after donor cell transfection is disclosed in U.S. Patent No. 5,646,008, which is incorporated herein by reference.

[0185] The iPSCs provided herein can be used to obtain any desired differentiated cell type. The therapeutic use of such differentiated human cells is unparalleled. For example, human hematopoietic stem cells can be used in medical procedures requiring bone marrow transplantation. Such procedures are used to treat many diseases, such as advanced cancers like ovarian cancer and leukemia, as well as diseases that impair the immune system. Hematopoietic stem cells can be obtained, for example, by fusing adult somatic cells from cancer or AIDS patients, such as epithelial cells or lymphocytes, with enucleated oocytes, such as bovine oocytes, to obtain germ cells or stem-like cells as described above, and culturing such cells under conditions favorable for differentiation until hematopoietic stem cells are obtained. Such hematopoietic cells can be used in the treatment of diseases including cancer and AIDS.

[0186] The methods of the present invention can also be used to treat, prevent, or stabilize neurological diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, or ALS, lysosomal storage disorders, multiple sclerosis, or spinal cord injury. For example, somatic cells can be obtained from an individual in need of treatment, reprogrammed to acquire pluripotency, and cultured to induce neuroectoderm cells that can be used to replace or assist the normal function of diseased or injured tissue.

[0187] For the treatment or prevention of endocrine conditions, reprogrammed cells that produce hormones such as growth factors, thyroid hormones, thyroid-stimulating hormone, parathyroid hormone, steroids, serotonin, epinephrine, or norepinephrine may be administered to mammals. In addition, reprogrammed epithelial cells may be administered to repair damage to body cavities or the inner lining of organs such as the lungs, intestines, exocrine glands, or urogenital tract. Furthermore, it is intended that iPSCs may be administered to mammals to treat cell damage or defects in organs such as the bladder, brain, esophagus, fallopian tubes, heart, intestines, gallbladder, kidneys, liver, lungs, ovaries, pancreas, prostate, spinal cord, spleen, stomach, testes, thymus, thyroid gland, trachea, ureters, urethra, or uterus.

[0188] A major advantage of this disclosure is that it provides an essentially unlimited supply of isogenic or syngenic human cells suitable for transplantation. Therefore, it prevents a significant problem associated with current transplantation methods, namely, the rejection of transplanted tissue that can occur due to host-to-graft or graft-to-host rejection. Conventionally, rejection is prevented or reduced by the administration of anti-rejection drugs such as cyclosporine. However, such drugs have significant adverse side effects, such as immunosuppression and carcinogenic properties, and are also very expensive. The present invention eliminates, or at least significantly reduces, the need for anti-rejection drugs such as cyclosporine, Imuran, FK-506, glucocorticoids, and rapamycin, and their derivatives.

[0189] Furthermore, iPSCs can be combined with a matrix to form tissues or organs in vitro or in vivo that can be used to repair or replace tissues or organs in recipient mammals. For example, iPSCs may be cultured in vitro in the presence of a matrix to produce tissues or organs of the urogenital system, such as the bladder, clitoris, corpus cavernosum, kidney, testicle, ureter, ureteral valve, or urethra, which may then be transplanted into a mammal (Atala, Curr. Opin. Urol. 9(6):517-526, 1999). In another transplantation application, artificial blood vessels are formed in vitro by culturing reprogrammed cells in the presence of a suitable matrix, and these vessels are then transplanted into mammals for the treatment or prevention of cardiovascular or circulatory conditions. For the generation of donor cartilage or bone tissue, iPSCs such as chondrocytes or osteocytes are cultured in vitro in the presence of a matrix under conditions that allow for the formation of cartilage or bone, and then the matrix containing the donor tissue is administered to the mammal. Alternatively, a mixture of cells and a matrix may be administered to a mammal for the in vivo formation of a desired tissue. Preferably, the cells are attached to the surface of the matrix or encapsulated by the matrix. Examples of matrices that may be used for the formation of donor tissue or organs include collagen matrices, carbon fibers, polyvinyl alcohol sponges, acrylamide sponges, fibrin-thrombin gel, hyaluronic acid-based polymers, and synthetic polymer matrices containing polyacid anhydrides, polyoltoesters, polyglycolic acid, or combinations thereof (see, for example, U.S. Patents 4,846,835, 4,642,120, 5,786,217, and 5,041,138).

[0190] iPSCs produced in accordance with this disclosure may be used to produce genetically modified differentiated cells or transgenic differentiated cells. Essentially, this is achieved by introducing a desired gene or a set of genes, or by removing all or part of one or more endogenous genes from an iPSC produced in accordance with the invention, and differentiating such cells into a desired cell type. A preferred method for achieving such modifications is homologous recombination, because such techniques can be used to insert, delete, or modify a gene or a set of genes at specific sites or sites in the stem-like cell genome.

[0191] This methodology can be used to replace defective genes, such as defective immune system genes or cystic fibrosis genes, or to introduce genes that result in the expression of therapeutically beneficial proteins such as growth factors, lymphakines, cytokines, and enzymes. For example, it is possible to introduce a gene encoding brain-derived growth factor into human embryonic cells or stem-like cells, differentiate those cells into nerve cells, and transplant those cells into Parkinson's disease patients to slow nerve cell loss during such diseases. Examples of mutations that can be rescued using these methods include mutations in cystic fibrosis genes, mutations associated with Dunningan disease such as R482W, R482Q, and R584H mutations in the lamin A gene, and mutations associated with the autosomal dominant form of Emery Deyfuss muscular dystrophy such as R249Q, R453W, and Q6STOP mutations in the lamin A gene. In the Q6STOP mutation, the Gln6 codon is mutated into a stop codon.

[0192] To date, cell types transfected with BDNF have varied from primary cells to immortalized cell lines, nerve-derived cells, or non-nerve-derived cells (myoblasts and fibroblasts). For example, astrocytes were transfected with the BDNF gene using a retroviral vector, and these cells were transplanted into a rat model of Parkinson's disease (Yoshimoto et al., Brain Research, 691:25-36, (1995)). This in vitro treatment reduced Parkinson's-like symptoms in rats by up to 45% on day 32 after transplantation. Similar results were obtained when the tyrosine hydroxylase gene was introduced into astrocytes (Lundberg et al., Develop. Neurol., 139:39-53 (1996) and the literature cited therein).

[0193] However, such in vitro systems have problems. In particular, the retroviral vectors currently in use are downregulated in vivo, and the transgenes are expressed only transiently (review by Mulligan, Science, 260:926-932 (1993)). Also, such studies have used astrocytes, which are primary cells with a finite lifespan and slow replication. These characteristics negatively affect the rate of transfection and hinder the selection of stably transfected cells. Furthermore, it is almost impossible to grow large populations of gene-targeted primary cells used in homologous recombination techniques.

[0194] In contrast, the difficulties associated with retroviral systems should be eliminated by the use of iPSCs, which are ES-like cells, as disclosed herein. iPSCs may be genetically engineered using known methods for introducing desired genes / mutations into ES cells, and the resulting genetically engineered cells may be differentiated into desired cell types, such as hematopoietic cells, nerve cells, pancreatic cells, chondrocytes, etc. Genes that can be introduced into iPSCs include, for example, epidermal growth factor, basic fibroblast growth factor, glial neurotrophic growth factor, insulin-like growth factor (I and II), neurotrophin 3, neurotrophin 4 / 5, ciliary neurotrophic factor, AFT-1, cytokine genes (interleukin, interferon, colony-stimulating factor, tumor necrosis factor (alpha and beta), etc.), genes encoding therapeutic enzymes, collagen, and human serum albumin.

[0195] In addition, one of the negative selection systems currently known in the art can be used to exclude therapeutic cells from the patient as needed. For example, donor cells transfected with the thymidine kinase (TK) gene lead to the creation of embryonic cells containing the TK gene. Differentiation of these cells leads to the isolation of target therapeutic cells that also express the TK gene. Such cells can be selectively excluded from the patient at any time during ganciclovir administration. Such a negative selection system is described in U.S. Patent No. 5,698,446, which is incorporated herein by reference.

[0196] Examples of diseases, disorders, or conditions that can be treated or prevented include neurological disorders, endocrine disorders, organic disorders, skeletal disorders, vascular disorders, urinary tract disorders, digestive disorders, skin disorders, hematological disorders, immune disorders, autoimmune disorders, inflammatory disorders, endocrine disorders, kidney disorders, bladder disorders, cardiovascular disorders, cancer, circulatory disorders, digestive disorders, hematopoietic disorders, and muscular disorders, disorders, and conditions. In addition, reprogrammed cells can be used for reconstructive purposes, such as repairing or replacing tissues or organs.

[0197] With respect to the therapeutic methods of this disclosure, the administration of iPSCs to mammals is not intended to be limited to any particular mode of administration, dosage, or frequency of administration, and this disclosure envisions all modes of administration, including intramuscular, intravenous, intra-articular, intrafocal, subcutaneous, or any other route sufficient to provide an appropriate dosage for the prevention or treatment of disease. iPSCs may be administered to mammals as a single dose or in multiple doses. When multiple doses are administered, these doses may be spaced apart, for example, by one week, one month, one year, or ten years. One or more growth factors, hormones, interleukins, cytokines, or other cells may also be administered before, during, or after the administration of cells to further bias them to a particular cell type.

[0198] The iPSCs of this disclosure may be used as an in vitro model of differentiation, particularly for studying genes involved in the regulation of early development. Differentiated cell tissues and organs using iPSCs may be used in drug research.

[0199] Furthermore, iPSCs produced in accordance with this disclosure can be introduced into animals, such as SCID mice, cattle, and pigs, for example, subcapsularly or intramuscularly, and used to create teratomas. These teratomas can be used to induce various histological types. Additionally, the internal cell mass produced by X-type nuclear transfer can be introduced together with a biodegradable, biocompatible polymer matrix that provides three-dimensional tissue formation. After tissue formation, the polymer degrades, ideally leaving only the donor tissue, such as the heart, pancreas, nerves, lungs, or liver. In some cases, it may be advantageous to include growth factors and proteins that promote angiogenesis. Alternatively, tissue formation can be achieved entirely in vitro using appropriate culture media and conditions, growth factors, and a biodegradable polymer matrix.

[0200] In certain more specific embodiments, a method for treating a subject requiring treatment is provided herein, comprising: (a) obtaining a population of cells isolated from the subject; (b) reprogramming γδT cells in the isolated cell population to produce iPSCs according to the method for producing iPSCs described herein; and (c) optionally differentiating the iPSCs into one or more desired cell types, and then administering the produced iPSCs or a pharmaceutical composition containing the produced iPSCs to the subject.

[0201] In a particular embodiment, the generated iPSCs are differentiated into one or more desired cell types and administered to a target.

[0202] For example, in some embodiments, given their remarkable multiseries differentiation and self-renewal capabilities, iPSCs can differentiate into T cells, providing a nearly unlimited supply of rejuvenated T cells and addressing a key problem that limits the effectiveness of T cells against tumors (i.e., T cell exhaustion) (Schietinger, A. & Greenberg, PD, Trends Immunol., 2015, 35(2):51-60). T cells exert effector function by binding to antigens via T cell receptors (TCRs). Nevertheless, sometimes TCR binding does not produce effector activity, especially under chronic infection conditions, leading to T cell exhaustion (Karagiannis, P., et al., Seminars in Immunology, 2015, 28(1):35-44). In such situations, adoptive cellular therapy (ACT) can be used as a compensatory mechanism, which includes either the in vitro expansion of T cells isolated from the patient's tumor microenvironment or the genetic modification of autologous T cell receptors to induce an immune response (Id.). Rejuvenating exhausted T cells by reprogramming them into iPSCs represents a promising solution to T cell exhaustion, as TCR rearrangement preserves certain specific antigen loci (Id.).

[0203] In some embodiments, compositions are provided herein comprising an isolated population or subpopulation of functionally enhanced derived immune cells differentiated from iPSCs produced according to the methods provided herein. In some embodiments, the iPSCs include one or more target gene edits that can be retained in the iPSC-derived immune cells, and the genetically engineered iPSCs and their derived cells are suitable for cell-based adoptive therapy. In one embodiment, the isolated population or subpopulation of genetically engineered immune cells comprises iPSC-derived pro-T cells or T cells. In one embodiment, the isolated population or subpopulation of genetically engineered immune cells comprises iPSC-derived pro-NK cells or NK cells. In one embodiment, the isolated population or subpopulation of genetically engineered immune cells comprises iPSC-derived immunomodulatory cells or myeloid-derived suppressor cells (MDSCs). In some embodiments, the iPSC-derived genetically engineered immune cells are further modified in vitro for improved therapeutic potential.

[0204] In a particular embodiment, the created iPSCs are administered to the subject without further differentiation.

[0205] In a particular embodiment, the subject is a human being.

[0206] In certain embodiments, the subject has a hyperproliferative disorder or a hematopoietic cancer. In some embodiments, the subject has a solid tumor. In some embodiments, the hematopoietic hyperproliferative disorder is polycythemia vera, essential thrombocythemia, myelofibrosis with myelo-metaplasia, or chronic myeloid leukemia.

[0207] In certain embodiments, the iPSCs produced herein or pharmaceutical compositions comprising the iPSCs produced herein can be used to treat cancer. Cancers that can be treated include tumors that are not angiogenic or are not substantially angiogenic, as well as angiogenic tumors. In some embodiments, cancer can be non-solid tumors (hematological malignancies, e.g., leukemia and lymphoma) or solid tumors. In some embodiments, types of cancer include, but are not limited to, carcinomas, blastomas, and sarcomas, as well as certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignant tumors, e.g., sarcomas, carcinomas, and melanomas. In some embodiments, adult tumors / cancers and pediatric tumors / cancers are also included.

[0208] In some embodiments, the iPSCs prepared herein or pharmaceutical compositions containing the iPSCs prepared herein are used to treat blood cancers. Blood cancers are cancers of the blood or bone marrow. Examples of blood (or hematogenic) cancers include acute leukemia (acute lymphoblastic leukemia, acute myelocytic leukemia, acute myelogenous leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia, etc.), chronic leukemia (chronic myelocytic (granulocyte) leukemia, chronic myeloid leukemia, chronic myelocytic leukemia) Leukemias include leukemia (and chronic lymphocytic leukemia, etc.), juvenile myelomonocytic leukemia, polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin lymphoma (low-grade and high-grade types), multiple myeloma, Waldenström's hypergammaglobulinemia, heavy chain disease, myelodysplastic syndrome, myeloid metaplasia of unknown cause, familial hemophagocytic lymphohistiocytosis, hairy cell leukemia, and spinal dysplasia.

[0209] In some embodiments, the subject has myeloma, non-Hodgkin lymphoma, Hodgkin lymphoma, chronic myeloid leukemia, chronic myelogenous leukemia, chronic granulocytic leukemia, acute lymphoblastic leukemia, acute non-lymphoblastic leukemia, or preleukemia.

[0210] In some embodiments, the prepared iPSCs or pharmaceutical compositions containing the prepared iPSCs described herein are used to treat solid tumors. Solid tumors are abnormal masses of tissue that do not typically contain cysts or fluid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named after the type of cells that form them (e.g., sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synoviomas, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid tumors, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic lung cancer, renal cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, and Wilms' tumor. Examples include ulcers, cervical cancer, testicular tumors, seminomas, bladder cancer, melanoma, and CNS tumors (gliomas (such as brainstem gliomas and mixed gliomas), gliablastomas (also known as pleomorphic gliablastomas), astrocytomas, CNS lymphomas, germ cell tumors, medulloblastomas, schwannomas, craniopharyngiomas, ependymomas, pineal glandomas, hemangioblastomas, acoustic neuromas, oligodendrogliomas, meningiomas, neuroblastomas, retinoblastomas, and brain metastases).

[0211] In some embodiments, the subjects have breast cancer, ovarian cancer, brain cancer, prostate cancer, lung cancer, colon cancer, skin cancer, liver cancer, pancreatic cancer, sarcoma, or chronic granulomatous disease.

[0212] In some embodiments, combination therapies comprising cells such as iPSCs provided herein and one or more additional agents are provided herein.

[0213] The iPSCs described herein or pharmaceutical compositions containing the iPSCs described herein are also provided for use in therapeutic applications. Furthermore, the iPSCs described herein or pharmaceutical compositions containing the iPSCs described herein are also provided for use in methods of treating hyperproliferative disorders or hematopoietic cancers in subjects requiring such treatment.

[0214] 5.7. Methods for identifying drugs that reprogram somatic cells or contribute to reprogramming them. In another embodiment, a method is provided herein for identifying agents that, either alone or in combination with one or more other agents, reprogram somatic cells (e.g., T cells) into a poorly differentiated state. The disclosure further provides agents identified according to the method provided herein.

[0215] In one embodiment, the method includes contacting somatic cells with an activated culture containing IL-15, zoledronic acid, and / or IL-2; contacting the somatic cells with a candidate drug; and then determining whether the presence of the candidate drug results in enhanced reprogramming (e.g., increased reprogramming rate and / or efficiency) compared to the reprogramming that would occur if the cells were not contacted with the candidate drug.

[0216] In some embodiments, a method is provided herein for identifying a drug that reprograms somatic cells (e.g., T cells) into a poorly differentiated state, either alone or in combination with one or more other drugs, comprising: (a) contacting an isolated cell population with an activated culture, the activated culture comprising IL-15 and zoledronic acid; (b) culturing the isolated cell population in the activated culture to enrich and / or activate γδT cells in the isolated cell population; (c) contacting the isolated cell population with a candidate drug; (d) transducing the γδT cells using one or more viral vectors encoding one or more reprogramming factors; (e) culturing the transduced γδT cells under conditions suitable for reprogramming mammalian somatic cells into a poorly differentiated state; and (f) determining whether at least a portion of the somatic cells have been reprogrammed into a poorly differentiated state. In some embodiments, the poorly differentiated state is a pluripotent state. In some embodiments, the poorly differentiated state is a pluripotent state. In certain embodiments, the activated culture further comprises one or more additional agents or compounds, for example, to improve the efficiency of activation or induction. In one embodiment, the activated culture further comprises interleukin-2 (IL-2).

[0217] In some embodiments, IL-15, zoledronic acid, and / or IL-2, along with the candidate drug, are present together in the cell culture medium, while in other embodiments, IL-15, zoledronic acid, and / or IL-2, along with the candidate drug, are not present together (e.g., cells are sequentially exposed to the drugs). In certain embodiments, cells are maintained in the culture for 1 to 20 days. In certain embodiments, cells are maintained in the culture for 1 to 17 days. In certain embodiments, cells are maintained in the culture for 1 to 15 days. In certain embodiments, cells are maintained in the culture for 1 to 13 days. In certain embodiments, cells are maintained in the culture for 1 to 11 days. In certain embodiments, cells are maintained in the culture for 1 to 9 days. In certain embodiments, cells are maintained in the culture for 1 to 7 days. In certain embodiments, cells are maintained in the culture for 1 to 5 days. In certain embodiments, cells are maintained in the culture for 1 to 3 days. In certain embodiments, cells are maintained in the culture for 12–72 hours. In certain embodiments, cells are maintained in the culture for 12–60 hours. In certain embodiments, cells are maintained in the culture for 12–48 hours. In certain embodiments, cells are maintained in the culture for 12–36 hours. In certain embodiments, cells are maintained in the culture for 12–24 hours. In certain embodiments, cells are maintained in the culture for 8–16 hours. In certain embodiments, cells are maintained in the culture for 4–8 hours. In certain embodiments, cells are maintained in the culture for 2–4 hours. Cells may be maintained in a culture for, for example, up to 13 days, up to 10 days, up to 9 days, up to 8 days, up to 7 days, up to 6 days, up to 5 days, up to 4 days, up to 3 days, up to 2 days, or up to 1 day, during which time they are exposed to IL-15, zoledronic acid, and / or IL-2 and candidate drugs for all or part of the time. In some embodiments, a drug is identified as a cell-reprogramming drug if, after the period, there are at least 2, 5, or 10 times more reprogrammed cells or colonies predominantly containing reprogrammed cells than if the cells were not exposed to the drug.

[0218] Candidate agents can be any molecule or supramolecular complex, such as peptides, small organic or inorganic molecules, polysaccharides, polynucleotides, etc., which are tested for their ability to reprogram cells, facilitate or enhance reprogramming. Candidate agents may be obtained from a wide variety of sources, including libraries of synthetic or natural compounds, as will be understood by those skilled in the art. In some embodiments, the candidate agent is a synthetic compound. Numerous techniques are available for the random and targeted synthesis of a wide variety of organic compounds and biomolecules. In some embodiments, the candidate modulator is provided as a mixture of natural compounds in the form of bacterial, fungal, plant and animal extracts, fermentation broths, conditioned media, etc., which are available or readily available.

[0219] In some embodiments, a library of compounds is screened. A library is typically a collection of compounds that can be presented or displayed so that they can be identified in a screening assay. In some embodiments, the compounds in the library are housed in individual wells (e.g., of a microtiter plate), containers, tubes, etc., to facilitate easy transfer to individual wells or containers for contact with cells, or for performing cell-free assays. A library may consist of molecules having common structural features that differ in the number or type of groups bound to the main structure, or it may be completely random. Examples of libraries include, but are not limited to, phage display libraries, peptide libraries, polysome libraries, aptamer libraries, synthetic small molecule libraries, natural compound libraries, and chemical libraries. Methods for preparing molecular libraries are well known in the art, and many libraries are available from commercial or non-commercial sources. Libraries of interest include synthetic organic combinatorial libraries. Libraries such as synthetic small molecule libraries and chemical libraries can include structurally diverse collections of chemical molecules. Small molecules often include organic molecules with multiple carbon-carbon bonds. The library may include cyclic carbon or heterocyclic structures, and / or aromatic or polyaromatic structures substituted with one or more functional groups. In some embodiments, the small molecules have 5 to 50 carbon atoms, e.g., 7 to 30 carbon atoms. In some embodiments, the compounds are macrocyclic. The libraries of interest also include peptide libraries, randomized oligonucleotide libraries, and the like. The library can be synthesized from peptoid and non-peptide synthetic portions. Such libraries containing non-peptide synthetic portions that are less susceptible to enzymatic degradation compared to their naturally occurring counterparts can be further synthesized. Small molecule combinatorial libraries may also be produced. Combinatorial libraries of organic small compounds may contain a collection of closely related analogues that differ from one or more in terms of diversity and are synthesized by organic techniques using multi-step processes.A combinatorial library can contain a vast number of organic microcompounds. As used herein, a “compound array” is a collection of compounds that are identifiable by their spatial addresses in Cartesian coordinates and are arranged such that each compound has a common molecular core and one or more variable structural diversity elements. Compounds in such a compound array are prepared in parallel in separate reaction vessels, and each compound is identified and tracked by its spatial address. In some embodiments, mixtures containing two or more compounds, extracts or other preparations obtained from natural sources (which may contain dozens or more compounds), and / or inorganic compounds are screened.

[0220] In one embodiment, the method of the present invention is used to screen for “approved drugs.” An “approved drug” is any compound (this term includes biomolecules such as proteins and nucleic acids) that has been approved for use in humans by the FDA or a similar government agency in another country for any purpose. This may be a particularly useful class of compounds for screening, as it represents a collection of compounds that are safe and, at least in the case of FDA-approved drugs, are thought to have therapeutic effects for at least one purpose. Thus, these drugs are likely to be safe for at least other purposes.

[0221] A representative example of a library that can be screened is DIVERSet™, available from ChemBridge Corporation, 16981 Via Tazon, San Diego, Calif. 92127. DIVERSet contains 10,000 to 50,000 manually synthesized drug-like small molecules. The compounds are pre-selected to form a “universal” library suitable for either high-throughput or lower-throughput screening, covering maximum pharmacophore diversity with a minimum number of compounds. For further library descriptions, see, for example, Tan, et al., Am. Chem Soc. 120, 8565-8566, 1998; Floyd CD, Leblanc C, Whittaker M, Prog Med Chem 36:91-168, 1999. Numerous libraries are commercially available from companies such as AnalytiCon USA Inc., POBox 5926, Kingwood, Tex. 77325; 3-Dimensional Pharmaceuticals, Inc., 665 Stockton Drive, Suite 104, Exton, PA. 19341-1151; and Tripos, Inc., 1699 Hanley Rd., St. Louis, Mo. 63144-2913. Examples of commercially available libraries include those based on quinic acid and shikimic acid, hydroxyproline, santonin, dianhydro-D-glucitol, hydroxypipecolic acid, androgravoride, piperazine-2-carboxylic acid-based libraries, and cytosine.

[0222] In some embodiments, the candidate drug is cDNA derived from a cDNA expression library prepared from cells, such as pluripotent cells. Such cells may include embryonic stem cells, oocytes, blastomeres, teratoma cells, embryonic germ cells, and inner cell mass cells.

[0223] It will be understood that the candidate reprogramming agents tested are typically those not present in the standard culture medium, or, if present, in amounts less than those used in the present invention. It will also be understood that a useful reprogramming agent or other form of reprogramming treatment does not need to be able to reprogram all types of somatic cells, nor does it need to be able to reprogram all somatic cells of a given cell type. A useful candidate agent is one that results in a population enriched with reprogrammed cells by 2, 5, 10, 50, 100, or more (i.e., the proportion of reprogrammed cells in the population is 2, 5, 10, 50, or 100 times greater than that in the starting population of cells treated in the same manner but without contact with the candidate agent).

[0224] In some embodiments, the screening method provided herein is used to identify a drug or combination of drugs that substitutes Klf4 when reprogramming cells to a pluripotent state. In some embodiments, the method is used to identify a drug that substitutes Sox2 when reprogramming cells to a pluripotent state. In some embodiments, the method is used to identify a drug that substitutes Oct3 / 4 when reprogramming cells to a pluripotent state. In some embodiments, the method is used to identify a drug that substitutes c-Myc when reprogramming cells to a pluripotent state. In some embodiments, the method is used to identify a drug that substitutes Lin28 when reprogramming cells to a pluripotent state. In some embodiments, the method is carried out using human cells. In some embodiments, the method is carried out using mouse cells. In some embodiments, the method is carried out using non-human primate cells.

[0225] In another embodiment, a method for identifying genes that activate the expression of endogenous pluripotency genes in somatic cells (e.g., T cells) is provided herein.

[0226] In some embodiments, the method includes (a) contacting an isolated cell population with an activated culture, the activated culture containing IL-15 and zoledronic acid; (b) culturing the isolated cell population in the activated culture to enrich and / or activate the γδT cells in the isolated cell population; (c) transducing the γδT cells using one or more viral vectors encoding one or more candidate reprogramming factors; (d) culturing the transduced γδT cells under conditions suitable for reprogramming mammalian somatic cells into a poorly differentiated state; and (e) determining whether at least a portion of the somatic cells have been reprogrammed into a poorly differentiated state. In some embodiments, the poorly differentiated state is a pluripotent state. In some embodiments, the poorly differentiated state is a pluripotent state.

[0227] In some more specific embodiments, the method includes (a) contacting an isolated cell population with an activated culture, the activated culture comprising IL-15 and zoledronic acid; (b) culturing the isolated cell population in the activated culture to enrich and / or activate the γδT cells in the isolated cell population; (c) transducing the γδT cells using one or more viral vectors encoding one or more candidate reprogramming factors; (d) culturing the transduced γδT cells under conditions suitable for reprogramming mammalian somatic cells into a pluripotent state; and (e) determining whether at least a portion of the somatic cells have been reprogrammed into a pluripotent state.

[0228] In certain embodiments, the activated culture further comprises one or more additional agents or compounds, for example, to improve the efficiency of activation or induction. In one embodiment, the activated culture further comprises interleukin-2 (IL-2).

[0229] Accordingly, in some embodiments, the method provided herein includes (a) contacting an isolated cell population with an activated culture comprising IL-15, zoledronic acid, and IL-2; (b) culturing the isolated cell population in the activated culture to enrich and / or activate γδT cells in the isolated cell population; (c) transducing the γδT cells using one or more viral vectors encoding one or more candidate reprogramming factors; (d) culturing the transduced γδT cells under conditions suitable for reprogramming mammalian somatic cells into a poorly differentiated state; and (e) determining whether at least a portion of the somatic cells have been reprogrammed into a poorly differentiated state.

[0230] In some more specific embodiments, the method provided herein includes (a) contacting an isolated cell population with an activated culture comprising IL-15, zoledronic acid, and IL-2; (b) culturing the isolated cell population in the activated culture to enrich and / or activate γδT cells in the isolated cell population; (c) transducing the γδT cells using one or more viral vectors encoding one or more candidate reprogramming factors; (d) culturing the transduced γδT cells under conditions suitable for reprogramming mammalian somatic cells into a pluripotent state; and (e) determining whether at least a portion of the somatic cells have been reprogrammed into a pluripotent state.

[0231] In other embodiments, the method includes culturing somatic cells, such as those provided herein, in the presence of, for example, IL-15, zoledronic acid, and / or IL-2; then transfecting the somatic cells of the disclosure with a cDNA library prepared from ES cells or oocytes; selecting cells expressing a first selection marker; and evaluating the expression of a first endogenous pluripotency gene in the transfected cells expressing the first selection marker. Expression of the first endogenous pluripotency gene indicates that the cDNA encodes a gene that activates the expression of an endogenous pluripotency gene in somatic cells.

[0232] This method is applicable to identify genes that activate the expression of at least two endogenous pluripotency genes in somatic cells. The somatic cells used in this method further include a second endogenous pluripotency gene linked to a second selection marker. This method can be modified to select transfected cells expressing both selection markers, in which the expression of the first and second endogenous pluripotency genes is evaluated. The expression of both the first and second endogenous pluripotency genes indicates that the cDNA encodes a gene that activates the expression of at least two pluripotency genes in somatic cells.

[0233] This method is further applicable to identify genes that activate the expression of at least three endogenous pluripotency genes in somatic cells. The somatic cells used in this method further include a third endogenous pluripotency gene linked to a third selection marker. The method is modified to select transfected cells expressing all three selection markers, in which the expression of all three endogenous pluripotency genes is evaluated. The expression of all three endogenous pluripotency genes indicates that the cDNA encodes a gene that activates the expression of at least three pluripotency genes in somatic cells.

[0234] The implementation of this invention utilizes, unless otherwise indicated, conventional techniques of mouse genetics, developmental biology, cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the scope of the techniques of the art. Such techniques are described in the literature. See, for example, Current Protocols in Cell Biology, ed. by Bonifacino, Dasso, Lippincott-Schwartz, Harford, and Yamada, John Wiley and Sons, Inc., New York, 1999; Manipulating the Mouse Embryos, A Laboratory Manual, 3rd Ed., by Hogan et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2003; Gene Targeting: A Practical Approach, IRL Press at Oxford University Press, Oxford, 1993; and Gene Targeting Protocols, Human Press, Totowa, NJ, 2000. All patents, patent applications, and references cited herein are incorporated by reference in their entirety.

[0235] 6. Embodiments The present invention provides the following non-limiting embodiments.

[0236] One set of embodiments (Embodiment Set A) provides the following: A1. A method for producing induced pluripotent stem cells (iPSCs), (a) Contacting an isolated cell population with an activated culture, wherein the activated culture contains IL-15 and zoledronic acid, (b) Culturing the isolated cell population in an activated culture to enrich and / or activate the γδT cells in the isolated cell population, (c) Transduction of γδT cells using a viral vector encoding one or more reprogramming factors, (d) A method comprising culturing transduced γδT cells under conditions suitable for reprogramming mammalian somatic cells into a pluripotent state. A2. The activated culture further comprises IL-2, as described in Embodiment A1. A3. The method according to Embodiment A1 or A2, wherein the viral vector is a Sendai virus (SeV) vector. A4. The method according to any one of embodiments A1 to A3, further comprising obtaining a population of cells isolated from the subject. A5. The method according to any one of Embodiments A1 to A4, wherein the isolated cell population is peripheral blood mononuclear cells (PBMCs). A6. The method according to any one of Embodiments A1 to A5, wherein the isolated cell population is terminally differentiated cells. A7. The method according to any one of embodiments A1 to A6, wherein the isolated cell population is mammalian cells. A8. The method according to any one of Embodiments A7, wherein the isolated cell population is human cells. A9. The method according to any one of Embodiments A1 to A8, wherein the isolated cell population is cultured in an activated culture for 1 to 20 days, 1 to 17 days, 1 to 15 days, 1 to 13 days, 1 to 11 days, 1 to 9 days, 1 to 7 days, 1 to 5 days, 1 to 3 days, 12 to 72 hours, 12 to 60 hours, 12 to 48 hours, 12 to 36 hours, 12 to 24 hours, 8 to 16 hours, 4 to 8 hours, or 2 to 4 hours. A10. The method according to Embodiment A9, wherein the isolated cell population is cultured in an activated culture for up to 13 days, up to 10 days, up to 9 days, up to 8 days, up to 7 days, up to 6 days, up to 5 days, up to 4 days, up to 3 days, up to 2 days, or up to 1 day. A11. The method according to Embodiment A10, wherein the isolated cell population is cultured in an activated culture for up to 3 days. A12. The method according to Embodiment A10, wherein the isolated cell population is cultured in an activated culture for 3 days. A13. After being cultured in the activated culture, the isolated cell population consisted of 5%-100% γδT cells, 5%-95% γδT cells, 5%-90% γδT cells, 5%-85% γδT cells, 5%-80% γδT cells, 5%-75% γδT cells, 5%-70% γδT cells, 5%-65% γδT cells, and 5%-60% γδT cells. The method according to any one of Embodiments A1 to A12, comprising 5% to 55% γδT cells, 5% to 50% γδT cells, 5% to 45% γδT cells, 5% to 40% γδT cells, 5% to 35% γδT cells, 5% to 30% γδT cells, 5% to 25% γδT cells, 5% to 20% γδT cells, or 5% to 15% γδT cells. A14. The method according to Embodiment A13, wherein the isolated cell population after being cultured in an activated culture contains less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 45%, less than 40%, less than 35%, or less than 30% γδT cells. A15. The method according to Embodiment A14, wherein the isolated cell population after being cultured in an activated culture contains less than 35% γδT cells. A16. The method according to any one of Embodiments A1 to A15, further comprising enriching the isolated cell population with γδT cells after step (b). A17. The method according to embodiment A16, wherein γδT cells are enriched by intercellular aggregate concentration. A18. At least a portion of γδ T cells are Vγ9 in step (b). + The method according to any one of embodiments A1 to A17, which is activated in γδT cells. A19. At least a portion of the γδ T cells are Vγ9δ2 in step (b). + The method according to any one of embodiments A1 to A17, which is activated in γδT cells. A20. The method according to any one of Embodiments A1 to A19, wherein one or more reprogramming factors are selected from the group consisting of OCT3 / 4, SOX2, KLF4, LIN28, and c-Myc. A21. The method according to any one of Embodiments A1 to A20, wherein in step (d), the transduced γδT cells are cultured in the presence of one or more supporting cell layers. A22. The method according to Embodiment A21, wherein in step (d), the transduced γδT cells are cultured in the presence of a monolayer of the supporting cell layer. A23. The supporting cell layer comprises mouse embryonic fibroblasts (MEFs) as described in Embodiment A21 or A22. A24. The method according to any one of Embodiments A1 to A23, further comprising isolating and / or purifying the prepared iPSCs. A25. The method according to Embodiment A24, further comprising administering to isolated iPSCs. A26. The method according to any one of Embodiments A1 to A24, further comprising differentiating iPSCs into cells of a desired cell type in vitro. A27. The method according to Embodiment A26, further comprising administering to differentiated cells. A28. The method according to any one of Embodiments A1 to A27, wherein the produced iPSCs are negative for the Sendai virus (SeV) vector. A29. The method according to any one of Embodiments A1 to A28, wherein the produced iPSC is derived from γδT cells. A30. The method according to any one of Embodiments A1 to A28, wherein the produced iPSC has rearrangement genes at the TRG locus and the TRD locus, and optionally the produced iPSC has a Vγ9 gene configuration and a Vδ2 gene configuration. A31. The method according to any one of Embodiments A1 to A28, wherein the produced iPSCs are not derived from αβT cells. A32. The method according to any one of Embodiments A1 to A28, wherein the produced iPSCs do not produce polymerase chain reaction (PCR) products from the TCRA and TCRB gene loci. A33. The method according to any one of Embodiments A1 to A32, wherein the produced iPSC is genomically stable without chromosome loss. A34. The method according to embodiment A33, wherein the genomic stability of the generated iPSCs is determined by karyotype analysis. A35. The method according to any one of Embodiments A1 to A34, wherein the produced iPSCs can be grown in a culture medium that does not contain supporting cells after adoption. A36. Induced pluripotent stem cells (iPSCs) prepared according to the method described in any one of Embodiments A1 to A35. A37. A pharmaceutical composition comprising the iPSC described in Embodiment A36 and a pharmaceutically acceptable excipient. A38. Differentiated cells prepared according to the method described in Embodiment A26. A39. A pharmaceutical composition comprising differentiated cells as described in Embodiment A38 and a pharmaceutically acceptable excipient.

[0237] Another set of embodiments (Embodiment Set B) provides the following: B1. A method of treating a subject that requires treatment, (i) Obtain a cell population including peripheral blood mononuclear cells (PBMCs) from the subject, (ii) Reprogramming γδT cells in a cell population into the generated iPSCs, (iii) optionally, administering the produced iPSCs or a pharmaceutical composition containing the produced iPSCs after differentiating the iPSCs into one or more desired cell types, Step (ii) is, (a) Contacting a cell population with an activated culture, wherein the activated culture contains IL-15 and zoledronic acid, (b) Culturing the cell population in an activated culture to enrich and / or activate the γδT cells in the cell population, (c) Transduction of γδT cells using a viral vector encoding one or more reprogramming factors, (d) A method comprising culturing transduced γδT cells under conditions suitable for reprogramming mammalian somatic cells into a pluripotent state. B2. The method according to Embodiment B1, wherein the activated culture further comprises IL-2. B3. The method according to Embodiment B1 or B2, wherein the viral vector is a Sendai virus (SeV) vector. B4. The method according to any one of Embodiments B1 to B3, wherein the cell population is cultured in an activated culture for 1 to 20 days, 1 to 17 days, 1 to 15 days, 1 to 13 days, 1 to 11 days, 1 to 9 days, 1 to 7 days, 1 to 5 days, 1 to 3 days, 12 to 72 hours, 12 to 60 hours, 12 to 48 hours, 12 to 36 hours, 12 to 24 hours, 8 to 16 hours, 4 to 8 hours, or 2 to 4 hours. B5. The method according to Embodiment B4, wherein the cell population is cultured in an activated culture for a maximum of 13 days, a maximum of 10 days, a maximum of 9 days, a maximum of 8 days, a maximum of 7 days, a maximum of 6 days, a maximum of 5 days, a maximum of 4 days, a maximum of 3 days, a maximum of 2 days, or a maximum of 1 day. B6. The method according to Embodiment B5, wherein the cell population is cultured in an activated culture for up to 3 days. B7. The cell population is cultured in an activated culture for 3 days, according to the method of Embodiment B5. B8. The method according to any one of Embodiments B1 to B7, wherein, after being cultured in an activated culture, the cell population comprises 5% to 100% γδT cells, 5% to 95% γδT cells, 5% to 90% γδT cells, 5% to 85% γδT cells, 5% to 80% γδT cells, 5% to 75% γδT cells, 5% to 70% γδT cells, 5% to 65% γδT cells, 5% to 60% γδT cells, 5% to 55% γδT cells, 5% to 50% γδT cells, 5% to 45% γδT cells, 5% to 40% γδT cells, 5% to 35% γδT cells, 5% to 30% γδT cells, 5% to 25% γδT cells, 5% to 20% γδT cells, or 5% to 15% γδT cells. B9. The method according to Embodiment B8, wherein, after being cultured in an activated culture, the cell population contains less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 45%, less than 40%, less than 35%, or less than 30% γδT cells. B10. The method according to Embodiment B9, wherein, after being cultured in an activated culture, the cell population contains less than 35% γδT cells. B11. The method according to any one of Embodiments B1 to B10, further comprising enriching the cell population with γδT cells after step (b). B12. The method according to embodiment B11, wherein γδT cells are enriched by intercellular aggregate concentration. B13. The method according to any one of embodiments B1 to B12, wherein at least a portion of the γδT cells are activated into Vγ9+ γδT cells in step (b). B14. The method according to any one of embodiments B1 to B12, wherein at least a portion of the γδT cells are activated into Vγ9δ2+ γδT cells in step (b). B15. The method according to any one of Embodiments B1 to B14, wherein one or more reprogramming factors are selected from the group consisting of OCT3 / 4, SOX2, KLF4, LIN28, and c-Myc. B16. The method according to any one of Embodiments B1 to B15, wherein in step (d), the transduced γδT cells are cultured in the presence of one or more supporting cell layers. B17. The method according to Embodiment B16, wherein in step (d), the transduced γδT cells are cultured in the presence of a monolayer of the supporting cell layer. B18. The method according to Embodiment B16 or B17, wherein the supporting cell layer comprises mouse embryonic fibroblasts (MEFs). B19. The method according to any one of Embodiments B1 to B18, further comprising isolating and / or purifying the prepared iPSCs. B20. The method according to any one of Embodiments B1 to B19, further comprising differentiating iPSCs into cells of a desired cell type in vitro. B21. The method according to any one of Embodiments B1 to B20, wherein the produced iPSCs are negative for the Sendai virus (SeV) vector. B22. The iPSC produced is derived from γδT cells, according to the method of any one of Embodiments B1 to B20. The generated iPSCs have rearranged genes at the TRG locus and the TRD locus, and optionally, the generated iPSCs have a Vγ9 gene configuration and a Vδ2 gene configuration, according to the method of any one of Embodiments B1 to B20. The generated iPSCs are not derived from αβ T cells, according to the method of any one of Embodiments B1 to B20. The generated iPSCs do not produce polymerase chain reaction (PCR) products from the TCRA locus and the TCRB locus, according to the method of any one of Embodiments B1 to B20. The generated iPSCs are genomically stable without chromosomal loss, according to the method of any one of Embodiments B1 to B25. The genomic stability of the generated iPSCs is determined by karyotype analysis, according to the method of Embodiment B26. The generated iPSCs can proliferate in a medium that does not contain feeder cells after adoption, according to the method of any one of Embodiments B1 to B27. The subject is a human, according to the method of any one of Embodiments B1 to B28. The subject has a proliferative disorder or a hematopoietic cancer, according to the method of any one of Embodiments B1 to B29.

[0238] In another set of embodiments (Embodiment Set C), the following are provided. An isolated population of induced pluripotent stem cells (iPSCs), wherein the isolated population of iPSCs contains pluripotent cells, and the pluripotent cells express one or more reprogramming factors and / or the pluripotent cells contain a nucleotide sequence encoding rearrangement of the TRG gene and the TRD gene, the isolated population of iPSCs. The reprogramming factor is selected from the group consisting of Oct3 / 4, Sox2, Klf4, c-Myc, and Lin28, for the isolated population of iPSCs according to Embodiment C1. C3. The isolated population of iPSCs is the isolated population of iPSCs described in Embodiment C1 or C2, which is negative for the Sendai virus (SeV) vector. C4. The isolated population of iPSCs is an isolated population of iPSCs described in any one of embodiments C1 to C3, derived from γδT cells. C5. The isolated population of iPSCs has rearranged genes at the TRG and TRD loci, and optionally, the isolated population of iPSCs has Vγ9 and Vδ2 gene configurations, as described in any one of embodiments C1 to C3. C6. The isolated population of iPSCs is an isolated population of iPSCs described in any one of embodiments C1 to C3, which is not derived from αβT cells. The isolated population of C7.iPSCs is the isolated population of iPSCs described in any one of embodiments C1 to C3, in which PCR products are not produced from the TCRA and TCRB gene loci. The isolated population of C8.iPSCs is an isolated population of iPSCs described in any one of embodiments C1 to C7, which is genomically stable without chromosome loss. The genomic stability of the isolated population of iPSCs is determined by karyotype analysis, as described in Embodiment C8. C10. An isolated population of iPSCs according to any one of embodiments C1 to C9, which can be grown and maintained in a culture medium that does not contain supporting cells after recruitment.

[0239] In yet another set of embodiments (Embodiment Set D), the following is provided: D1. A method for producing induced pluripotent stem cells (iPSCs), (a) A step for carrying out the function of enriching and / or activating γδT cells in an isolated cell population, (b) A method comprising the steps for performing a function of reprogramming γδT cells into a pluripotent state. D2. The method according to Embodiment D1, comprising the steps of carrying out the function of enriching and / or activating γδT cells in an isolated cell population, wherein the steps include contacting the isolated cell population with an activated culture, the activated culture comprising IL-15 and zoledronic acid, and culturing the isolated cell population in the activated culture to enrich and / or activate γδT cells in the isolated cell population. D3. The activated culture further comprises IL-2, according to Embodiment D2. D4. The method according to any one of embodiments D1 to D3, further comprising a step for performing the function of obtaining a population of cells isolated from a subject. D5. The method according to any one of embodiments D1 to D4, wherein the isolated cell population is peripheral blood mononuclear cells (PBMCs). D6. The method according to any one of embodiments D1 to D5, wherein the isolated cell population is terminally differentiated cells. D7. The method according to any one of embodiments D1 to D6, wherein the isolated cell population is mammalian cells. D8. The method according to embodiment D7, wherein the isolated cell population is human cells. D9. The method according to any one of embodiments D2 to D8, wherein the isolated cell population is cultured in an activated culture for 1 to 20 days, 1 to 17 days, 1 to 15 days, 1 to 13 days, 1 to 11 days, 1 to 9 days, 1 to 7 days, 1 to 5 days, 1 to 3 days, 12 to 72 hours, 12 to 60 hours, 12 to 48 hours, 12 to 36 hours, 12 to 24 hours, 8 to 16 hours, 4 to 8 hours, or 2 to 4 hours. D10. The method according to Embodiment D9, wherein the isolated cell population is cultured in an activated culture for up to 13 days, up to 10 days, up to 9 days, up to 8 days, up to 7 days, up to 6 days, up to 5 days, up to 4 days, up to 3 days, up to 2 days, or up to 1 day. D11. The method according to Embodiment D10, wherein the isolated cell population is cultured in an activated culture for up to 3 days. D12. The method according to Embodiment D10, wherein the isolated cell population is cultured in an activated culture for 3 days. D13. After culturing in the activated culture, the isolated cell population consisted of 5%-100% γδT cells, 5%-95% γδT cells, 5%-90% γδT cells, 5%-85% γδT cells, 5%-80% γδT cells, 5%-75% γδT cells, 5%-70% γδT cells, 5%-65% γδT cells, and 5%-60% γδT cells. The method according to any one of Embodiments D2 to D12, comprising 5% to 55% γδT cells, 5% to 50% γδT cells, 5% to 45% γδT cells, 5% to 40% γδT cells, 5% to 35% γδT cells, 5% to 30% γδT cells, 5% to 25% γδT cells, 5% to 20% γδT cells, or 5% to 15% γδT cells. D14. The method according to Embodiment D13, wherein the isolated cell population after being cultured in an activated culture contains less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 45%, less than 40%, less than 35%, or less than 30% γδT cells. D15. The method according to Embodiment D14, wherein the isolated cell population after being cultured in an activated culture contains less than 35% γδT cells. D16. At least some of the γδT cells are Vγ9 + The method according to any one of embodiments D1 to D15, which is activated in γδT cells. D17. At least some of the γδT cells are Vγ9δ2 + The method according to any one of embodiments D1 to D15, which is activated in γδT cells. D18. The method according to any one of Embodiments D1 to D17, comprising the steps for carrying out the function of reprogramming γδT cells into a pluripotent state, which include transducing γδT cells using a viral vector encoding one or more reprogramming factors, and culturing the transduced γδT cells under conditions suitable for reprogramming mammalian somatic cells into a pluripotent state. D19. The method according to embodiment D18, wherein the viral vector is a Sendai virus (SeV) vector. D20. The method according to Embodiment D18 or D19, wherein one or more reprogramming factors are selected from the group consisting of OCT3 / 4, SOX2, KLF4, LIN28, and c-Myc. D21. The method according to any one of embodiments D18 to D20, wherein transduced γδ T cells are cultured in the presence of one or more supporting cell layers. D22. The method according to Embodiment D21, wherein transduced γδT cells are cultured in the presence of a monolayer of supporting cells. D23. The supporting cell layer comprises mouse embryonic fibroblasts (MEFs) as described in Embodiment D21 or D22.

[0240] In yet another set of embodiments (Embodiment Set E), the following is provided: E1. Induced pluripotent stem cells (iPSCs) produced according to the method, the method is: (a) A step for carrying out the function of enriching and / or activating γδT cells in an isolated cell population, (b) iPSCs comprising a step for performing a function to reprogram γδT cells into a pluripotent state. E2. The iPSC according to Embodiment E1, comprising the steps for carrying out the function of enriching and / or activating γδT cells in an isolated cell population, wherein the steps include contacting the isolated cell population with an activated culture, the activated culture comprising IL-15 and zoledronic acid, and culturing the isolated cell population in the activated culture to enrich and / or activate γδT cells in the isolated cell population. E3. The activated culture further comprises IL-2, the iPSC according to Embodiment E2. E4. The iPSC according to any one of Embodiments E1 to E3, further comprising a step for performing the function of obtaining a population of cells isolated from a subject. E5. The isolated cell population is a peripheral blood mononuclear cell (PBMC), iPSC according to any one of Embodiments E1 to E4. E6. The isolated cell population is a terminally differentiated cell, iPSC as described in any one of Embodiments E1 to E5. E7. The isolated cell population is a mammalian cell, iPSC as described in any one of Embodiments E1 to E6. E8. The isolated cell population is human cells, iPSC as described in any one of Embodiments E7. E9. The iPSC according to any one of Embodiments E2 to E8, wherein the isolated cell population is cultured in an activated culture for 1 to 20 days, 1 to 17 days, 1 to 15 days, 1 to 13 days, 1 to 11 days, 1 to 9 days, 1 to 7 days, 1 to 5 days, 1 to 3 days, 12 to 72 hours, 12 to 60 hours, 12 to 48 hours, 12 to 36 hours, 12 to 24 hours, 8 to 16 hours, 4 to 8 hours, or 2 to 4 hours. E10. The iPSCs according to Embodiment E9, wherein the isolated cell population is cultured in an activated culture for up to 13 days, up to 10 days, up to 9 days, up to 8 days, up to 7 days, up to 6 days, up to 5 days, up to 4 days, up to 3 days, up to 2 days, or up to 1 day. E11. The isolated cell population is cultured in an activated culture for up to 3 days, iPSC as in Embodiment E10. E12. The isolated cell population is cultured in an activated culture for 3 days, iPSC as in Embodiment E10. E13. After being cultured in the activated culture, the isolated cell population consisted of 5%-100% γδT cells, 5%-95% γδT cells, 5%-90% γδT cells, 5%-85% γδT cells, 5%-80% γδT cells, 5%-75% γδT cells, 5%-70% γδT cells, 5%-65% γδT cells, and 5%-60% γδT cells. iPSC according to any one of Embodiments E2 to E12, comprising 5% to 55% γδT cells, 5% to 50% γδT cells, 5% to 45% γδT cells, 5% to 40% γδT cells, 5% to 35% γδT cells, 5% to 30% γδT cells, 5% to 25% γδT cells, 5% to 20% γδT cells, or 5% to 15% γδT cells. E14. The iPSC according to Embodiment E13, wherein the isolated cell population after being cultured in an activated culture contains less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 45%, less than 40%, less than 35%, or less than 30% γδT cells. E15. The iPSCs described in Embodiment E14, wherein the isolated cell population after being cultured in an activated culture contains less than 35% γδT cells. At least a part of the E16.γδ T cells is the iPSC according to any one of Embodiments E1 to E15, which is activated into Vγ9+ γδ T cells. At least a part of the E17.γδ T cells is the iPSC according to any one of Embodiments E1 to E15, which is activated into Vγ9δ2+ γδ T cells. For the process of implementing the function of reprogramming γδ T cells into a pluripotent state, the steps include transducing the γδ T cells using a viral vector encoding one or more reprogramming factors, and culturing the transduced γδ T cells under conditions suitable for reprogramming mammalian somatic cells into a pluripotent state. The iPSC is according to any one of Embodiments E1 to E17. The E19. viral vector is a Sendai virus (SeV) vector, and the iPSC is according to Embodiment E18. The E20. one or more reprogramming factors are selected from the group consisting of OCT3 / 4, SOX2, KLF4, LIN28, and c-Myc, and the iPSC is according to Embodiment E18 or E19. The E21. transduced γδ T cells are cultured in the presence of one or more feeder cell layers, and the iPSC is according to any one of Embodiments E18 to E20. The E22. transduced γδ T cells are cultured in the presence of a single layer of feeder cell layer, and the iPSC is according to Embodiment E21. The E23. feeder cell layer includes mouse embryonic fibroblasts (MEF), and the iPSC is according to Embodiment E21 or E22. An isolated population of induced pluripotent stem cells (iPSCs) containing pluripotent cells, wherein the pluripotent cells include means for expressing one or more reprogramming factors, and / or the pluripotent cells include means for encoding rearrangement of the TRG gene and the TRD gene. An isolated population of induced pluripotent stem cells (iPSCs). The E25. reprogramming factors are selected from the group consisting of Oct3 / 4, Sox2, Klf4, c-Myc, and Lin28, and the isolated population of iPSCs is according to Embodiment E24. E26. An isolated population of iPSCs according to Embodiment E24 or E25, wherein the isolated population of iPSCs is negative for the Sendai virus (SeV) vector. E27. An isolated population of iPSCs is an isolated population of iPSCs according to any one of embodiments E24 to E26, derived from γδT cells. E28. An isolated population of iPSCs having rearranged genes at the TRG and TRD loci, and optionally, an isolated population of iPSCs having Vγ9 and Vδ2 gene configurations, as described in any one of Embodiments E24 to E27. E29. An isolated population of iPSCs that does not originate from αβT cells, as described in any one of Embodiments E24 to E28. E30. The isolated population of iPSCs is the isolated population of iPSCs described in any one of Embodiments E24 to E29, in which PCR products are not produced from the TCRA and TCRB gene loci. E31. An isolated population of iPSCs is a population of iPSCs described in any one of Embodiments E24 to E30, which is genomically stable without chromosome loss. E32. The genomic stability of the isolated population of iPSCs is determined by karyotype analysis, as described in Embodiment E31. E33. An isolated population of iPSCs according to any one of Embodiments E24 to E32, wherein the isolated population of iPSCs can be grown and maintained in a culture medium that does not contain supporting cells after recruitment. [Examples]

[0241] 7. Examples The following describes various methods and materials used in the research. These are described to provide a complete disclosure and explanation of the methods of preparation and use of the present invention to those skilled in the art, and are not intended to limit the scope of what the inventors consider to be their invention, nor are they intended to represent all experiments that have been performed and are not feasible. Illustrative descriptions written in the present tense should be understood as not necessarily performed, but rather as potentially performed to generate data, etc., related to the teachings of the present invention. While efforts have been made to ensure accuracy of the numerical values ​​used (e.g., quantities, percentages, etc.), some experimental error and deviation should be taken into account.

[0242] 7.1. Example 1: Selective activation and enrichment of γδT cells from PBMC cultures To generate γδT cell-derived iPSCs, whole PBMCs from healthy individuals were cultured for various periods (3 days, 8 days, and 13 days) using zoledronic acid monohydrate (Zol), interleukin-2, and interleukin-15 (Zol + IL-2 + IL-15). To determine the ideal timing for inducing iPSCs from Zol-stimulated PBMCs, intercellular aggregates were concentrated from PBMC cultures on day 3, day 8, or day 13 (Figure 1, top row). Intercellular aggregates or blast cells containing activated cells could be observed from day 3 after Zol stimulation of PBMCs.

[0243] Vγ9 + Due to the selective nature of Zol in the activation and / or expansion of γδT cells, intercellular aggregates or blast cells are largely Vγ9 + It was hypothesized that the cells were composed of γδT cells. Flow cytometry analysis revealed that the intercellular aggregates consisted of approximately 35%, 91%, and 93% γδT cells of the total PBMCs on days 3, 8, and 13 of the culture period, respectively (Figure 1, top row). Among the γδT cells, TCRVγ9 +The cells comprised approximately 35%, 96%, and 98% of γδT cells on days 3, 8, and 13 of the culture period, respectively (Figure 1, bottom row). However, not all healthy donors respond similarly to Zol-mediated stimulation. Therefore, screening and identifying donors that respond rapidly to Zol-mediated stimulation helps in finding the optimal donor for reprogramming.

[0244] 7.1.1. Selective activation of Vγ9+ γδT cells from all PBMCs On days 3, 8, and 13, the vials of frozen PBMCs were rapidly thawed and diluted by adding them to 49 mL of warm complete RPMI medium in a 50 mL Falcon tube. The complete RPMI medium contained RPMI (catalog no. 61870-036, Gibco), 10% FBS (catalog no. 10099-141, Gibco), and 1x Pen / Strep (catalog no. 15070-063, Gibco). The PBMCS were centrifuged at 1500 rpm for 5 minutes. The cells were washed once by resuspending them in 35 mL of complete RPMI medium (RPMI + 10% FBS + 1x Pen / Strep). The cell pellet was resuspended in complete RPMI medium and the cells were counted. Alternatively, PBMCs were isolated from whole blood samples by density gradient centrifugation and the cells were counted using a hemocytometer. The cell density was 1.0 × 10⁶ in complete RPMI medium. 6 The concentration was adjusted to the number of cells / mL.

[0245] Meanwhile, the prepared γδ T cell culture medium (RPMI-10%, RPMI supplemented with 10% FBS and 1x Pen / Strep) was supplemented with recombinant human IL-2 (rhIL-2) (catalog number 202-IL, R&D Systems) to a final concentration of 1000 IU / mL, recombinant human rhIL-15 (catalog number 247-ILB-025, R&D Systems) to a final concentration of 10 ng / mL, and zoledronic acid to a final concentration of 5 μM. The cell density in the prepared γδ T cell culture medium was increased to 1 × 10⁶ 6 Prepared to the number of cells / mL. 10 × 10 6The cells were seeded in 10 mL of culture medium in a T-75 flask. (1) For Zol-activated PBMCs on day 3, cell enrichment was performed between day 3 and day 0 without adding any additional culture medium. (2) For Zol-activated PBMCs on day 8, 10 mL of culture medium containing 2x concentrations of IL-2 (200 IU) and IL-15 (20 ng / mL) was added on day 5 to bring the final concentrations of IL-2 and IL-15 to 100 IU and 10 ng / mL, respectively. Cell blast formation was observed under a microscope. Furthermore, on day 3, the cells were spun down at 1500 rpm for 5 minutes at room temperature. The cell pellet was resuspended on day 3 of culture in 40 mL of culture medium (RPMI + 10% FBS + 1x Pen / Strep) containing 100 IU of IL-2 and 10 ng / mL of IL-15. (3) For Zol-activated PBMCs on day 13, 10 mL of culture medium containing 2x concentrations of IL-2 (200 IU) and IL-15 (20 ng / mL) was added on day 11. Cell blast formation was observed under a microscope. On day 7, the cells were spun down at 1500 rpm for 5 minutes at room temperature. The cell pellet was resuspended on day 7 of culture in 40 mL of culture medium (RPMI + 10% FBS + 1x Pen / Strep) containing 100 IU of IL-2 and 10 ng / mL of IL-15. On day 5, the cells were spun down at 1500 rpm for 5 minutes at room temperature. The cell pellet was resuspended on day 5 of culture in 40 mL of culture medium (RPMI + 10% FBS + 1x Pen / Strep) containing 100 IU of IL-2 and 10 ng / mL of IL-15. Furthermore, on day 3 of culture, the cells were spun down at 1500 rpm for 5 minutes at room temperature. The cell pellet was resuspended on day 3 of culture in 40 mL of culture medium (RPMI + 10% FBS + 1x Pen / Strep) containing 100 IU IL-2 and 10 ng / mL IL-15.

[0246] 7.1.2. Enrichment of γδT cells Activated γδ T cells were indirectly enriched via intercellular aggregate concentration. On day 0, T-75cm contained Zol-cultured PBMCs (for 3, 8, and 13 days, respectively). 2 The cell culture flask was tilted at a 45-degree angle, and the supernatant was aspirated using a 10 mL sterile pipette without disturbing the settled cells. The remaining settled population consisted mostly of clumps of activated cells. Vγ9 in total PBMCs + γδT cells were selectively activated with Zol. The activated cells formed intercellular aggregates or blast cells. These cell aggregates had a higher sedimentation rate compared to single cells, although the supernatant consisted mainly of single cells.

[0247] The EasySep® Human γδ T Cell Isolation Kit (Stemcell Technologies) was used according to the manufacturer's instructions to enrich a pure population of γδ T cells.

[0248] PBMCs cultured with Zol on day 3 or day 8 were harvested and spun down at 1500 rpm for 5 minutes at room temperature. γδT cells enriched from Zol-stimulated PBMCs on day 3 or day 8 were used for transduction with a SeV vector.

[0249] The cells were washed once by resuspending them in plain RPMI (without FBS, or 1x Pen / Strep) medium and spun down at 1500 rpm for 5 minutes. The cell pellet was resuspended in 1 mL of EasySep® buffer (catalog no. 20144, STEMCELL Technologies), and the cells were counted using a hemocytometer.

[0250] The cell density of the settled cell aggregates was measured using 1 mL of EasySep buffer in a 5 mL polystyrene round-bottom tube, with a measurement of 50 × 10⁻¹⁴. 6Cells were prepared. 50 μL of the biotinylated cocktail was added to the resuspended cells, mixed, and incubated at room temperature for 15 minutes. Before the end of the incubation period, EasySep magnetic particles were vortexed for 30 seconds to uniformly disperse them. After the incubation period, 50 μL of EasySep magnetic particles were added to 1 mL of resuspended cells, mixed, and incubated at room temperature for 10 minutes.

[0251] After the incubation period, 1.5 mL of EasySep® buffer was added to the tube containing the cells, and the cells were resuspended by gently mixing them up and down. The tube was placed on a magnetic stand and incubated at room temperature for 5 minutes.

[0252] At the end of the incubation period, the culture medium containing the concentrated cell suspension was collected by inverting the magnet containing the tubes in a single continuous motion. In multiple tube scenarios, the supernatant was collected from the tubes without disturbing the bound magnetic particles by using a 1 mL pipette hand while the tubes were on the magnetic stand.

[0253] 37 μl of vortexed magnetic particles were added to the concentrated cell suspension, then mixed and incubated at room temperature for 5 minutes. The tube containing the cell suspension was placed on a magnetic stand and incubated at room temperature for 5 minutes. The concentrated cells were collected by transferring the cell suspension to a new 15 mL tube by inverting the magnet containing the tube in a single continuous motion. The tube was then refilled with 10 mL of complete RPMI medium (RPMI + 10% FBS + 1x Pen / Strep).

[0254] The cells were spun down at 1500 rpm for 5 minutes at room temperature and washed again by adding 10 mL of complete RPMI medium. The cells were spun down at 1500 rpm for 5 minutes at room temperature, resuspended in 1 mL of complete RPMI medium, and counted using a hemocytometer. The purity of the concentrated γδ T cells was checked by flow cytometry by staining the cells with mAbs for TCRγδ, TCRαβ, and TCR Vγ9.

[0255] 7.2. Example 2: Generation of human γδT cell-derived iPSCs from PBMC cultures Intercellular aggregates (or blast cells) were subjected to transduction with Sendai virus (SeV) vectors encoding OCT3 / 4, SOX2, KLF4, and c-Myc reprogramming factor. SeV vector-transduced cells were grown under conditions that did not include a mitotically inactivated MEF-supporting cell layer, T cell-depleted autologous PBMCs, and / or supporting cells, as described in sections 7.2.1 to 7.2.5 below.

[0256] Colonies began to be observed around 20 days after SeV transduction, and the number of colonies reached its maximum around 26 days. Surprisingly, only PBMC cultures stimulated with Zol+IL-2+IL-15 for 3 days produced a significant number of colonies 20 days after SeV transduction. In contrast, PBMC cultures stimulated with Zol+IL-2+IL-15 for 8 or 13 days did not produce a significant number of colonies after SeV transduction.

[0257] This surprising observation suggests that less concentrated cells (3 days) are far more susceptible to reprogramming compared to more concentrated cells (8 or 13 days).

[0258] As shown in Figures 2A and 2B, undifferentiated iPSC colonies of different clones (clones A-C) on the MEF supporting cell layer were identified as round colonies with smooth, dense boundaries and dense cells within dense boundaries that lacked heterotrophic centers. Clones A-C originated from the same donor.

[0259] In contrast, none of the experimental groups of SeV-transduced PBMCs (concentrated for 3, 8, or 13 days) grown on laminin-511 coated plates (support cell-free culture) or co-cultured with mitotically inactivated T-cell-depleted auto-PBMCs (support cell-based culture) produced colonies. Concentrated γδ T cells derived from 8-day PBMC cultures grown on MEF-supported cell layers also produced no colonies whatsoever.

[0260] Furthermore, mRNA or episome-mediated reprogramming was investigated as an alternative to Sendai virus (SeV)-mediated reprogramming. Since Zol-activated PBMCs were not resistant to repeated electroporation-mediated mRNA transfection, further research on mRNA-mediated reprogramming could not be conducted. Episome-mediated reprogramming resulted in very few colonies. Therefore, SeV-mediated reprogramming was observed to be the most efficient method for reprogramming Zol-activated γδ T cells.

[0261] 7.2.1. Coating of cell culture plates with extracellular matrix Gelatin coating 2 mL of 0.1% gelatin solution (catalog number ES-006-B, Merck) was added to the wells of a 6-well cell culture plate and incubated at room temperature for 2 hours or at 37°C for 1 hour.

[0262] At the end of the incubation period, the gelatin solution was aspirated using a vacuum-based aspirator, taking care to ensure that the surface area of ​​the wells did not come into contact with the aspirator. Cells (e.g., mouse embryonic fibroblasts) were added to the wells without delay.

[0263] iMatrix-511 coating A stock concentration of iMatrix-511 (catalog number 892011, Nippi / Matrixome) solution at 0.5 mg / mL was diluted with sterile DPBS (catalog number 14190-136, Gibco). The dish was then diluted to 0.5 μg / cm³. 2 Coated with a diluted iMatrix-511 of the specified concentration. One well (9.6 cm) of a 6-well plate. 2 For each well, 9.6 μL of iMatrix-511 (4.8 μg) was added to 1.99 mL of sterile DPBS and incubated overnight at 4°C, for 1 hour at 37°C, or for 3 hours at room temperature.

[0264] After the incubation period, the diluted iMatrix-511 was aspirated from the wells, taking care to prevent the well surface from coming into contact with the aspirator and to prevent the wells from drying out in the air.

[0265] To prevent the plate from drying out, the required culture medium was immediately added to the wells. The medium should be dripped into the wells along the wall. Rinsing is not necessary between aspiration and cell seeding. Cells were immediately plated at the desired density. The plate was returned to the incubator.

[0266] Vitronectin coating A vial of 0.5 mg / mL stock concentration of vitronectin (catalog number A14700, Gibco) was thawed at room temperature. 60 μL of vitronectin aliquots were prepared in polypropylene tubes. These aliquots were either used immediately or frozen at -80°C.

[0267] To coat the wells of a 6-well plate, two 60 μL aliquots of vitronectin were removed from a -80°C storage chamber and thawed at room temperature. Two 60 μL aliquots were required for each 6-well plate.

[0268] 120 μL of thawed vitronectin was added at room temperature to a 15 mL conical tube containing 12 mL of sterile DPBS without calcium or magnesium, and the diluted vitronectin was gently resuspended by pipetting up and down. This yielded a working concentration of 5 μg / mL of vitronectin (i.e., a 1:100 dilution).

[0269] 2 mL of diluted vitronectin solution was added to each well of a 6-well plate. (6-well plate (10 cm)) 2 When used to coat with 2 mL / well, the final concentration of vitronectin is 0.5 μg / cm³. 2 The coated plates were incubated at 37°C for 3 hours (or at room temperature for 1 hour, according to the vitronectin manufacturer).

[0270] Coated plates can be wrapped in laboratory film and used or stored at 2-8°C for up to one week. However, coated plates cannot be allowed to dry out. Before use, preheat the coated plates to room temperature for at least one hour.

[0271] The vitronectin solution was aspirated and discarded. Rinsing the coated plate after the removal of vitronectin is not necessary. Cells can be directly subcultured onto the vitronectin-coated plate.

[0272] 7.2.2. Preparation of mitotically inactivated mouse embryonic fibroblast (MEF) cell cultures Mouse embryonic fibroblast (MEF) resuscitation The MEF(CF-1) (catalog number SCRC-1040, ATCC) cell line was obtained from ATCC in a frozen vial. The frozen vial of MEF cells was rapidly thawed in a 37°C water bath. The thawed contents were added dropwise to a 50 mL Falcon tube containing 40 mL of MEF medium preheated to 37°C, which included DMEM (catalog number 11965-092, Gibco), 15% FBS, and 1% Pen / Strep. The MEF cells were spun down at 1500 rpm for 5 minutes at room temperature, and the supernatant was discarded. The cells were washed once with 30 mL of MEF medium (DMEM + 15% FBS + 1% Pen / Strep) and spun down at 1500 rpm for 5 minutes at room temperature. The cells were then counted using a hemocytometer with trypan blue (catalog number TCL046, Himedia).

[0273] MEF seeding and culture MEF to T-75cm 2 0.8 × 10 in a flask 6 Cells were seeded in 20-25 mL of MEF medium (DMEM + 15% FBS + 1% Pen / Strep) at a cell density. Alternatively, MEF was seeded in a T-150 flask at a density of 1 × 10⁶. 6 Cells were seeded in 40 mL of MEF medium at a given cell density. The MEF was incubated at 37°C in a humidified incubator with 7.5–10% CO2. To grow MEF at 7.5–10% CO2, the medium should contain 3.7 g / L of sodium bicarbonate. When the cell culture reached a concentration of 60–70%, the cells were subcultured (as shown below).

[0274] MEF subculture The MEF culture medium was removed and discarded. The cell layer was then rinsed with 20 mL (T-75 flask) or 40 mL (T-175 flask) of sterile DPBS. 4 mL (T-75 flask) or 10 mL (T-175 flask) of preheated trypsin-EDTA (catalog no. 25200-056, Gibco) was added, and the mixture was incubated for 2–5 minutes. The MEF was separated from the flask as observed under a microscope and dissociated to form a single-cell suspension. The trypsin was neutralized by adding twice the volume of complete MEF culture medium (DMEM + 15% FBS + 1% Pen / Strep) to the flask.

[0275] All cells were transferred to a centrifuge tube and centrifuged at 1500 rpm for 5 minutes at room temperature. After discarding the supernatant, 10 mL of complete growth medium was added to the cell pellet. Single-cell suspensions were prepared by gently suspending the cells with a 10 mL pipette. Further MEF culture medium was added to the single-cell suspension as needed for subculturing.

[0276] For subculturing, MEF cells were divided in a 1:2 or 1:3 ratio as needed. The cells were incubated in flasks in a humidified incubator at 37°C containing 7.5–10% CO2. When MEFs are used as a supporting cell layer for iPSCs, it is not recommended to use them beyond passage 6 (P6).

[0277] MEF freezing MEF cells were frozen in 1 mL of cryovial culture medium (complete MEF medium supplemented with an additional 40% FBS and 10% (v / v) DMSO (catalog no. D2650, Sigma)) at a cell density of 5 million cells / cryovial.

[0278] The cryovial was stored overnight at -80°C in a step cooler, and then transferred to liquid nitrogen the following day.

[0279] MEF cells undergo mitotic inactivation. Frozen MEF vials (<passage 5) were rapidly thawed in a 37°C water bath and added dropwise to 49 mL of preheated complete MEF medium (DMEM + 15% FBS + 1x Pen Strep) in a 50 mL Falcon tube. Cells were centrifuged at 1500 rpm for 5 minutes at room temperature and washed once with phosphate-buffered saline (PBS). One million MEF cells were placed at T-150 cm. 2 The cells were seeded in 40 mL of MEF medium (DMEM + 15% FBS + 1% Pen / Strep) in a flask and incubated at 37°C in a humidified incubator with 7.5% CO2. To grow MEF cells at 7.5% CO2, the DMEM medium should contain 3.7 g / L of sodium bicarbonate. On day 2 of culture, the medium was replaced with fresh MEF medium. After 3–3.5 days of culture, the MEF cells reached the subconfluent stage (approximately 70% concentration).

[0280] Mitomycin C master stock was prepared at a concentration of 1 mg / mL in redistilled H2O (dd H2O). Mitomycin C was added to MEF culture medium (DMEM + 15% FBS + 1x Pen / Strep) to a final working concentration of 10 μg (10 μl) per 1 ml. A T-150 cm² containing logarithmic passage 3 MEF was used. 2 For the flask, 400 μL of mitomycin C (1 mg / mL stock concentration) was added to 40 mL of MEF culture medium (DMEM + 15% FBS + 1x Pen / Strep). The mixture was incubated in the flask at 37°C for 2 hours in a humidified incubator with 7.5% CO2. After the incubation period, the medium containing mitomycin C was aspirated. The cells were washed 10 times repeatedly with 40 mL of plain DMEM medium. Vigorous washing is necessary to remove any remaining mitomycin C, as its presence would hinder the growth of the co-cultured cells. As a final wash, the cells were washed once with 40 mL of DPBS.

[0281] 10 mL of 1× trypsin-EDTA was added to washed T-150 cm containing MEF cells.2 The solution was added to a flask. It was incubated at 37°C for 3-5 minutes in a humidified incubator with 7.5% CO2. After incubation, trypsin was neutralized by adding 20 mL of complete MEF medium (DMEM + 15% FBS + Pen / Strep). The cells were spun down at 1500 rpm for 5 minutes at room temperature. After washing, the mitomycin C-treated MEF was used directly or frozen for downstream application.

[0282] 7.2.3. Sendai virus (SeV) vector-mediated reprogramming of γδ T cells Day 0: Sendai virus infection Cells were counted using trypan blue. 500,000 intercellular aggregates of concentrated PBMCs or γδT cells were pipetted into wells of a low-adhesion 24-well plate. Low-adhesion plates are important to prevent any cells, including PBMCs, from adhering to each other during or after SeV infection.

[0283] A pure population of γδT cells was enriched only from PBMCs (Plant-Based Microorganisms) collected on days 8 and 13 of PBMC culture using Zol+IL-2+IL-15 culture, without any contamination by αβT cells or other cells.

[0284] Cytotune® 2.0 tubes (catalog number A16517, Thermo Fischer Scientific) were removed from -80°C and rapidly thawed one by one for 5-10 seconds in a 37°C water bath. After thawing, these tubes were placed on ice.

[0285] The calculated volumes of Sendai virus particles containing KOS, hc-Myc, and hKlf4 were added to cells in 0.3 mL of complete RPMI medium (RPMI + 10% FBS) at infection multiplicities of 5, 5, and 3, respectively (MOI, calculated based on titers specific to each lot of the Cyotune® 2.0 kit). The complete culture medium was supplemented with 100 IU of IL-2 and 10 ng / mL of IL-15. Polyblen (catalog number TR-1003-G, Millipore) was added to the virus-containing cell suspension at a concentration of 4 μg / mL.

[0286] Day 1: Change the culture medium and culture the cells. The cells and culture medium were removed from the culture plate and transferred to a 15 mL Falcon tube. The wells in the plate were gently rinsed with 1 mL of complete RPMI medium (RPMI + 10% FBS + 1x Pen / Strep) to ensure that the majority of the cells were collected. The complete culture medium was supplemented with 100 IU of IL-2 and 10 ng / mL of IL-15.

[0287] Cytotune® 2.0 Sendai virus was removed from the cell suspension by rotating the cells at 200 × g for 10 minutes at room temperature. The medium was aspirated, and the cell pellet was resuspended in 0.5 mL of complete RPMI medium in a low-adhesion 24-well plate.

[0288] The cells were cultured for 2 days at 37°C in a humidified incubator with 5% CO2 in complete RPMI medium (RPMI + 10% FBS + 1% Pen / Strep).

[0289] Day 3: Transplantation of transduced cells - under Meteorological Elevation Field (MEF) conditions The supporting cell layer was mitotically arrested using mitomycin C (catalog number M4287, Millipore). A detailed protocol for treating MEFs with mitomycin C is described in Section 7.2.2. The mitotically arrested MEFs were plated onto gelatin-coated wells of a 6-well plate. It is desirable to plate the mitotically arrested MEFs one or two days before seeding transduced cells onto the MEFs. Whether mitomycin C arrested the cell cycle of the MEFs was checked by measuring the culture medium consumption by the mitotically arrested MEFs over 7 days at 37°C and 5% CO2. Whether mitomycin C actually arrested the cell cycle of the MEFs should always be checked.

[0290] SeV-transduced intercellular aggregates (PBMCs) or γδT cells were counted and seeded at various cell densities (10,000 to 100,000 cells / well) on a MEF monolayer in 2 mL of complete RPMI medium in a 6-well plate. The complete culture medium was supplemented with 100 IU of IL-2 and 10 ng / mL of IL-15.

[0291] Day 3: Transplantation of transduced cells - under conditions without supporting cells (on iMatrix-511 coated plates) On the day that SeV-transduced intercellular aggregates-enriched PBMCs or γδT cells were seeded, 6-well plates were coated with laminin 511-E8 fragments (a detailed protocol for coating plates with iMatrix-511 is described in Section 7.2.1).

[0292] SeV-transduced PBMCs or γδT cells were seeded at various cell densities (10,000 to 100,000 cells / well in a 6-well plate) on wells coated with laminin 511-E8 fragments in 2 mL of complete RPMI culture medium.

[0293] Day 5, Day 7, Day 9, and Day 11: Change of culture medium Half (1 mL) of the used complete RPMI medium was removed without disturbing the cells, and the wells were replenished with 1 mL of fresh StemFit Basic 2.0 (catalog number SFB500, AJINOMOTO) containing 100 ng / mL of basic fibroblast growth factor (bFGF) (catalog number AMS-480-100, Amsbio). The final working concentration of bFGF was 50 ng / mL.

[0294] On days 7, 9, and 11, bFGF was added at a double concentration, i.e., 100 ng / mL, while replenishing the culture medium with fresh medium.

[0295] Days 13, 15, 17, 19, and 21: Complete replenishment of culture medium. 1.5 mL of old medium was removed, and the wells were refilled with 2 mL of StemFit Basic 2.0 medium containing 50 ng / mL of bFGF. Colonies were observed daily, and developing colonies were marked with a marker pen and tracked.

[0296] 7.2.4. Colony Pickup and Propagation Once the colonies became prominent, around day 23, we prepared for manual colony pickup. The 6-well plate containing the iPSC colonies was removed from the incubator, and the cells were observed under a microscope.

[0297] I marked the undifferentiated colonies with a marker pen. For reference, I took one or two photographs of these colonies. Once the colonies had been identified, I transferred the plate to a laminar hood.

[0298] The used culture medium was aspirated from the wells using a vacuum pump. For support cell-based culture, 800 μL of TrypLE (catalog no. 12563-029, Gibco) dissociation reagent, pre-diluted 1:1 in sterile DPBS, was added to the wells of a 6-well plate. The plate was incubated at 37°C for 4 minutes in a humidified incubator containing 5% CO2.

[0299] For cultures without supporting cells, 800 μL of TrypLE dissociation reagent, pre-diluted 1:1 in sterile DPBS, was added to the wells of a 6-well plate. The plate was incubated at 37°C for 1 minute in a humidified incubator containing 5% CO2.

[0300] At the end of the incubation period, the plate was removed from the incubator and gently tilted. All of the TrypLE reagent present in the wells was aspirated using a 1 mL pipette. 2 mL of StemFit Basic 2.0 medium containing 10 μM ROCK inhibitor (catalog no. SCM075, Merck Millipore) and bFGF were added to the wells.

[0301] For cultures without supporting cells, 2 mL of mTeSR medium (catalog number 85850, STEMCELL Technologies) containing 10 μM of ROCK inhibitor was added to each well (without bFGF).

[0302] For colony pickup, plates containing StemFit Basic 2.0 (support cell-based culture) or mTeSR (support cell-free) medium were transferred to an inverted microscope (OLYMPUS, model CKX53SF, serial number 8M44621). Meanwhile, the inverted microscope was moved into a laminar hood. It is important to ensure that the microscope is clean and disinfected before moving it into the laminar hood. The microscope was adjusted to focus on pre-marked colonies scraped back and forth using a gel-loading tip with a narrow opening attached to a 200 μL pipette. The colonies were divided by scraping them into small square clumps.

[0303] Colonies can be collected with minimal scraping to prevent contamination from adjacent colonies, or entire colonies can be collected by 200 μL of chips and transferred to wells in a 96-well plate containing either 200 μL of StemFit Basic 2.0 medium containing a ROCK inhibitor and bFGF for support cell-based cultures, or mTeSR medium containing a ROCK inhibitor but without bFGF for non-support cell cultures.

[0304] Using a 200 μL pipette, the scraped mass was collected and immediately transferred to either an irradiated MEF monolayer seeded plate (support cell-based culture) in StemFit Basic 2.0 medium containing 10 μM ROCK inhibitor and 100 ng / mL bFGF, or a laminin-511 / vitronectin coated plate (support cell-free culture) in mTeSR medium containing 10 μM ROCK inhibitor, for only 24 hours on day 0, after which no further ROCK inhibitor was added.

[0305] Irradiated MEF was obtained from ATCC as a frozen vial (catalog number SCRC-1040.1, ATCC) and thawed as described by ATCC. The thawed cells were counted and seeded onto gelatin-coated plates at a density of 500,000 cells / well in 6-well plates of MEF culture medium one day before transferring iPSC colonies onto them (this density may vary depending on different conditions). Unlike mitomycin C-treated MEF monolayers, irradiated MEF monolayers lasted only 5–7 days.

[0306] The entire colony pickup procedure needed to be carried out quickly. Prolonged exposure of colonies to the external environment can lead to improper adhesion and / or differentiation of cells within the colony.

[0307] The plates were arranged in a figure-eight pattern and transferred to a humidified incubator at 37°C containing 5% CO2. Colonies were cultured for the next 48 hours without any disturbance. During the 48 hours of cell culture, the used medium in one well of the 6-well plate was replaced every 24 hours using 2 mL of fresh medium until the next passaging.

[0308] The passage interval depended on the growth of the iPSC colony, which could vary from donor to donor. Typically, each passage took 3–5 days. Numerous differentiated cells were observed up to the end of the first four passages. From the fifth passage onward, undifferentiated colonies were observed.

[0309] The procedure described above can be repeated for each new passage cycle. The plates were observed daily.

[0310] 7.2.5. Freezing and Thawing iPSC Colonies Freezing of iPSC colonies The plate was removed from the incubator, and the cells were observed under an inverted microscope. Undifferentiated colonies (colonies with smooth, dense boundaries, and dense cells within dense boundaries that lacked heterotrophic centers) were marked with a marker pen. For reference, one or two photographs of these colonies were taken. Once colony identification was complete, the plate was transferred to a laminar hood.

[0311] The used culture medium was aspirated from the wells using a vacuum pump. For support cell-based culture, 800 μL of TrypLE dissociation reagent, pre-diluted 1:1 with sterile DPBS, was added to the wells of the 6-well plate. The plate was incubated in a humidified incubator containing 5% CO2 at 37°C for 2–4 minutes (for support cell-based culture) or 1 minute (for culture without support cells).

[0312] At the end of the incubation period, the plate was removed from the incubator and gently tilted. All TrypLE reagent present in the wells was aspirated using a vacuum pump. The wells were washed with plain medium. 2 mL of StemFit Basic 2.0 medium (for support cell-based cultures) or mTeSR medium (for cultures without support cells) was added to the wells.

[0313] The plate was placed under a microscope for colony pickup. All differentiated colonies were selectively removed using a gel loading tip with a narrow opening attached to a 200 μL pipette. The plate was washed with 2 mL of StemFit Basic 2.0 medium (for support cell-based cultures) or mTeSR medium (for cultures without support cells), and the medium was aspirated with a vacuum pump. The washing process was repeated once more. After these two washes, the majority of the removed differentiated colonies were removed from the plate.

[0314] In support cell-based cultures, undifferentiated colonies were collected in Eppendorf tubes as described in Section 7.2.4. In cultures without support cells, the remaining undifferentiated colonies were scraped from the wells using a scraper and collected in 1.5 mL Eppendorf tubes.

[0315] The cells were spun down at 100 × g for 30 seconds at room temperature. The supernatant was discarded using a 1 mL pipette. The cell pellet was gently resuspended in 1 mL of frozen medium (ES-FBS + 10% DMSO) or, alternatively, in a knockout serum substitute containing 10% DMSO as the frozen medium (KnockOut® SR, catalog no. 10828028, Thermo Fisher Scientific).

[0316] The vials were stored in a step cooler, left at -80°C overnight, and then transferred to liquid nitrogen the following day.

[0317] Thawing iPSC colonies Frozen iPSCs (derived from support cell-based or support cell-free cultures) were rapidly thawed in a 37°C water bath. The contents of the vial were added dropwise to a 50 mL conical tube containing 25 mL of StemFit Basic 2.0 medium (for support cell-based cultures) or mTeSR medium (for support cell-free cultures) containing 10 μM ROCK inhibitor. It is important to ensure that cells are added dropwise, as sudden addition of cells to the medium can cause osmotic shock.

[0318] The cells were spun down at 1200 rpm for 5 minutes at room temperature. The supernatant was discarded, and the cells were gently resuspended in 2 mL of StemFit Basic 2.0 medium containing 10 μM ROCK inhibitor and 100 ng / mL bFGF (for support cell-based cultures) or mTeSR medium containing 10 μM ROCK inhibitor (for cultures without support cells). The ROCK inhibitor in the culture medium helps prevent spontaneous differentiation of iPSCs and improves their survival during thawing.

[0319] The resuspended iPSC clumps were seeded into 6-well plates pre-seed with irradiated MEFs (as described in Section 7.2.4) or pre-coated with vitronectin (as described in Section 7.2.1), for support cell-based culture and support cell-free culture, respectively.

[0320] The plates were incubated at 37°C for 24 hours in a humidified incubator containing 5% CO2. After 24 hours of incubation, the culture medium containing the ROCK inhibitor was replaced with fresh medium containing bFGF but without the ROCK inhibitor. No substantial difference was observed even when the ROCK inhibitor was continued to be used throughout the culture.

[0321] The culture medium used was replaced every 24 hours with 2 mL of fresh medium per well of the 6-well plate until the next subculturing.

[0322] The passage interval depended on the recovery and propagation of iPSC colonies. Colony propagation varied from donor to donor. However, colony recovery depended on how well the iPSC colonies were frozen and thawed. Typically, each passage took an average of 3–5 days.

[0323] Damaged colonies may require a longer recovery time (7-10 days). The culture medium needs to be replaced with fresh medium every 24 hours until the iPSCs have fully recovered.

[0324] The recovered colonies initially produced many differentiated cells (up to passage 4 or 5). From passage 5 onward, a significant number of undifferentiated colonies can be observed.

[0325] 7.3. Example 3: Characterization of TRG gene rearrangement and TRD gene rearrangement in γδT cell-derived iPSCs iPSC colonies derived from Zol-activated PBMCs on day 3 were examined for rearrangement at the TRG and TRD loci. In the first step, genomic DNA from all five iPSC colonies and the 22Rv1 cell line was isolated as described in Section 7.3.1. Genomic PCR was performed using primers (for the TRG locus) from the IdentiClone® T cell receptor gamma gene rearrangement assay kit (catalog number 1-207-0101, Invivoscribe), and gene rearrangement at the TRG locus was evaluated according to the procedure described in Section 7.3.2.

[0326] As detailed in Section 7.3.2, when amplicons obtained from genomic PCR were subjected to capillary electrophoresis, amplicons of the desired size of approximately 191 and 192 bp were observed in all clones, confirming a specific Vγ9 gene rearrangement at the TRG locus (Figure 3). However, Figure 3 also shows additional amplicons of approximately 180 and 182 bp in clones A, B, and C, suggesting the possibility of other Vγ gene rearrangements in these clones.

[0327] Considering that the T cell receptor gamma gene rearrangement assay did not produce conclusive results and did not produce any artifacts associated with the ABI detection system (detailed in Section 7.3.2), genomic PCR was performed using publicly available primers specific to the variable region (Vγ9) and ligation regions (JP1 / JP2, JP) of the TRG locus and the variable region (Vγ2) and ligation regions (Jδ1 and Jδ3) of the TRD locus. The publicly available primer sequences for genomic PCR analysis of TRG and TRD gene rearrangements are shown in Table 2 below.

[0328] [Table 2]

[0329] In the genomic PCT analysis performed, all iPSC colonies from clones A, B, and C showed rearrangements of the TRG and TRD genes. The gene rearrangements were identified as single bands representing Vγ9-JP and Vδ2-Jδ1 or Jδ3 recombination, indicating that these colonies possess rearranged Vγ9 Vδ2-TCR genes. For all three clones, the TRG gene rearrangement was detected as a single band representing Vγ9-JP. For clone A, the TCRδ gene rearrangement was detected as Vδ2-Jδ1. For clones B and C, Vδ2-Jδ3 recombination was detected, indicating that these clones possess Vγ9 and Vδ2 gene rearrangements (Figure 4). GAPDH amplification was observed in all three clones as a housekeeping control gene.

[0330] No amplification was observed in genomic DNA isolated from the 22Rv1 cell line, reaffirming the specific properties of the genomic primers used in this study (Figure 4). Furthermore, Figure 4 also shows that no amplification was observed in the genomic DNA of clones A, B, and C when amplified using primers for TCRα and TCRβ, confirming that these colonies were not derived from αβT cells. In addition, sequencing of the amplicons and BLAST of the sequences against the whole human genome confirmed the successful placement of the Vγ9 and Vδ2 genes at the TRG and TRD loci in all clones (Figure 5 for clone B, and Figures 6A-6D for clones A, C, D, and E). The amplicons were run on a 1% agarose gel only for clones A, B, and C, but all five clones were subjected to genomic PCR and sequencing using specific primers (Figures 5 and 6A-6D).

[0331] 7.3.1. Isolation of Genomic DNA Genomic DNA from iPSC colonies and 22Rv1 cells was isolated using the GenElute® Mammalian Genomic DNA Miniprep Kit (catalog number G1N70-1Kt, Sigma) as detailed in the following protocol.

[0332] Undifferentiated iPSC colonies were picked as described in Section 7.2.4. The cells were pelleted at 200 × g for 30 seconds at room temperature. The culture medium was carefully removed using a 1 mL pipette until no medium remained in the tube. The cells were rapidly frozen in liquid nitrogen and stored at -80°C until future use.

[0333] The cell pellet was slowly thawed on ice for 10-20 minutes and completely resuspended in 200 μL of resuspension solution. 20 μL of RNase A solution was added, and the mixture was incubated at room temperature for 2 minutes. 20 μL of proteinase K solution was added to the sample, followed by 200 μL of lysis solution C (B8803). The mixture was vortexed thoroughly for approximately 15 seconds and incubated at 70°C for 10 minutes. A homogeneous mixture is essential for efficient lysis.

[0334] 500 μL of column preparation solution, designed to maximize DNA binding to the membrane and provide more consistent yields, was added to each pre-assembled GenElute® Miniprep Binding Column. The columns were centrifuged at 12,000 × g for 1 minute using a benchtop Eppendorf centrifuge. The flow-through solution was discarded.

[0335] 200 μL of ethanol (95-100%) was added to the solution mixture, and the mixture was thoroughly mixed by vortexing for 5-10 seconds. A homogeneous solution is essential.

[0336] To reduce DNA shear during transfer, the entire contents of the tube were transferred to the treated conjugated column using a wide-mouth pipette tip. The column was centrifuged at ≥6500 × g for 1 minute. The collection tube containing the flow-through solution was discarded. The conjugated column was placed in a new 2 mL collection tube.

[0337] Before initial use, the washing solution concentrate was diluted with ethanol according to the manufacturer's instructions. 500 μL of the washing solution was added to the bound column and centrifuged at ≥6,500 × g for 1 minute. The collection tube containing the flow-through solution was discarded. The bound column was placed in a new 2 mL collection tube.

[0338] Another 500 μL of washing solution was added to the binding column, and the column was dried by centrifugation at maximum speed (12,000–16,000 × g) for 3 minutes. The binding column must be ethanol-free before eluting DNA. If residual ethanol was observed, the column was centrifuged at maximum speed for an additional minute. The collection tube containing the flow-through solution was discarded. The binding column was placed in a new 2 mL collection tube.

[0339] 200 μL of elution solution was pipettered directly to the center of the binding column, and the DNA was eluted by centrifugation at ≥6,500 × g for 1 minute. To increase elution efficiency, the binding column was incubated at room temperature for 5 minutes after adding the elution solution, and then centrifuged.

[0340] 7.3.2. T cell receptor gamma gene rearrangement assay 2.0 The IdentiClone® T-cell receptor gamma gene rearrangement assay 2.0 PCR assay uses multiple consensus DNA primers that target conserved gene regions within the T-cell receptor gamma chain gene. Genomic DNA was isolated from a given clone, and the region was subsequently amplified using the IdentiClone® T-cell receptor gamma gene rearrangement assay 2.0 kit. This kit consists of a single master mix containing primers (conjugated to 6-FAM fluorescent dye) that target the Vγ2, Vγ3, Vγ4, Vγ5, Vγ8, Vγ9, Vγ10, and Vγ11 as well as the Jγ1 / Jγ2, JγP, and JγP1 / JγP2 regions. This was followed by fractionation by capillary electrophoresis and analysis using GeneMapper software (Eurofins). The PCR amplicons had an expected size range of 159–207 base pairs.

[0341] PCR was performed using genomic DNA isolated from iPSC colonies under the following PCR conditions. First, a temperature of 95°C was applied for 3 minutes. Next, the following cycle was applied 25 times: 95°C for 30 seconds, 65°C for 30 seconds, and 72°C for 45 seconds. Finally, the temperature was maintained at 72°C for 5 minutes, and then maintained at 25°C until the mixture was removed. The PCR mixture (30 μL) consisted of the following components: 15 μL of 2×Pwo Master (catalog number 03789403001, Roche), 100 ng of DNA template, 0.5 μL each of two primers (100 μM), and water to make 30 μL.

[0342] Initially, only the genomic DNA from clone B was used for PCR as a test to confirm that the system was functioning. The PCR sample was submitted to a company called Eurofins (Bengalru, India) for analysis and processing according to the kit manufacturer's protocol. Details of this sample are summarized in Table 3 below.

[0343] [Table 3]

[0344] In the analysis of the results, based on the presence of peaks at specific sizes of DNA fragments, it was determined that test sample B (clone B) was positive for rearrangement of the Vγ9 Vδ2 gene.

[0345] The remaining genomic DNA samples were used to perform PCR in the same manner as for clone B, and were again subjected to analysis in Eurofins. Details of these samples are summarized in Table 4 below.

[0346] [Table 4]

[0347] During the analysis, some of the test samples (clones) showed positivity for the Vγ9 Vδ2 rearrangement, but this was only considered a preliminary result. Sequencing was performed to confirm these clones. For sequencing, PCR was performed using two pairs of primers to amplify the γ and δ regions, respectively. The sequences of these primers are summarized in Table 5 below.

[0348] [Table 5]

[0349] Sample processing and analysis were performed using Eurofin (Bengalru, India) according to the kit manufacturer's instructions. PCR products were labeled with 6-FAM. Size standards (ROX or LIZ) and Hi-Di formamide were added according to the pre-coagulation protocol.

[0350] Regarding ABI fluorescence detection, leading peaks were frequently observed, which were artifacts attributable to the detection method used by the ABI platform. These leading peaks were sometimes distorted and had bases sloping to the right towards the true peak. This was particularly evident in the sample control-size ladder master mix, where the 96 bp peak had a leading peak at 84 bp.

[0351] In a new microcentrifuge tube, appropriate amounts of PCR reaction product (10 μL for ROX size standard and 9.5 μL for LIZ size standard) were mixed with Hi-Di formamide and the ROX or LIZ size standard by thoroughly vortexing the mixture.

[0352] In a new 96-well PCR plate, 10 μl of Hi-Di formamide with ROX or LIZ size standards was added to each individual well for each PCR.

[0353] Each 1 μL of PCR reaction product was transferred to a well containing Hi-Di formamide and a ROX or LIZ size standard. Only one sample was added per well and mixed by pipetting up and down. The PCR plate was then capped or covered.

[0354] The sample was thermally denatured at 95°C for 2 minutes, and then rapidly cooled on ice for 5 minutes.

[0355] Sample sheets and injection lists were prepared for each sample. The samples were electrophoresed using an ABI3100 / 3130 capillary electrophoresis system according to its user manual. The data were automatically displayed as size and color-specific peaks.

[0356] 7.3.3. Genome PCR Genomic PCR was performed to evaluate rearrangements at the TRG and TRD loci. Genomic DNA isolated from iPSC clones was used as a template (as described above).

[0357] The amplicons were identified by 1% agarose gel electrophoresis. In a second series of experiments, DNA was extracted from the dominant band on the agarose gel, cloned into a Topo vector, and sequenced using a 3730xl DNA analyzer (catalog number 3730XL, Thermo Fisher Scientific). The amplicon sequences were analyzed for sequence homology against the human whole genome using the BLAST program (https: / / blast.ncbi.nlm.nih.gov / BlastAlign.cgi).

[0358] 7.4. Example 4: Evaluation of the presence of SeV transgenicity in γδT cell-derived iPSCs After confirming rearrangement at the TRG and TRD loci in all five clones, the presence of the Sendai virus transgene (SeV Tg) was examined in all clones by RT-PCR as described below. Around the 10th passage, almost all colonies were negative for the SeV vector. This observation was consistent with the fact that higher passage colonies do not contain the SeV transgene. The RT-PCR primer sequences are summarized in Table 6 below.

[0359] [Table 6]

[0360] 7.4.1. RT-PCR RT-PCR was performed using the primers listed in Table 6 above to evaluate the expression of pluripotency genes (Oct3 / 4, Nanog, Sox2, Lin28) and to verify the presence or absence of the Sendai virus transgene (SeV Tg) among all five iPSC clones. Total RNA was isolated from iPSC colonies and all PBMCs using the RNeasy Plus mini-kit (catalog no. 74134, Qiagen) according to the manufacturer's instructions. Isolated RNA was quantified using nanodrops. cDNA synthesis was performed using the Primescript 1st strand cDNA synthesis kit (catalog no. 6110B, Takara) according to the manufacturer's instructions.

[0361] The obtained cDNA was used as a template, and RT-PCR was performed using the primers listed in Table 6 above. At the end of RT-PCR, the amplicons were subjected to 1% agarose gel electrophoresis to visualize bands with a DNA ladder at one end. cDNA prepared from all PBMCs was used as a template-negative control, and primers for TCRα and TCRβ were used as negative primer controls.

[0362] 7.5. Example 5: Evaluation of pluripotency markers in γδT cell-derived iPSCs Pluripotency markers of γδ T cell-derived iPSC colonies were evaluated by RT-PCR as described in Section 7.4.1 (Figure 7A), by immunohistochemistry as described in Section 7.5.1 (Figure 7B), and by flow cytometry as described in Section 7.5.2 (Figure 7C). The RT-PCR results showed that these colonies expressed mRNA encoding the pluripotency transcription factors Oct3 / 4, Nanog, Sox2, and Lin28. All PBMCs were used as negative controls to demonstrate that the primers were specific to the pluripotency markers (RT-PCR primer list and sequences are shown in Table 6).

[0363] Further supporting these results, immunohistochemical data confirmed the presence of master transcription factors (Nanog, Oct3 / 4, Sox2) in all five iPSC colonies (see Figure 7B). Figure 7B shows that Nanog and Oct3 / 4 expression was uniform across all clones. However, Sox2 was expressed differently in all five clones (variing from 20-40%). Furthermore, as shown in Figure 7C, flow cytometry data from single suspensions of iPSC clones reaffirmed the IHC observations, showing that they expressed high levels of surface SSEA-4 and TRA1-60, as well as nuclear Oct-3. Interestingly, consistent with the IHC data, flow cytometry analysis also showed different levels of Sox2 expression among all five clones (Figure 7C).

[0364] 7.5.1. Immunohistochemistry (IHC) Seeding of iPSCs on a coverslip The coverslips were cut to the desired size and placed in the wells of a 24-well plate. The coverslips were then coated with vitronectin by immersion in 1 mL of PBS containing vitronectin, thereby adapting the iPSCs from support cell conditions to support cell-free conditions. Thus, the coverslips were coated using vitronectin.

[0365] A 24-well plate was incubated at 37°C for 2 hours. After incubation, PBS containing vitronectin was aspirated, and the wells were washed once with DPBS. The iPSC mass was immediately plated onto a vitronectin-coated coverslip in mTeSR culture medium containing 10 μM ROCK inhibitor.

[0366] 24-well plates were incubated in a humidified incubator at 37°C containing 5% CO2. After 24 hours of incubation, the culture medium containing the ROCK inhibitor was replaced with fresh mTeSR medium without the ROCK inhibitor. At this stage, colonies began to adhere to the coverslip. No substantial difference was observed even when the ROCK inhibitor was continued to be used during incubation.

[0367] The culture was continued for another two days in a culture medium replenished with fresh medium, or until the colonies grew to the desired size.

[0368] Intranuclear staining On the day of IHC staining, the culture medium was aspirated from the 24-well plate containing the coverslip on which the iPSC colonies were seeded, and washed twice with 0.5 mL of DPBS.

[0369] iPSC-seeded coverslips were fixed by incubation in 200 μL of 4.2% PFA at room temperature for exactly 2 minutes. After incubation, the PFA was aspirated from the wells containing the coverslips seeded with iPSC colonies. The coverslips containing the fixed cells were washed twice with 400 μL of 1x BD Perm wash buffer (catalog no. 51-2091KZ, BD Biosciences).

[0370] Blocking was performed by incubating coverslips containing fixed cells in 400 μL of blocking buffer (10% donkey serum and 0.35% Triton X-100) at room temperature for 1 hour. After the incubation period, the cells were washed once with 400 μL of 1x BD Perm wash buffer.

[0371] Next, the cells were permeabilized by incubation in 400 μL of 1x fixation / permeabilization solution at 4°C for 1 hour in a refrigerator. The fixation / permeabilization solution was from the eBioscience® Foxp3 / Transcription Factor Staining Buffer Set. One portion of the fixation / permeabilization concentrate was diluted with three portions of fixation / permeabilization diluent. Since Nanog, Oct3 / 4, and Sox2 are transcription factors, they were detected using the above reagents with nuclear permeabilization. Instead of the permeabilization buffer from the eBioscience® Foxp3 / Transcription Factor Staining Buffer Set, 1x BD Perm wash was used due to the presence of a mild detergent in the former.

[0372] The cells were washed twice with 400 μL of 1x BD Perm wash buffer. The permeabilized cells were stained in 400 μL of wash buffer containing either an unbound or fluorescent dye-conjugated primary antibody against human Nanog (catalog no. AF1997, R&D Systems), Oct3 / 4 (catalog no. 130-117-821 for the fluorescent dye-conjugated antibody, Miltenyi Biotec, and catalog no. AF1759 for the unbound antibody), and Sox2 (catalog no. 130-121-129, Miltenyi Biotec).

[0373] Cells were incubated in the dark at room temperature for 1 hour. After incubation, the wash buffer containing the antibody was aspirated and the wells were washed twice with 400 μL of wash buffer. When cells were probed with an unbound primary antibody, the signal was detected using a fluorescently labeled secondary antibody. The goat anti-human Oct3 / 4 and Nanog antibodies were unbound, while the anti-Sox 2 antibody was conjugated to fluorescein isothiocyanate (FITC).

[0374] Next, the cells were stained in 400 μL of washing buffer containing a secondary antibody at room temperature in the dark for 1 hour.

[0375] Anti-goat IgG NorthernLights® fluorescent 557-conjugated antibody (catalog number NL001, R&D Systems) was used as a secondary antibody in IHC studies. It is resistant to photobleaching and therefore ideal for multiplexed IHC studies.

[0376] After the incubation period, the coverslips containing the probed cells were washed twice in 400 μL of 1x BD Perm wash buffer. The coverslips containing the fixed and stained cells were collected from the 24-well plate using fine-tipped tweezers.

[0377] All residual wash buffer was removed from the coverslip by pressing one corner of it against a paper towel. It was necessary to ensure that there were no wash buffer droplets on the coverslip.

[0378] A droplet of VECATSHIELD Antifade mounting medium containing DAPI (catalog number H-1200, Vector Laboratories) was added to a coverslip containing fixed and stained cells. Holding the corner of the coverslip with tweezers, it was inverted in a single motion and placed on a microscope slide. The slide with the coverslip was left to stand in the dark at room temperature for 30 minutes. Excess exudate mounting medium was removed using a paper towel. The edges of the coverslip were sealed with nail polish.

[0379] The slides were imaged using a fluorescence microscope (Carl-Zeiss Vert.A1 AXIO). After imaging, the sealed slides were stored in a freezer at -20°C. If it is necessary to image them again, the slides are thawed at room temperature in a dark place for 1 hour. After wiping away any water droplets formed by condensation, imaging can be performed.

[0380] analysis Immunohistochemical (IHC) images were acquired using a fluorescence microscope. Individual channels in the images were saved and exported as TIFF format files. These TIFF files were exported to different computers, including one with ImageJ software. ImageJ software was used to generate the overlay image. Briefly, the TIFF images were converted to an 8-bit format, and the images to be overlaid were selected with the appropriate R, G, and B channels. The composite image was generated in RGB color, and the image was saved as a TIFF / JPEG format file.

[0381] 7.5.2. Flow Cytometry To characterize pluripotency markers mediated by flow cytometry, iPSC colonies were first isolated into single cells. The cells were spun down at 1800 rpm for 5 minutes at room temperature in a V-bottom 96-well plate. The supernatant was aspirated, and the cell pellet was resuspended in 200 μL of DPBS containing 5 μL of Live / Dead fixable violet dead cell line (catalog no. L34955, Thermo Fisher Scientific) and anti-Fc antibody.

[0382] The cells were incubated at 4°C for 30 minutes. After incubation, the cells were spun down at 1800 rpm for 5 minutes at room temperature and washed once with 200 μL of FACS buffer.

[0383] About surface staining Cells were surface-stained in 100 μL of FACS buffer (DPBS + 2% FBS) containing fluorescent dye-conjugated antibodies against SSEA-4 (catalog number 330418, BioLegend) and Tra1-60 (catalog number A25617, Thermo Fisher Scientific) at 4°C for 30 minutes. After the incubation period, the cells were spun down and aspirated with FACS buffer (DPBS + 2% FBS) containing mAbs. The cells were gently washed twice with 200 μL of FACS buffer.

[0384] Next, the cells were fixed by resuspending them in 100 μL of BD Cytofix (catalog number 554655, BD Biosciences) in a refrigerator at 4°C for 30 minutes. After the incubation period, the cells were spun down at 1800 rpm at room temperature for 5 minutes and resuspended in 150 μL of FACS buffer.

[0385] Flow cytometry data was obtained from cells on the same day or after fixation.

[0386] Regarding intracellular staining Cells were permeabilized by incubation in 200 μL of fixation / permeabilization solution at 4°C for 30 minutes. The fixation / permeabilization solution was from the eBioscience® Foxp3 / transcription factor staining buffer set. One portion of the fixation / permeabilization concentrate was diluted with three portions of fixation / permeabilization diluent.

[0387] After the incubation period, the fixation / permeabilization solution was aspirated. The cells were washed twice with 200 μL of 1x BD Perm wash buffer.

[0388] Next, the cells were probed intracellularly with 100 μL of washing buffer containing fluorescent dye-conjugated antibodies against Oct3 / 4 and Sox2, and incubated in the dark at 4°C for 30 minutes.

[0389] After the incubation period, 100 μL of wash buffer was added to the stained cells to a total volume of 200 μL. The cells were spun down at 1800 rpm for 5 minutes at room temperature. The supernatant was discarded, and the cells were washed once with 200 μL of wash buffer. The cells were then resuspended in 150 μL of FACS buffer (DPBS + 2% FBS). Flow cytometry data were obtained from the cells on the same day or after fixation.

[0390] analysis For surface phenotype profiling experiments, cells were first sorted based on FSC-H (forward scattering-height) versus SSC-H (side scattering-height). Live cells were gated in, and other cells were excluded. Doublets were then excluded from live cells by gating cells with the FSC-A (forward scattering-area) versus FSC-H (forward scattering-height) parameter. Live cells were gated for pluripotency markers such as surface expression of SSEA-4 and Tra1-60, and intracellular expression of Oct3 and Sox2. Fluorescence minus one (FMO) controls were used for each marker to define specific gates.

[0391] 7.6. Example 6: Evaluation of genomic stability of iPSC clones To investigate the genomic stability of these iPSC colonies, karyotyping was performed on clones A, B, and C at passage 19, and on clones D and E at passage 9, as described below. As shown in Figures 8A to 8D, karyotyping of clones B, C, D, and E showed normal chromosome banding patterns using G-banding technology, confirming that the colonies were normal without any abnormal patterns. Karyotyping data for clone A was not available.

[0392] Karyotyping was performed to evaluate the genomic stability of the iPSC colonies. Undifferentiated iPSC colonies were collected from colonies A, B, and C, and D and E, at passages 19 and 9, respectively, as described above. Single cells were isolated from the colonies, added to StemFit Basic02 medium, and sent to Human Health (Bengalru, India) for karyotyping.

[0393] 7.7. Example 7: Adoption of γδT cell-derived iPSCs into culture conditions without supporting cells As described above in Example 2, iPSC colonies under support cell-free conditions were not observed in any of the T cell reprogramming experiments. After verifying the colonies for Vγ9 gene rearrangement, Vδ2 gene rearrangement, and pluripotency markers, iPSC colonies (passage 14 for clones A, B, and C, and passage 4 for clones D and E) were recruited under support cell-free conditions by testing various combinations of matrix and culture medium. Surprisingly, after recruitment, all colonies were found to be maintained and proliferated under support cell-free conditions in vitronectin combined with mTeSR® medium (see Figure 9).

[0394] From the above, it will be understood that specific embodiments are described herein for illustrative purposes, but various modifications can be made without departing from the spirit and scope of what is provided herein. All references mentioned above are incorporated herein by reference in their entirety. Various embodiments of the present invention are shown below. 1. A method for producing induced pluripotent stem cells (iPSCs), (a) Contacting an isolated cell population with an activated culture, wherein the activated culture contains IL-15 and zoledronic acid, (b) Culturing the isolated cell population in the activated culture to enrich and / or activate the γδT cells in the isolated cell population, (c) Transducing the γδT cells using a viral vector encoding one or more reprogramming factors, (d) A method comprising culturing the transduced γδT cells under conditions suitable for reprogramming mammalian somatic cells into a pluripotent state. 2. The method according to 1 above, wherein the activated culture further comprises IL-2. 3. The method according to 1 or 2 above, wherein the viral vector is a Sendai virus (SeV) vector. 4. The method according to any one of 1 to 3 above, further comprising obtaining the isolated cell population from the subject. 5. The method according to any one of 1 to 4 above, wherein the isolated cell population is peripheral blood mononuclear cells (PBMCs). 6. The isolated cell population is terminally differentiated cells, according to any one of the methods described in 1 to 5 above. 7. The method according to any one of 1 to 6 above, wherein the isolated cell population is mammalian cells. 8. The method according to 7 above, wherein the isolated cell population is human cells. 9. The method according to any one of 1 to 8 above, wherein the isolated cell population is cultured in the activated culture for a maximum of 13 days, a maximum of 10 days, a maximum of 9 days, a maximum of 8 days, a maximum of 7 days, a maximum of 6 days, a maximum of 5 days, a maximum of 4 days, a maximum of 3 days, a maximum of 2 days, or a maximum of 1 day. 10. The method according to 9 above, wherein the isolated cell population is cultured in the activated culture for a maximum of 3 days. 11. The method according to 9 above, wherein the isolated cell population is cultured in the activated culture for 3 days. 12. The method according to any one of 1 to 11 above, wherein, after being cultured in the activated culture, the isolated cell population contains less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 45%, less than 40%, less than 35%, or less than 30% γδT cells. 13. The method according to 12, wherein, after culturing in the activated culture, the isolated cell population contains less than 35% γδT cells. 14. The method according to any one of 1 to 13 above, further comprising enriching the γδT cells in the isolated cell population after step (b). 15. The method according to 14 above, wherein the γδT cells are concentrated by intercellular aggregate concentration. 16. At least a portion of the γδT cells are Vγ9 in step (b). + The method described in any of items 1 to 15 above, which is activated in γδT cells. 17. At least a portion of the γδT cells are Vγ9δ2 in step (b). + The method described in any of items 1 to 15 above, which is activated in γδT cells. 18. The method according to any one of items 1 to 17 above, wherein the one or more reprogramming factors are selected from the group consisting of OCT3 / 4, SOX2, KLF4, LIN28, and c-Myc. 19. The method according to any one of 1 to 18 above, wherein in step (d), the transduced γδT cells are cultured in the presence of one or more supporting cell layers. 20. The method according to 19, wherein in step (d), the transduced γδT cells are cultured in the presence of a monolayer of supporting cells. 21. The method according to 19 or 20 above, wherein the supporting cell layer comprises mouse embryonic fibroblasts (MEFs). 22. The method according to any one of 1 to 21 above, further comprising isolating and / or purifying the iPSCs prepared. 23. The method according to 22, further comprising administering the isolated iPSC to the subject. 24. The method according to any one of items 1 to 22 above, further comprising differentiating the iPSC into cells of a desired cell type in vitro. 25. The method according to 24, further comprising administering the differentiated cells to the target. 26. The method according to any one of items 1 to 25 above, wherein the produced iPSC is negative for the Sendai virus (SeV) vector. 27. The iPSC produced is derived from γδT cells, according to any one of the methods described in 1 to 26 above. 28. The method according to any one of 1 to 26 above, wherein the produced iPSC has rearrangement genes at the TRG locus and the TRD locus, and optionally the produced iPSC has a Vγ9 gene configuration and a Vδ2 gene configuration. 29. The iPSC produced is not derived from αβT cells, according to any of the methods described in 1 to 26 above. 30. The method according to any one of items 1 to 26 above, wherein the iPSCs produced are not subjected to polymerase chain reaction (PCR) products from the TCRA and TCRB gene loci. 31. The method according to any one of items 1 to 30 above, wherein the produced iPSC is genomically stable without chromosome loss. 32. The method according to 31, wherein the genome stability of the produced iPSC is determined by karyotype analysis. 33. The method according to any one of items 1 to 32 above, wherein the produced iPSC can be grown in a culture medium that does not contain supporting cells after adoption. 34. Induced pluripotent stem cells (iPSCs) produced according to any of the methods described in 1 to 33 above. 35. A pharmaceutical composition comprising the iPSC described in 34 above and a pharmaceutically acceptable excipient. 36. Differentiated cells prepared according to the method described in item 24 above. 37. A pharmaceutical composition comprising differentiated cells as described in 36 above and a pharmaceutically acceptable excipient. 38. A method of treating an object that needs to be treated, (i) Obtain a cell population including peripheral blood mononuclear cells (PBMCs) from the subject, (ii) Reprogramming γδT cells in the cell population to produce iPSCs according to any of the methods described in 1 to 33 above, (iii) A method comprising, optionally, differentiating the iPSC into one or more desired cell types, and then administering the produced iPSC or a pharmaceutical composition containing the produced iPSC to the subject. 39. The method described in 38 above, wherein the subject is a human. 40. The method described in 38 or 39 above, wherein the subject has hyperproliferative disorder or hematopoietic cancer. 41. An isolated population of induced pluripotent stem cells (iPSCs), wherein the isolated population of iPSCs comprises pluripotent cells, the pluripotent cells express one or more reprogramming factors, and / or the pluripotent cells contain nucleotide sequences encoding rearrangements of the TRG and TRD genes, and optionally, the iPSCs have Vγ9 and Vδ2 gene configurations. 42. A method for producing induced pluripotent stem cells (iPSCs), (a) A step to perform the function of enriching and / or activating γδT cells in an isolated cell population, (b) A method comprising the step of performing a function to reprogram the γδT cells into a pluripotent state. 43. Induced pluripotent stem cells (iPSCs) prepared according to the method described in 42 above. 44. An isolated population of induced pluripotent stem cells (iPSCs) comprising pluripotent cells, wherein the pluripotent cells include means for expressing one or more reprogramming factors, and / or the pluripotent cells include means for encoding the rearrangement of the TRG gene and the TRD gene.

Claims

1. A method for producing human induced pluripotent stem cells (iPSCs), (a) A population of isolated cells containing γδ T cells is cultured in contact with an activated culture to activate and enrich the Vγ9Vδ2 γδ T cells in the isolated cell population, wherein the activated culture contains IL-15, zoledronic acid, and IL-2. (b) After step (a), γδT cells enriched with Vγ9Vδ2 are purified from the isolated cell population, (c) Transduction of purified Vγ9Vδ2-enriched γδT cells using a viral vector encoding one or more reprogramming factors, (d) Culture transduced cells under conditions suitable for reprogramming mammalian somatic cells into a pluripotent state, thereby producing iPSCs, The generated iPSCs have rearranged TRG and TRD genes, The generated iPSC has a TCR rearrangement including the Vγ9 and Vδ2 gene segments.

2. The method according to claim 1, wherein the viral vector is a Sendai virus (SeV) vector.

3. The method according to any one of claims 1 to 2, wherein the isolated cell population is peripheral blood mononuclear cells (PBMCs).

4. The method according to any one of claims 1 to 3, wherein the isolated cell population is cultured in the activated culture for 3 to 13 days.

5. The method according to any one of claims 1 to 3, wherein, after culturing in the activated culture in step (a), the isolated cell population contains 5% to 90% γδ T cells.

6. The method according to claim 5, wherein, after culturing in the activated culture in step (a), the isolated cell population contains 5% to 35% γδT cells.

7. The method according to any one of claims 1 to 3, wherein the purification step (b) further concentrates γδT cells in which Vγ9Vδ2 is concentrated in the isolated cell population by intercellular aggregate concentration.

8. The method according to any one of claims 1 to 3, wherein the one or more reprogramming factors are selected from the group consisting of OCT3 / 4, SOX2, KLF4, LIN28, and c-Myc.

9. The method according to any one of claims 1 to 3, wherein in step (d), the transduced cells are cultured in the presence of one or more supporting cell layers.

10. The method according to claim 9, wherein in step (d), the transduced cells are cultured in the presence of a monolayer of the supporting cell layer.

11. The method according to claim 9 or 10, wherein the supporting cell layer comprises mouse embryonic fibroblasts (MEFs).

12. The method according to any one of claims 1 to 3, further comprising isolating the prepared iPSC.

13. The method according to claim 12, further comprising differentiating the iPSC into cells of a desired cell type in vitro.

14. The method according to any one of claims 1 and 3, wherein the iPSC produced is negative for the Sendai virus (SeV) vector.

15. The method according to any one of claims 1 to 3, wherein the produced iPSC is genomically stable without chromosome loss.

16. The method according to claim 15, wherein the genome stability of the produced iPSC is determined by karyotype analysis.

17. The method according to any one of claims 1 to 3, wherein the prepared iPSC can be grown in a culture medium that does not contain supporting cells after being adapted to the culture medium that does not contain supporting cells.

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