Novel and efficient method for reprogramming blood cells into induced pluripotent stem cells

Abstract

We provide an improved technique for establishing highly efficient and reproducible reprogramming, including the generation of induced pluripotent stem cells (iPSCs), using non-integrating episomal plasmid vectors. [Solution] A method for generating induced pluripotent stem cells derived from blood cells, comprising providing a quantity of blood cells, delivering a quantity of reprogramming factors to the blood cells, and culturing the blood cells in a reprogramming medium for at least four days, wherein the method generates induced pluripotent stem cells derived from blood cells by delivering the reprogramming factors and culturing in the reprogramming medium.

Description

[Technical Field]

[0001] Field of Invention This specification describes methods and compositions relating to regenerative medicine, including the induction of induced pluripotent stem cells (iPSCs) derived from whole blood and peripheral blood, which provide regenerative transplantable materials. [Background technology]

[0002] background Pluripotent stem cells ("pSCs") offer a wide range of opportunities to generate therapeutic materials for use in regenerative medicine, while also providing a valuable in vitro model for studying disease onset and progression. Induced pluripotent stem cells ("iPSCs"), a category of pSCs, possess the characteristic stem cell properties of self-renewal (i.e., immortality) and the ability to differentiate into cells derived from all three embryonic germ layers (i.e., pluripotency). These cells can be obtained through "reprogramming," which involves the dedifferentiation of cells from non-embryonic origins, such as adult somatic cells. The reprogramming process offers the additional advantage of allowing potential patient-specific immunological incompatibility while eliminating potential ethical concerns associated with other types of embryonic raw materials like pSCs, such as embryonic stem cells ("ESCs").

[0003] In addition to establishing robust reprogramming techniques, the full realization of the therapeutic goals of stem cell regenerative medicine requires further consideration of host cell types that can function as sources for regenerative materials. Ideally, cells would not only possess the plasticity and stability in subsequent proliferation necessary for stable reprogramming, but also offer advantages in clinical aspects such as ease of isolation, storage, stability, and maintenance. In this regard, blood cells represent an attractive option for such applications, given their widely used medical diagnostic and highly accessible source for cell reprogramming. While fibroblasts are a widely used cell source for many reprogramming experiments conducted over the past decade, this raw material may not be the best choice for directional reprogramming. Skin biopsies to obtain fibroblasts are an invasive and non-sterile procedure and require proliferation of cells harvested before the experiment. Most importantly, skin cells have more mutations due to environmental damage such as UV irradiation than cells from within the body, such as blood.

[0004] Given the ultimate therapeutic goal of generating patient-specific, immunocompatible biological materials, there is a critical need in this field to identify the raw materials for therapeutic materials suitable for final clinical application, as well as to establish robust and reproducible methods for reprogramming cells. Such improved methods would need to possess high reprogramming efficiency, consistent replication, generate genomically stable cells, and be easily scalable to various cell types.

[0005] This specification describes improved techniques for establishing highly efficient and reproducible reprogramming using non-integrated episomal plasmid vectors, including the generation of iPSCs from non-T cell and non-B cell components in blood samples. These described approaches enable the use of blood as a readily available source for cell reprogramming, exhibiting superior genomic and karyotype stability. [Overview of the project]

[0006] Described herein is a method for generating induced pluripotent stem cells derived from blood cells, comprising preparing a certain amount of blood cells, delivering a certain amount of reprogramming factors to the blood cells, and culturing the blood cells in a reprogramming medium for at least four days, wherein the delivery of the reprogramming factors and the culturing in the reprogramming medium generate induced pluripotent stem cells derived from blood cells. In other embodiments, the delivery of a certain amount of reprogramming factors includes nucleofection. In other embodiments, the reprogramming factors include one or more factors selected from the group consisting of Oct-4, Sox-2, Klf-4, c-Myc, Lin-28, SV40 large T antigen ("SV40LT"), and short hairpin RNA targeting p53 ("shRNA-p53"). In other embodiments, the reprogramming factors are encoded in one or more oriP / EBNA1-derived vectors. In other embodiments, one or more oriP / EBNA1-derived vectors include pEP4 E02S ET2K, pCXLE-hOCT3 / 4-shp53-F, pCXLE-hSK, pCXLE-hUL, and pCXWB-EBNA1. In other embodiments, after delivering the reprogramming factor to blood cells, the blood cells are seeded on the treated cell culture surface and cultured on the treated cell culture surface in reprogramming medium. In other embodiments, the treated cell culture surface includes seeding of mouse embryonic feeder cells (MEFs). In other embodiments, the treated cell culture surface includes extracellular matrix proteins. In other embodiments, the extracellular matrix proteins include laminin. In other embodiments, the laminin includes L-521. In other embodiments, the reprogramming medium includes embryonic stem cell (ESC) medium. In other embodiments, the ESC medium includes basic fibroblast growth factor (bFGF). In other embodiments, the reprogramming medium includes E7 medium. In other embodiments, the reprogramming medium comprises E7 medium containing L-ascorbic acid, transferrin, sodium bicarbonate, insulin, sodium selenite and / or bFGF.In other embodiments, the culture of hematopoietic cells in reprogramming medium is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 days. In other embodiments, the culture of hematopoietic cells in reprogramming medium is at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. Further described herein are cell lines comprising induced pluripotent stem cells derived from hematopoietic cells produced by the aforementioned methods. In other embodiments, hematopoietic cells are isolated from subjects having disease mutations. In other embodiments, disease mutations are associated with neurodegenerative diseases, disorders, and / or conditions. In other embodiments, disease mutations are associated with inflammatory bowel disease, disorders, and / or conditions. In other embodiments, hematopoietic cells are mononuclear cells that are non-T cells and non-B cells. In other embodiments, hematopoietic cells are samples obtained from human subjects. In other embodiments, the sample is whole blood. In other embodiments, the sample is peripheral blood. In other embodiments, the sample comprises isolated components of mononuclear cells that are non-T cells and non-B cells. Further described herein are induced pluripotent stem cell lines derived from blood cells.

[0007] Further described herein is a method for generating induced pluripotent stem cells, comprising preparing a certain amount of blood cells, delivering a certain amount of reprogramming factors to the blood cells, seeding the blood cells on a treated cell culture surface, and culturing the blood cells in a reprogramming medium for at least four days, wherein the delivery of the reprogramming factors and the culturing in the reprogramming medium generate blood cell-derived induced pluripotent stem cells. In other embodiments, the delivery of a certain amount of reprogramming factors includes nucleofection, the reprogramming factors comprising one or more factors selected from the group consisting of Oct-4, Sox-2, Klf-4, c-Myc, Lin-28, SV40 large T antigen ("SV40LT"), and p53-targeting short hairpin RNA ("shRNA-p53") encoded by one or more oriP / EBNA1-derived vectors. In other embodiments, one or more oriP / EBNA1-derived vectors include pEP4 E02S ET2K, pCXLE-hOCT3 / 4-shp53-F, pCXLE-hSK, pCXLE-hUL, and pCXWB-EBNA1. In other embodiments, the treated cell culture surface includes seeding of mouse embryonic feeder cells (MEFs). In other embodiments, the treated cell culture surface includes extracellular matrix proteins. In other embodiments, the extracellular matrix proteins include laminin. In other embodiments, the laminin includes L-521. In other embodiments, the reprogramming medium includes embryonic stem cell (ESC) medium containing basic fibroblast growth factor (bFGF). In other embodiments, the reprogramming medium includes E7 medium containing L-ascorbic acid, transferrin, sodium bicarbonate, insulin, sodium selenite, and / or bFGF. In other embodiments, the culture of blood cells in reprogramming medium is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 days. In other embodiments, the culture of blood cells in reprogramming medium is at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days.In other embodiments, the blood cells are a sample obtained from a human subject. In other embodiments, the sample is whole blood. In other embodiments, the sample is peripheral blood. In other embodiments, the sample comprises isolated components of mononuclear cells that are non-T cells and non-B cells. Further described herein are induced pluripotent stem cell lines derived from blood cells. [Invention 1001] To prepare a certain amount of blood cells, To deliver a certain amount of reprogramming factor to the blood cells, The blood cells are cultured in reprogramming medium for at least 4 days. A method for generating induced pluripotent stem cells derived from blood cells, including, The method comprising delivering the reprogramming factor and culturing in a reprogramming medium to generate induced pluripotent stem cells derived from blood cells. [Invention 1002] The method of the present invention 1001, comprising delivering a certain amount of reprogramming factor, including nucleofection. [Invention 1003] The method of the present invention 1001, wherein the reprogramming factor comprises one or more factors selected from the group consisting of Oct-4, Sox-2, Klf-4, c-Myc, Lin-28, SV40 large T antigen ("SV40LT"), and short hairpin RNA targeting p53 ("shRNA-p53"). [Invention 1004] The method of the present invention 1003, wherein the reprogramming factor is encoded in one or more oriP / EBNA1-derived vectors. [Invention 1005] The method of the present invention 1004, wherein the one or more oriP / EBNA1-derived vectors include pEP4 E02S ET2K, pCXLE-hOCT3 / 4-shp53-F, pCXLE-hSK, pCXLE-hUL, and pCXWB-EBNA1. [Invention 1006] The method of the present invention 1001, comprising delivering a reprogramming factor to the blood cells, seeding the blood cells on a treated cell culture surface, and culturing the blood cells on the treated cell culture surface in a reprogramming medium. [Invention 1007] The method of the present invention 1006, wherein the treated cell culture surface includes seeding of mouse embryonic feeder cells (MEFs). [Invention 1008] The method of the present invention 1006, wherein the treated cell culture surface contains extracellular matrix proteins. [Invention 1009] The method of the present invention 1008, wherein the extracellular matrix protein comprises laminin. [Invention 1010] The method of the present invention 1009, wherein laminin contains L-521. [Invention 1011] The method of the present invention 1001, wherein the reprogramming medium includes embryonic stem cell (ESC) medium. [Invention 1012] The method of the present invention 1011, wherein the ESC medium contains basic fibroblast growth factor (bFGF). [Invention 1013] The method of the present invention 1001, wherein the reprogramming medium comprises E7 medium. [Invention 1014] The method of the present invention 1013, wherein the reprogramming medium comprises E7 medium containing L-ascorbic acid, transferrin, sodium bicarbonate, insulin, sodium selenite and / or bFGF. [Invention 1015] The method of the present invention 1001, wherein the blood cells are cultured in a reprogramming medium for at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 days. [Invention 1016] The method of the present invention 1001, wherein the blood cells are cultured in a reprogramming medium for at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. [Invention 1017] A cell line comprising blood cell-derived induced pluripotent stem cells, which is produced by the method of the present invention 1001. [Invention 1018] The method of the present invention 1001, wherein the blood cells are isolated from a subject having a disease mutation. [Invention 1019] The method of the present invention 1018, wherein the disease mutation is associated with a neurodegenerative disease, disorder, and / or condition. [Invention 1020] The method of the present invention 1018, wherein the disease mutation is associated with an inflammatory bowel disease, disorder, and / or condition. [Invention 1021] The method of the present invention 1001, wherein the blood cells are mononuclear cells that are non-T cells and non-B cells. [Invention 1022] The method of the present invention 1001, wherein the blood cells are a sample collected by blood drawing from a human subject. [Invention 1023] The method of the present invention 1022, wherein the sample is whole blood. [Invention 1024] The method of the present invention 1022, wherein the sample is peripheral blood. [Invention 1025] The method of the present invention 1022, wherein the sample is an isolated component of mononuclear cells that are non-T cells and non-B cells. [Invention 1026] A blood cell-derived induced pluripotent stem cell line. [BRIEF DESCRIPTION OF THE DRAWINGS]

[0008] [Figure 1] Figures 1A to 1B Separation of blood components. Figure 1A is a schematic diagram showing the separated components. Figure 1B shows an image of the separated components. [Figure 2A]Figures 2A-2E Quality control: iPS cells. Various measurement methods for quality control of generated cells are shown, including: Figure 2A Microscopic examination and staining of pluripotent stem cell markers. Figure 2B Description of pluripotency of stem cells. Figure 2C Karyotype analysis. Figure 2D RT-PCR of pluripotency and lineage-specific markers for different cell lines. Figure 2E Pluripotent stem cells capable of differentiating into all three embryonic lineages. [Figure 2B] See the explanation in Figure 2A. [Figure 2C] See the explanation in Figure 2A. [Figure 2D] See the explanation in Figure 2A. [Figure 2E] See the explanation in Figure 2A. [Figure 3] PBMC isolation and cryopreservation process. This process shows how to obtain blood cells that can be used as raw materials for reprogramming and iPSC generation. [Figure 4] Peripheral blood mononuclear cells (PBMCs). This diagram illustrates the various components of blood, including the population and relative population ratios of PBMCs. [Figure 5] This shows a T-cell receptor clonality assay of iPSCs derived from blood. [Figure 6A] Figures 6A-6B show ideograms illustrating chromosomal changes observed in iPSC lines, specifically the Cedars-Sinai iPSC Core. Each colored bar represents the occurrence of one chromosomal change in a single cell line. Cytogenetic changes are color-coded as follows: (Marooon, monosomy, total chromosome loss). BC-iPSCs are superior to fibroblast-derived iPSCs in maintaining a more stable karyotype. Figures 6A and 6B show individual chromosome-level ideograms of abnormal karyotypes derived from fibroblast-derived iPSCs and PBMC-derived iPSCs in our laboratory. The abnormal iPSC karyotypes represented in the ideograms are (Figure 6A) fib-iPSC (n:59) / karyotypes of all 258 fib-iPSC cultures, and (Figure 6B) BC-iPSC (n:4) / all BC-iPSCss karyotypes (n=106). [Figure 6B] See the explanation in Figure 6A. [Figure 7A]Figures 7A-7B show karyotypes. Figure 7A shows the distribution of abnormal karyotypes related to passage number. Figure 7B shows the distribution of abnormal karyotypes (Marooon, monosomy, and total chromosomal loss) related to the age of the tissue donor. [Figure 7B] See the explanation in Figure 7A. [Figure 8A]Figures 8A-8F: Reliable episomal reprogramming of PBMCs into iPSCs. Figure 8A: Schematic diagram showing the episomal reprogramming process and the timeline of iPSC generation from PBMCs. Figure 8B: Bright-field images of reprogrammed iPSC colonies derived from PBMCs were obtained from two healthy volunteers, one using the non-T cell (03iCTR-NTn1) reprogramming method and the other using the T cell (80iCTR-Tn1) reprogramming method. These showed a high nucleus:cytoplasmic ratio, were alkaline phosphatase positive, and immunopositive for surface antigens SSEA4, TRA-1-60, TRA-1-81, and nuclear pluripotency markers OCT3 / 4, SOX2, and NANOG. Scale bar: 75 μm. Figure 8C: Pluripotency levels of BC-iPSCs were confirmed by PluriTest based on Illumina microarray gene chips and bioinformatics. Figure 8D: PBMC-iPSC strains maintained a normal G-band karyotype, as shown in two representative lines. Figure 8E: Clonality assay of non-T cell or T cell. Figure 8F: Quantitative RT-PCR analysis of POU5F1(OCT4), SOX2, LIN28, L-MYC, and KLF4 expression in 49iCTR and 84iSMA LCL-iiPSCs compared to H9 hESC. Figure 8G: T cell receptor gene rearrangement assays show that TCRG gene rearrangements occurring in BC-iPSCs derived from the T cell method were detected using different sets of PCR primers, while PCR products in the 145–255 bp range were not detected in non-T cell iPSC clones. Clonal controls are included. Figure 8F: The TaqMan hPSC Scorecard table shows the differentiation potential of three representative BC-iPSC lines based on genes selected from four gene groups (self-renewal / pluripotency, ectoderm, endoderm, and mesoderm) compared to spontaneous in vitro EB differentiation of EB cells derived from PBMC-iPSC lines. Figure 8G: Neuronal differentiation directionality of representative fibroblast-derived iPSCs (n:26) and PBMC-derived iPSCs (n:12) showing an equivalent number of ectoderm cells (Nkx6.1+ / β3 tubulin+ neurons). Scale bar: 75 μm.Figure 8H: The 5p method (n=28 for T cells, n=13 for non-T cells) shows higher iPSC reprogramming efficiency for cryopreserved PBMCs compared to the published 4p method (n=7 for both T cells and non-T cells). Reprogramming success rate is calculated as the proportion of 10 total attempts (success or failure) of PBMC reprogramming. ["T cells, 5p vs 4p (***): z-value = 3.74, two-sided p-value = 0.00018, z-test of 2 ratios", "Non-T cells, 4p vs 5p (****): z-value = 7.18, two-sided p-value < 0.0001, z-test of 2 ratios"]. ND: Not detected. Furthermore, higher reprogramming efficiency was observed for both T cell and non-T cell populations compared to the 4p method. Reprogramming efficiency is based on the individual iPSC colonies identified and normalized to the total number of nucleofected PBMCs after thawing. ["T cells, 4p vs 5p (***): two-sided p-value = 0.0004, unpaired t-test with Welch's correction", "Non-T cells, 4p vs 5p (***): two-sided p-value < 0.0001, unpaired t-test with Welch's correction"]. ND: Not detected. Figures 8I and 8J: Ideogram representations of individual chromosome-level abnormal karyotypes derived from fibroblast-derived iPSCs (upper) and PBMC-derived iPSCs (lower) from the inventors' laboratory. The abnormal iPSC karyotypes represented in the ideograms are (upper) fib-iPSCs (n=61) out of a total n=260 fib-iPSC cultures analyzed for karyotyping, and (lower) PBMC-iPSCs (n=4) out of a total n=206 PBMC-iPSC strains analyzed for karyotyping. The analysis includes chromosome testing of at least 20 cells per culture. The datasets in this specification include only abnormalities that conform to the ISCN2013 definition of clonality. The letters above the translocation (dark blue bars) categories are coded to correspond between the chromosomal locations where each translocation abnormality occurred. The width of the bars in the idiogram for each chromosome was expanded in proportion to the number of samples examined for fib-iPSC and PBMC-iPSC. Therefore, the bars indicating abnormalities found in PBMC-iPSC strains are 1.26 times (260 / 206) wider than the bars indicating abnormalities found in fib-iPSC strains. [Figure 8B] See the explanation in Figure 8A. [Figure 8C] See the explanation in Figure 8A. [Figure 8D] See the explanation in Figure 8A. [Figure 8E] See the explanation in Figure 8A. [Figure 8F] See the explanation in Figure 8A. [Figure 8G] See the explanation in Figure 8A. [Figure 8H] See the explanation in Figure 8A. [Figure 8I] See the explanation in Figure 8A. [Figure 8J] See the explanation in Figure 8A. [Figure 9] Figures 9A-9D: BC-iPSCs are more stable for long-term culture compared to fib-iPSCs. Figure 9A: Abnormal G-band karyotype rates observed in iPSC strains relative to donor age of fibroblasts and PBMCs. Figure 9B: Abnormal G-band karyotype rates in repeated karyotype analyses of iPSC strains evaluated in the first karyotype analysis after reprogramming and in long-term cell culture. The dotted line represents the overall average karyotype abnormality rate for fib-iPSCs (orange) and BC-iPSCs (blue). Figure 9C: Abnormality rates in the first G-band karyotype analysis evaluated for fib-iPSC clones and PBMC-iPSC clones with respect to the passage number of iPSCs. Figure 9D: Abnormality rates in the 2nd to 4th (repeated) G-band karyotype analyses evaluated for fib-iPSC clones and PBMC-iPSC clones with respect to the passage number of iPSCs. [Figure 10-1]Figures 10A-10E: Comparative genome hybridization confirmed that BC-iPSCs were relatively smaller and had fewer minute cytogenetic abnormalities compared to fib-iPSCs. (Figures 10A-10B) Average size of new acquired amplifications and deletions in Mb detected by aCGH in fib-iPSCs (n:6) and PBMC-iPSCs (n:7) when comparing the parental cells of each iPSC strain. The iPSC strains used in aCGH had abnormal and normal G-band karyotypes. Abnormal: fib-iPSCs (n:3) and PBMC-iPSCs (n:2). Normal: fib-iPSCs (n:3) and PBMC-iPSCs (n:5). (Figures 10C-10D) Average number of new acquired amplifications and deletions detected by aCGH in the Mb of fib-iPSC (n:6) and PBMC-iPSC (n:7) compared to the parental cells of each iPSC strain. Figure 10E: List of the most recurrent, minute amplifications / deletions observed in fib-iPSC or PBMC-iPSC strains in the inventors' laboratory. Figure 10F: All data shown here are based on iPSC strains induced in the inventors' laboratory in Cedars-Sinai. fib-iPSCs were reprogrammed using the 3p method, and PBMC-iPSCs were reprogrammed using the 5p method. Percentage of abnormal G-band karyotypes observed in iPSC strains relative to donor age of fibroblasts and PBMCs. ["For fib-iPSC, the donor age of 21-40 versus 0-20 or 51-60 or 41-50 or 61-90 is not significant (ns), z-test of the two proportions."] fib-iPSC: n=10 / 63 (0-20), n=17 / 50 (21-40), n=4 / 28 (41-50), n=10 / 41 (51-60), n=7 / 29 (61-90). ["fib-iPSC vs. PBMC-iPSC: Age group 0-20: ns, Age group 21-40 (**): z-value = 2.9, two-sided p-value = 0.003, Age group 41-50 (*): z-value = 2.4, two-sided p-value = 0.015, Age group 51-60 (***): z-value = 3.5, two-sided p-value = 0.0005, Age group 61-90 (****): z-value = 4.02, two-sided p-value < 0.0001, z-test of proportion 2"].PBMC-iPSC: n=2 / 34 (0-20), n=0 / 19 (21-40), n=0 / 39 (41-50), n=1 / 56 (51-60), n=0 / 62 (61-90). ND: Not detected. Abnormal G-band karyotype rate in repeated iPSC strains evaluated in the first karyotype after reprogramming and in long-term cell culture. The dotted line represents the overall average karyotype abnormality rate for fib-iPSC (black) and PBMC-1 iPSC (gray). [“Initial karyotype, fib-iPSC vs. PBMC-iPSC (****): z-value = 5.59, two-sided p-value < 0.0001, 2-proportional z-test”, “Repeated (2nd to 4th) karyotype, fib-iPSC vs. PBMC-iPSC (***): z-value = 3.72, two-sided p-value = 0.0002, 2-proportional z-test”, “fib-iPSC and PBMC-iPSC, initial karyotype vs. repeated karyotype were tested as not statistically significant by the 2-proportional z-test”). fib-iPSC: n=46 / 205 (initial karyotype) and n=15 / 55 (karyotype in repeated). PBMC-iPSC: n=2 / 143 (initial karyotype) and n=2 / 63 (karyotype in repeated). Abnormality rates in the initial G-band karyotype evaluated for the passage number of iPSCs for fib-iPSC clones and PBMC-iPSC clones. [“fib-iPSC vs. PBMC-iPSC - passages 1-8 (****): z-value = 4.31, two-sided p-value < 0.0001, passages 9-23 (***): z-value = 3.21, two-sided p-value = 0.001, 2-proportion z-test”]. fib-iPSC: n=20 / 102 (passages 1-8) and n=12 26 / 103 (passages 9-23). ​​PBMC-iPSC: n=1 / 99 (passages 1-8) and n=1 / 42 (passages 9-23). ​​Abnormality rates in the 2nd to 4th (repeated) G-band karyotypes evaluated for the passage number of iPSCs in fib-iPSC clones and PBMC-iPSC clones. [“fib-iPSC vs. PBMC-iPSC: Low passage count 1-14: ns, z-value = 1.53, two-tailed p-value = 0.12, z-test with 2 proportions}]. [“fib-iPSC vs. PBMC-iPSC: High passage count ≥ 15 (**): z-value = 2.61, two-tailed p-value = 0.009, z-test with 2 proportions”]. fib-iPSC: n=7 / 12 (low passage count 1-14) and n=8 / 43 (high passage count ≥ 15). PBMC-iPSC: n=0 / 2 (low passage count 1-14) and n=2 / 61 (high passage count ≥ 15).ND: Not detected. Figure 10G: Average size of new acquired amplifications and deletions in Mb of fib-iPSC (n=6) and PBMC-iPSC (n=8) detected by aCGH, compared to parental cells of each iPSC strain. ["fib-iPSC vs PBMC-iPSC (**): two-sided p-value = 0.0015, unpaired t-test"]. The iPSC strains used for aCGH amplification / deletion analysis had abnormal and normal G-band karyotypes. Abnormal: fib-iPSC (n=3) and PBMC-iPSC (n=2). Normal: fib-iPSC (n=3) and PBMC-iPSC (n=6). Average number of new acquired amplifications and deletions in Mb of fib-iPSC (n=6) and PBMC-iPSC (n=8) detected by aCGH, compared to parental cells of each iPSC strain. [fib-iPSC vs. PBMC-iPSC(*): Two-sided p-value = 0.0369, unpaired t-test]. iPSC strains were used to analyze the number of new duplications / deletions using fib2 iPSCs (n=3) and PBMC-iPSCs (n=2) with one abnormal karyotype, as well as fib-iPSCs (n=3) and PBMC3 iPSCs (n=6) with a normal karyotype. A list of the most recurrent minute amplifications / deletions identified in iPSC strains observed in the inventors' laboratory. Figure 10H: Frequency of various karyotypic abnormalities at the individual chromosome level in fibroblast-derived iPSCs (fib-iPSCs) observed by G-band karyotyping analysis. Rearrangements - structural rearrangements including translocations, inversions, and derivatives. Cells shaded in gray indicate chromosomes in which no karyotypic abnormalities were observed in fib-iPSC cultures. Cells shown in orange represent the top three karyotype abnormalities for each overall category of chromosomal abnormalities, including chromosomal deletions or structural rearrangements. [Figure 10-2] See the explanation in Figure 10-1. [Figure 10-3] See the explanation in Figure 10-1. [Figure 10-4] See the explanation in Figure 10-1. [Figure 10-5] See the explanation in Figure 10-1. [Figure 11A]Figures 11A-11C: Transgene-free BC-iPSC. Figure 11A: Absence of plasmid-based EBNA gene expression in BC-iPSC genomic DNA using assay. Figure 11B: Relative normalized gene expression measured by quantitative RT-PCR analysis using primers to detect endogenous POU5F1 (OCT4), SOX2, LIN28, L-MYC, and KLF4 expression (coding DNA sequence, CDS). Figure 11C: Plasmid-derived expression (Pla) that is similarly undetectable in iPSC strains. [Figure 11B] See the explanation in Figure 11A. [Figure 11C] See the explanation in Figure 11A. [Figure 12A] Figures 12A-12B: Chromosomal ideograms of lymphoblastoid cell line (LCL)-iPSCs and epithelial cell-derived iPSCs. Ideogram representations of individual chromosome-level abnormal karyotypes derived from LCL-derived iPSCs (Figure 12A) and epithelial cell-derived iPSCs (Figure 12B) in the inventors' laboratory. The abnormal iPSC karyotypes represented by the ideograms are (Figure 12A) LCL-iPSC (n:15) / all 60 LCL-iPSC culture karyotypes, and (Figure 12B) epithelial-iPSC (n:7) / all BC-iPSC karyotypes (n:26). [Figure 12B] See the explanation in Figure 12A. [Figure 13A] Figures 13A-13B: Three-series differentiation potential of PBMC-derived iPSCs. TaqMan hPSC Scorecard table showing the three-series differentiation potential of representative PBMC-derived iPSCs generated in our laboratory. Figure 13A: Expression of selected genes in four groups (self-replicating / pluripotent, ectoderm, endoderm, and mesoderm) is compared with Figure 13B: in vitro differentiation of spontaneous embryoid bodies (EBs) derived from each PBMC-iPSC strain 14-18 days after the iPSC stage. The negative score for self-replicating reflects the differentiation state of the EBs, while the positive scores for each of the three germ layers indicate a relatively similar trend among PBMC-derived iPSC strains for generating ectoderm, mesoderm, and endoderm without directional differentiation. [Figure 13B] See the explanation in Figure 13A. [Figure 14A]Figures 14A-14C: Transgenes are absent in PBMC-iPSCs reprogrammed with episomal plasmids. Figure 14A: Absence of plasmid-based EBNA1 gene in the genomic DNA of PBMC-iPSCs. Figures 14B and 14C: Relative normalized gene expression measured by quantitative RT-PCR analysis using primers to detect endogenous POU5F1(OCT4), SOX2, LIN28, L-MYC, and KLF4 expression (coding DNA sequence, CDS) and plasmid-derived expression (Pla), which is undetectable in the PBMC-iPSC system. [Figure 14B] See the explanation in Figure 14A. [Figure 14C] See the explanation in Figure 14A. [Figure 15] Figures 15A-15B: PBMC-iPSCs reprogrammed using the 5p method lack integration and expression of the SV40LT gene. Figure 15A shows the absence of plasmid-based EBNA1 and SV40LT gene presence in genomic and episomal DNA isolated from PBMC-iPSCs, H9-hESCs, an integrated iPSC control strain, and a positive control (nucleofected using 5p in PBMCS for 30 days). Figure 15B: Gene expression measured by quantitative RT-PCR analysis in the same cell lines using primers that detect the expression of EBNA1, SV40LT, and GAPDH. Compared to the nucleofected positive control, SV40LT gene expression is not detected in the reprogrammed PBMC-iPSC strain. 32 PCR cycles were used for all primer sets. [Figure 16A] Figures 16A-16B: Chromosomal ideograms of lymphoblastoid cell line (LCL)-iPSCs and epithelial cell-derived iPSCs. Figure 16A shows idiograms at the chromosome level of abnormal karyotypes derived from LCL-derived iPSCs (top) and Figure 16B shows idiograms of epithelial-derived iPSCs (bottom) from the inventors' laboratory. The abnormal iPSC karyotypes represented by the idiograms are (top) LCL-iPSC (n=15) / all 60 LCL-iPSC culture karyotypes, and (bottom) epithelial-iPSC (n=7) / all PBMC-iPSC karyotypes (n=26). [Figure 16B]See the explanation in Figure 16A. [Figure 17] p53 analysis. Somatic cell variants (those appearing only in iPSC clones) are identified using rearranged and recalibrated sequence files (.bam) generated by the GATK toolkit and MuTect package. This package receives processed sequence reads from paired normal samples (in this case, blood-derived DNA) and somatic cell samples (in this case, iPSC clone DNA), then applies a Bayesian classifier to identify variants occurring only in somatic cell samples, and then additional quality control steps identify the variants as germline or somatic. The identified variants are then annotated in a large SNP database of common variations identified in the population (dbSNP). [Figure 18A] Figures 18A-18B: Additional characterization of reprogramming techniques in fibroblasts using the 3P and 5P methods. Figure 18A: 22 days after nucleofection. Figure 18B: 28 days after nucleofection. [Figure 18B] See the explanation in Figure 18A. [Figure 19A] Figures 19A-19B: Additional characterization of reprogramming techniques in fibroblasts using the 3P and 5P methods. Figure 19A: AP staining-3 plasmid conditions. Figure 19B: AP staining-5 plasmid conditions. [Figure 19B] See the explanation in Figure 19A. [Figure 20] Frequency of various karyotypic abnormalities at the individual chromosome level in fibroblast-derived iPSCs (fib-iPSCs) observed by G-band karyotyping analysis. Structural rearrangements including translocations, inversions, and derivatives. Cells shaded in gray indicate chromosomes in fib-iPSC cultures in which no karyotypic abnormalities were observed. Cells shown in orange indicate the highest-ranking (top 3) karyotypic abnormalities for each category of overall chromosomal abnormalities, including chromosome addition, subtraction, or structural rearrangement. [Modes for carrying out the invention]

[0009] Detailed explanation All references cited herein are incorporated by reference as if they were whole. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. Allen et al., Remington: The Science and Practice of Pharmacy 22 nd ed.,Pharmaceutical Press(September 15,2012), Hornyak et al.,Introduction to Nanoscience and Nanotechnology,CRC Press(2008), Singleton and Sainsbury,Dictionary of Microbiology and Molecular Biology 3 rd ed., revised ed., J. Wiley & Sons (New York, NY 2006), Smith, March's Advanced Organic Chemistry Reactions, Mechanisms and Structure 7 th ed., J. Wiley & Sons (New York, NY 2013), Singleton, Dictionary of DNA and Genome Technology 3 rd Wiley-Blackwell (November 28, 2012), and Green and Sambrook, Molecular Cloning: A Laboratory Manual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012) provide a general explanation of most of the terms used in this application. For references on antibody preparation methods, see Greenfield, Antibodies A Laboratory Manual 2 ndSee ed., Cold Spring Harbor Press (Cold Spring Harbor NY, 2013), Kohler and Milstein, Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion, Eur.J.Immunol. 1976 Jul, 6(7):511-9, Queen and Selick, Humanized immunoglobulins, US Patent No. 5, 585, 089 (1996 Dec), and Riechmann et al., Reshaping human antibodies for therapy, Nature 1988 Mar 24, 332(6162):323-7.

[0010] Those skilled in the art will understand that many of the methods and materials described herein, or their equivalents, can be used in carrying out the present invention. In fact, the present invention is by no means limited to the methods and materials described herein. For the purposes of the present invention, the following terms are defined below.

[0011] As used throughout this specification and the appended claims, “a,” “an,” and “the” include multiple references unless the context clearly indicates otherwise. Similarly, unless the context clearly indicates otherwise, “in” includes “in” and “on.”

[0012] As described, patient-specific induced pluripotent stem cells ("iPSCs") are expected to have many applications, including disease modeling to elucidate the mechanisms involved in disease causes, drug screening, and ultimately, regenerative medicine treatments. The practical application of human iPSCs is contingent on maintaining quality, genomic integrity, and stability. Several laboratories have shown that karyotype abnormalities originating from various tissue sources occur in approximately 10–25% of human iPSC lines, and recurrent genomic abnormalities have been observed during long-term culture.

[0013] Maintaining the genomic integrity and stability of hiPSC lines is essential for reliable disease modeling and the safe clinical application of stem cells, such as in regenerative medicine. Abnormal cytogenetic errors occurring during somatic cell reprogramming and / or during the maintenance, expansion, and long-term culture of hiPSCs affect the accuracy of in vitro disease modeling or the in vivo practicality of iPSCs for regenerative medicine. Given that several studies have reported chromosomal aneuploidy, translocations, duplications and deletions, and point mutations in iPSCs, it is crucial that iPSCs for clinical use are free from cancer-associated genomic abnormalities. Highly aneuploid human embryonic carcinoma (EC) stem cells, malignant analogs of normal hESCs, typically include amplified regions on the short arm of chromosome 12, as well as augmentation of chromosomes 1, 17, and the X chromosome.

[0014] Increased subchromosomal copy number variations (CNVs) due to differences in early, mid, or late passages, deletions associated with tumor suppressor genes, and oncogene duplication have all been reported in iPSCs. In most of these studies, genomic abnormalities in iPSC lines have resulted from embedded reprogramming methods. However, to date, there have been no reports of systematically and adequately controlling recurrent subchromosomal abnormalities specific to numerous fibroblast-derived or blood-derived hiPSC lines (using similar reprogramming and stem cell culture methods). We have developed an iPSC core facility with extensive experience in the production, characterization, and differentiation of human iPSC lines. This biostorage currently houses more than 160 well-characterized individual donor human iPSC lines, each with multiple clones (2-6) per cell line. iPSC Core systematically monitors the genomic stability of hiPSCs from 450 independent iPSC cultures across multiple passages derived from fibroblasts, peripheral blood mononuclear cells, immortalized lymphoblastoid cell lines, and primary epithelial cells, all originating from the Core.

[0015] The most frequently used starting material for reprogramming cells has been dermal fibroblasts isolated by skin biopsy. However, as described, iPSCs derived from skin punch biopsy are more invasive and require an extended growth period of 2-3 weeks in culture before reprogramming. Most importantly, there are more mutations associated with epidermal exposure to UV light, raising concerns about the safety of skin-derived iPSC cells. The problems associated with common fibroblast starting materials would be avoided by using peripheral blood, which is readily available as a patient tissue source for reprogramming. Peripheral blood is the most readily available adult tissue, and a large number of frozen samples already stored in blood banks would be available. Furthermore, many repositories store peripheral blood or lymphoblasts in specific genotypes.

[0016] Blood is widely used in clinical diagnosis and appears to be a favorable alternative for iPSC induction due to its standardized and less invasive collection methods compared to skin biopsies. The main components of peripheral blood (PB) are red blood cells and platelets. White blood cells number 3.6–11 × 10⁶. 6 These are nucleated cells in PB at a concentration of / ml. Mature T cells and primary progenitor cells in PB can be readily proliferated using established methods, and these are one of the best-used sources for reprogramming. T cells are the most abundant cells in PB, following granulocytes (20-30%), and T cells can be readily proliferated with IL-2 and anti-CD3 / CD28 microbeads. Reprogramming of T cells to pluripotency has been reported by many laboratories using different approaches to develop alternatives to senescent T cells for immunotherapy. However, mature T cells retain a single T cell receptor (TCR) after somatic recombination and lose the ability to generate a T cell repertoire of limitless potential.

[0017] Here, the inventors have developed a reliable protocol for efficiently reprogramming blood cells (BCs), including peripheral mononuclear blood cells (PBMCs), into iPSCs (BC-iPSCs), and demonstrate that these iPSC strains are superior in terms of cytogenetic stability compared to their fibroblast-derived iPSC (Fib-iPSC) strains obtained from public storage or local clinics. The inventors describe a method and cytogenetic stability for inducing BC-iPSCs from both lymphoid T cell and myeloid non-T cell populations. The inventors found that hiPSC strains generated from PBMCs using the non-integration method had a significantly lower incidence of genomic abnormalities than hiPSC strains generated by the integration method. Both fib-iPSCs and BC-iPSCs derived from the same somatic cells contained a comparable number of novel CNVs. The inventors' results demonstrate that freshly isolated human PBMCs can be faithfully reprogrammed into pluripotency with greater cytogenetic stability using episomal plasmids. Thus, PBMCs are a preferred somatic cell source for iPSC reprogramming, minimizing any impact of acquired genomic abnormalities, and are also considered an ideal cell source for regenerative medicine.

[0018] In contrast to mature T or B cells, an alternative source of hematopoietic progenitor cells includes intact genomes. Furthermore, they can be grown under culture conditions favorable to the proliferation of myeloid or erythrocyte cells. Hematopoietic stem cells / progenitor cells express the surface marker CD34 and reside in the stem cell niche. However, only about 1% of stem cells / progenitor cells enter circulation daily, and consequently, only 0.01–0.1% of cells in the PB are CD34+ cells. This population can be enriched by magnetically activated cell sorting (MACS), or CD34+ cells can be grown to a purity of 5–20% by culturing MNCs for several days, allowing them to be used for reprogramming without further purification.

[0019] Other nucleated peripheral blood cells include granulocytes (primarily neutrophils), monocytes, T lymphocytes, B lymphocytes, and a small number of progenitor cells. Concentration of these blood components can be achieved by depleting erythrocytes and platelets using multiple centrifuges following lysis buffer. Ficoll gradient centrifugation can also be used to deplete both erythrocytes and granulocytes and enrich mononuclear cells (MNCs). Against this backdrop, reprogramming by exogenously expressed factors is, as is well known, inefficient, and multiple cell cycles are required to achieve pluripotency. Thus, primary granulocytes, monocytes, and B lymphocytes are among the most difficult cells to reprogram due to the lack of reliable protocols for proliferation. Major successes in this field include the reprogramming of Epstein-Barr virus immortalized lymphoblastic B cells into pluripotency, which readily proliferates in ex vivo culture.

[0020] Considering the above, the reprogramming of the non-T cell component of blood is of paramount importance. With existing techniques, it is virtually impossible to reprogram this population from isolated peripheral blood mononuclear cells (PBMCs). More specifically, PBMCs are any peripheral blood cells with a round nucleus. These include lymphocytes (T cells, B cells, NK cells), monocytes, and dendritic cells. Lymphocytes are small (5-10 μm) and medium (10-18 μm) in size and make up 70-90% of PBMCs. These cells include 70-85% CD3+ T cells (40-70% of PBMCs), CD4 helper T cells (25-60% of PBMCs), typically with a CD4-to-CD8 ratio of 2:1, and CD8 "cytotoxic" T cells (5-30% of PBMCs). The remaining segments include 5-20% B cells (up to 15% of PBMCs) and 5-20% NK cells (up to 15% of PBMCs). Monocytes are 16-25 μm in size and make up 10-30% of PBMCs (macrophages). Dendritic cells make up 1-2% of PBMCs.

[0021] Here, the inventors describe a novel and reliable non-integration episomal plasmid reprogramming method for fresh (non-proliferating) peripheral blood mononuclear cells (PBMCs) into iPSCs (PBMC-iPSCs). The inventors demonstrate that PBMC-iPSCs generated using this method exhibit excellent chromosomal-level karyotype stability with an abnormality rate of only 1.9% and minimal new copy number variations in subchromosomes. In contrast, iPSC lines episomal reprogrammed from pre-proliferated dermal fibroblasts, lymphoblast cell lines, or epithelial cells (fib-iPSC, LCL-iPSC, or epi-iPSC, respectively) exhibit more than 10-fold karyotype instability (23-27%). These findings demonstrate that iPSCs reprogrammed and maintained from non-proliferating PBMCs using the method described herein possess excellent cytogenetic stability and minimal genomic abnormalities, suggesting that this is a preferred method for iPSCs in research and therapeutic clinical applications.

[0022] These described approaches enable the use of peripheral blood as an readily available resource for cell reprogramming, possessing superior genomic and karyotype stability, while avoiding environmental damage that would otherwise cause mutations or other forms of structural changes present in therapeutic materials derived from peripheral blood.

[0023] As described herein, the inventors have established an improved technique for highly efficient and reproducible reprogramming using non-integrated episomal plasmid vectors, including the generation of iPSCs from blood cells, including whole blood and peripheral blood, resulting in reprogrammed pluripotent cells referred to herein as "BC-iPSC".

[0024] Generally, different approaches to non-integration reprogramming include at least (1) delivery of integration-deficient viruses, (2) episomal delivery, (3) direct RNA delivery, (4) direct protein delivery, and (5) chemical induction. As further described herein, the use of episomal vectors, which substantially do not involve the vectors used for their production, is due to the fact that the episomal vectors or similar vectors enabling the generation of iPSCs do not encode a viral genome sufficient to produce an infectious or replicating competent virus. At the same time, these vectors actually possess some degree of self-renewal ability in the initial somatic host cell. This self-renewal ability provides some degree of sustained expression, which is understood to be beneficial in enabling the dedifferentiation process for initiation of colonization into the target host cell.

[0025] One example of a plasmid vector that meets these criteria is the Epstein Barr oriP / Nuclear Antigen-1 ("EBNA1") combination, which is known to be able to restrict self-replication and function in mammalian cells. Because it contains two elements derived from the Epstein-Barr virus, oriP and EBNA1, the binding of the EBNA1 protein to the viral replicon region oriP allows the plasmid to maintain its existence as an episome in mammalian cells for a relatively long period. This feature of the oriP / EBNA1 vector makes it ideal for generating non-integrated iPSCs.

[0026] More specifically, sustained expression of reprogramming factors encoded by the oriP / EBNA1 vector occurs over multiple cell division cycles. Sufficiently high levels of reprogramming factors across several cell divisions enable good reprogramming even after a single infection. While sustained expression of reprogramming factors is understood to be beneficial during the initial programming phase, unrestrained constitutive expression would interfere with subsequent stages of the reprogramming process. For example, undiluted expression of reprogramming factors would interfere with subsequent host cell proliferation, development, and fate determination.

[0027] Simultaneously, a further advantage is the removal of the final reprogramming factor transgene, as a small portion of the episome is lost with each cell cycle. This is due to the asymmetric replication capabilities of the host cell genome and episomal self-renewal, and it is estimated that about 0.5% of the vector is lost per generation. The gradual depletion of plasmids during each cell division is unavoidable if proliferation continues to result in a population of unintegrated iPSCs. Persistent but ultimately halted reprogramming factor expression on oriP / EBNA1 is very consistent with the needs for various stages of the reprogramming process, as has been attempted through the use of transposons and excisable polycistronic lentiviral vector elements, and does not require further manipulation steps to remove the reprogramming factors. While oriP / EBNA1 has been applied by others in reprogramming studies, the reported efficiencies are very low (only 3-6 colonies per million nucleofected cells), which may be due in part to the large plasmid (e.g., over 12kb) encoding multiple reprogramming factors, which negatively impacts transfection efficiency.

[0028] In addition to these choices in vector design, the specific combinations of reprogramming factors used in the literature have varied. As mentioned earlier, the reprogramming factors used include the pluripotency-related genes Oct-4, Sox-2, Lin-28, Nanog, Sall4, Fbx-15, and Utf-1. These factors are typically expressed early in development and are understood to be involved in maintaining the pluripotency of a subset of cells that make up the inner cell mass of preimplantation and postimplantation-proprietary embryos. Their ectopic expression is thought to enable the establishment of an embryo-like transcriptional cascade that initiates and propagates another dormant endogenous nuclear pluripotency program within the host cell. Certain other reprogramming determinants have been applied, such as Tert, Klf-4, c-Myc, SV40 large T antigen ("SV40LT"), and short hairpin RNA targeting p53 ("shRNA-p53"). These determinants may not be pluripotency determinants themselves, but have been reported to be beneficial in reprogramming. For example, TERT and SV40LT are thought to enhance cell proliferation to promote survival during reprogramming, while others, such as short hairpin targeting of p53, suppress or eliminate reprogramming barriers such as senescence and apoptosis mechanisms. In each case, an increase in both the rate and efficiency of reprogramming is observed. Furthermore, microRNAs ("miRNAs") are also known to influence pluripotency and reprogramming, and some miRNAs derived from the miR□290 cluster have been applied to reprogramming studies. For example, it has been reported that introducing miR 291 3p, miR 294, or miR 295 into fibroblasts, in addition to pluripotency-related genes, increases reprogramming efficiency.

[0029] While various vectors and reprogramming factors in this field appear to present multiple components capable of establishing reprogramming within cells, a high degree of complexity arises when considering the stoichiometric expression levels required to maintain good reprogramming. For example, it has been reported that somatic cell reprogramming efficiency is four times higher when Oct-4 and Sox2 are encoded in a 1:1 ratio on a single vector, in contrast to delivering the two factors in separate vectors. In the latter case, the uptake rates of the two factors become more difficult to control, negatively impacting reprogramming efficiency. One approach to address these barriers is the use of polycistronic vectors, such as those incorporating internal ribosome translocation sites ("IRES"), which are provided upstream of the transgene(s) distal to the transcription promoter. This mechanism allows one or more transgenes to be provided in a single reprogramming vector, and various inducible or constitutive promoters to be combined as an expression cassette to transmit transcriptional regulation to multiple transgenes at a more granular level. These more specific levels of control offer significant advantages to the reprogramming process, allowing separate expression cassettes on a vector to be designed accordingly under the control of separate promoters.

[0030] While there are advantages to delivering such factors via a single or small number of vectors, there are upper size limitations on the final vector size, which can hinder attempts to facilitate their delivery to host target cells. For example, early reports on the use of polycistronic vectors noted very low reprogramming efficiencies, sometimes occurring in less than 1% of cells, more typically less than 0.1%. Part of these barriers are due to certain target host cells, such as low tolerance to large constructs (e.g., fibroblasts) or insufficient processing of IRES sites by the host cell. Furthermore, the arrangement of factors in the vector expression cassette affects both their stoichiometric and temporal expression, leading to further variations that influence reprogramming efficiency. Thus, some improved techniques can rely on multiple vectors, each encoding one or more reprogramming factors in various expression cassettes. Under these designs, a less refined but relatively direct pathway is provided for modulating expression levels in target cells by changing the amount of a particular vector for delivery.

[0031] A further advantage of the techniques described herein is the use of prescribed medium conditions for the reprogramming process, including the use of ESC medium and / or E7 medium. Certain additives may be present to stimulate the reprogramming process, but serum or animal components are not used (e.g., L-ascorbic acid, transferrin, sodium bicarbonate, insulin, sodium selenite and / or bFGF). In some cases, they may offer further advantages by altering the chemical and / or atmospheric conditions under which the reprogramming takes place. For example, since pre-implantation embryos are not angiogenic and are hypoxic (similar to the bone marrow stem cell niche), further opportunities to enhance reprogramming efficiency may be provided by reprogramming under hypoxic conditions of 5% O2 instead of the 21% O2 of the atmosphere. Similarly, chemical induction techniques have been used in combination with reprogramming techniques widely used in different reprogramming studies, particularly histone deacetylase (HDAC) inhibitor molecules, such as valproic acid (VPA). Simultaneously, other small molecules such as the MAPK kinase (MEK)-ERK ("MEK") inhibitor PD0325901, the transforming growth factor β ("TGF-β") type I receptor ALK4, ALK5, and ALK7 inhibitor SB431542, and the glycogen synthase kinase-3 ("GSK3") inhibitor CHIR99021 are applied to activate differentiation induction pathways (e.g., BMP signaling) in combination with the modulation of other pathways (e.g., inhibition of the MAPK kinase (MEK)-ERK pathway) to sustain self-renewal. Other small molecules such as Rho-related coiled-coil-containing protein kinase ("ROCK") inhibitors, such as Y-27632 and thiazovibin ("Tzv"), are applied to promote survival and reduce the vulnerability of pSCs to cell death, particularly in single-cell dissociation.

[0032] In addition to the selection of delivery vectors, combinations of reprogramming factors, and reprogramming conditions, further modifications must consider the properties of the host target cells for reprogramming. To date, a wide range of cells, including fibroblasts, gastric and hepatocyte cultures, human keratinocytes, adipocytes, and frozen human monocytes, have served as sources for reprogramming. Clearly, there is broad and robust potential for dedifferentiation across many tissue sources. Nevertheless, it is widely understood that reprogramming is achieved with different efficiencies and dynamics depending on the donor cell type. For example, while fibroblasts are the most common donor cell type for reprogramming studies, other cell types, such as human primary keratinocytes transduced with Oct-4, Sox-2, Klf-4, and c-Myc, have been reported to reprogram with more than 100-fold efficiency and twice as fast. Furthermore, some other cell types, such as umbilical cord blood cells, may require only a subset of reprogramming factors such as Oct-4 and Sox-2 for dedifferentiation for implantation, while neural progenitor cells may require only Oct-4. While not bound by any particular theory, differences in the reprogramming efficiency and / or required reprogramming factors of specific host cells are thought to be due to certain reprogramming factors and / or endogenous epigenetic states with higher endogenous levels that are better suited to reprogramming.

[0033] While many other raw materials have been used in research for iPSC generation, peripheral blood (PB)-derived mononuclear cells (MNCs) are a very attractive host cell candidate due to their convenience and characteristics as a nearly limitless source for cell reprogramming. PB cells, in particular, are relatively easy to isolate (e.g., blood collection) compared to isolation from other raw materials such as fibroblasts (e.g., skin biopsy). These cells do not require cumbersome culture and proliferation before reprogramming, thereby shortening the overall time to obtain reprogrammed iPSCs.

[0034] Following successful reprogramming, clonal selection enables the generation of pluripotent stem cell lines. Ideally, such cells possess the required morphology (i.e., dense colonies, a high nucleus-to-cytoplasmic ratio, and prominent nucleoli), the ability to self-replicate for unlimited proliferation in culture (i.e., immortality), and the ability to differentiate into all three germ layers (e.g., endoderm, mesoderm, and ectoderm). Further techniques for characterizing the pluripotency of a given cell population include injection into immunocompromised animals, such as severe combined immunodeficiency ("SCID") mice, for the formation of teratomas characteristic of cells or tissues of all three germ layers.

[0035] As an example above, approximately 5 × 10 per nucleofection 6 Cellular PBMCs were nucleofected with either plasmid mixture 4p or plasmid mixture 5p using program V-024 in the Amaxa Nucleofector 2D Device with the Amaxa Human T-cell Nucleofector® Kit. Subsequently, approximately 1 × 10⁻⁶ cells were nucleofected. 6The cells were either covered with a mitomycin-treated mouse embryonic feeder (MEF) layer or seeded in the wells of a 6-well plate coated with 10 μg / ml Laminin-521 (L-521, BioLamina). Each episomal plasmid (pEP4 E02S ET2K, pCXLE-hOCT3 / 4-shp53-F, pCXLEhUL, pCXLE-hSK, and pCXLE-EBNA1), (Addgene) expressed seven factors (OCT4, SOX2, KLF4, LMYC, LIN28, SV40LT, and p53shRNA). Since exogenously introduced genes are not integrated but instead transiently expressed by episomes, this method has a significant advantage over viral transduction. Cells were seeded in 2 ml of either αβ T cell medium (X-vivo10 supplemented with 30 U / ml IL-2 and 5 ul / well Dynabeads Human T-activator CD3 / CD28) or non-T cell medium (αMEM supplemented with 10% FBS, 10 ng / ml IL-3, 10 ng / ml IL-6, 10 ng / ml G-CSF, and 10 ng / ml GM-CSF). Two days after nucleofection, an equal volume of primate ESC medium (ReproCell) containing 5 ng / ml bFGF (for MEF conditions) or E7 medium (for L-521 conditions) was added to the wells without aspirating the old medium. From day 4 onwards, the medium was gently aspirated from each well, and 2 ml of appropriate fresh reprogramming medium was added to each well. The medium was changed every other day. Approximately 18 days after nucleofection, individual colonies were observed in all wells under each condition. Approximately 25 days after nucleofection, individual colonies were isolated and subcloned into one well of a 12-well plate containing appropriate substrate and medium. These nucleofected cells were seeded in feeder-independent BD Matrigel® growth factor-reduced matrix (Corning / BD Biosciences, #354230). All cultures were maintained at 20% O2 throughout the reprogramming process.Individual PBMC-iPSC colonies exhibiting ES / iPSC-like morphology appeared between 25 and 32 days, and those with the best morphology were mechanically isolated and transferred to 12-well plates with fresh Matrigel® matrix and maintained in mTeSR® 1 medium. For further analysis, iPSC clones were further grown and scaled up. All fibroblasts were reprogrammed using the 3p reprogramming plasmid mixture as described.

[0036] This specification describes compositions of induced pluripotent stem cells ("BC-iPSCs") derived from blood cells. In certain embodiments, a composition of induced pluripotent stem cells derived from blood cells comprises preparing a certain amount of blood cells, delivering a certain amount of reprogramming factors to the blood cells, and culturing the blood cells in a reprogramming medium for at least four days to produce cells, wherein the delivery of reprogramming factors and culturing produce induced pluripotent stem cells derived from blood cells. In certain embodiments, the blood cells are T cells. In other embodiments, the blood cells are non-T cells. In other embodiments, the blood cells are mononuclear cells (MNCs), including, for example, peripheral blood mononuclear cells (PBMCs). In other embodiments, the cells are primary granulocytes, monocytes, and B lymphocytes.

[0037] In certain embodiments, the reprogramming factors are Oct-4, Sox-2, Klf-4, c-Myc, Lin-28, SV40 large T antigen ("SV40LT"), and p53-targeting short hairpin RNA ("shRNA-p53"). In other embodiments, these reprogramming factors are encoded in a combination of vectors containing pEP4 E02S ET2K, pCXLE-hOCT3 / 4-shp53-F, pCXLE-hSK, pCXLE-hUL, and pCXWB-EBNA1. This includes, for example, the use of approximately 0.5–1.0 ug of pCXLE-hOCT3 / 4-shp53, 0.5–1.0 ug of pCXLE-hSK, 0.5–1.0 ug of pCXLE-UL, approximately 0.25–0.75 ug of pCXWB-EBNA1, and 0.5–1.0 ug of pEP4 E02S ET2K. This includes, for example, the use of 0.83 ug of pCXLE-hOCT3 / 4-shp53, 0.83 ug of pCXLE-hSK, 0.83 ug of pCXLE-UL, 0.5 ug of pCXWB-EBNA1, and 0.83 ug of pEP4 E02S ET2K, where the stoichiometric ratio of SV40LT (encoded in pEP4 E02S ET2K) to EBNA-1 (encoded in pCXWB-EBNA1) supports the reprogramming of the non-T cell component of blood, including peripheral blood mononuclear cells. In certain other embodiments, the reprogramming medium includes PD0325901, CHIR99021, HA-100, and A-83-01. In other embodiments, the culture of blood cells in the reprogramming medium is 4 to 30 days. In various embodiments, blood cells are seeded on a treated cell culture surface after delivery of a certain amount of reprogramming factors. In various embodiments, the treatment involves seeding feeder cells, such as mouse embryonic fibroblasts. In other embodiments, the treatment involves coating with an extracellular matrix protein. In various embodiments, the extracellular matrix protein includes laminin.

[0038] In various embodiments, BC-iPSCs can be continuously passaged as cell lines. In various embodiments, BC-iPSCs possess genomic stability. Genomic stability can be confirmed by various techniques known in the art. For example, G-band karyotype analysis can identify abnormal cells lacking genomic stability, where the abnormal cells have approximately 10% or more mosaicism or one or more balanced translocations larger than approximately 5, 6, 7, 8, 9, 10 or more Mb. Alternatively, genomic stability can be measured using comparative genomic hybridization (aCGH) microarrays, for example, by comparing BC-iPSCs with iPSCs derived from non-blood cell sources such as fibroblasts. Genomic stability may include copy number variations (CNVs), duplications / deletions, and unbalanced translocations. In various embodiments, BC-iPSCs include amplifications and deletions with an average size of approximately 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, or 20 Mb or less. In various embodiments, BC-iPSCs do not exhibit amplification and deletions with an average size of approximately 20-30 Mb or less. In various embodiments, BC-iPSCs do not exhibit amplification and deletions with an average size of approximately 30-40 Mb or less. In various embodiments, BC-iPSCs exhibit amplification and deletions with an average size of approximately 40-50 Mb or less. In various embodiments, the average number of new amplifications and deletions acquired in BC-iPSCs is approximately 5, 4, 3, 2, or less than 1. For example, new amplifications and deletions in fib-iPSCs are at least twice as large as in PBMC-iPSCs.

[0039] In different embodiments, the reprogramming factor may also include one or more of the following: Oct-4, Sox-2, Klf-4, c-Myc, Lin-28, SV40LT, shRNA-p53, nanog, Sall4, Fbx-15, Utf-1, Tert, or Mir-290 cluster microRNAs such as miR□291□3p, miR□294, or miR□295. In different embodiments, the reprogramming factor is encoded by a vector. In different embodiments, the vector may be, for example, a non-integrated episomal vector, a minicircle vector, a plasmid, a retrovirus (integrated and non-integrated), and / or other genetic elements known to those skilled in the art. In different embodiments, the reprogramming factor is encoded by one or more oriP / EBNA1-derived vectors. In different embodiments, a vector may encode one or more reprogramming factors, and a combination of vectors can be used simultaneously to deliver one or more Mir-290 cluster microRNAs such as Oct-4, Sox-2, Klf-4, c-Myc, Lin-28, SV40LT, shRNA-p53, nanog, Sall4, Fbx-15, Utf-1, Tert, or miR□291□3p, miR□294, or miR□295. For example, oriP / EBNA1 is an episomal vector that can encode vector combinations of multiple reprogramming factors such as pCXLE-hUL, pCXLE-hSK, pCXLE-hOCT3 / 4-shp53-F, pEP4 EO2S T2K, and pCXWB-EBNA1.

[0040] In other embodiments, the reprogramming factor is delivered by techniques known in the art, such as nucleofection, transfection, transduction, electrofusion, electroporation, microinjection, and cell fusion, among others. In other embodiments, the reprogramming factor is provided as RNA, linear DNA, peptides or proteins, or as a cell extract of pluripotent stem cells.

[0041] In various embodiments, the reprogramming medium comprises embryonic stem cell (ESC) medium. In various embodiments, the reprogramming medium comprises bFGF. In various embodiments, the reprogramming medium is E7 medium. In various embodiments, the reprogramming E7 medium comprises L-ascorbic acid, transferrin, sodium bicarbonate, insulin, sodium selenite and / or bFGF. In different embodiments, the reprogramming medium comprises at least one chemically derived small molecule. In different embodiments, the at least one chemically derived small molecule includes PD0325901, CHIR99021, HA-100, A-83-01, valproic acid (VPA), SB431542, Y-27632, or thiazovibin ("Tzv"). In different embodiments, BC culture in reprogramming medium is for at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days.

[0042] In certain embodiments, BC-iPSCs are derived from blood cells previously isolated from a subject, for example, by taking a blood sample from the subject. In other embodiments, blood cells are isolated from a subject having disease mutations. For example, a subject having any number of mutations, such as autosomal dominant, recessive, or X-linked mutations, can serve as a source of blood cells for generating BC-iPSCs having the aforementioned mutations. In other embodiments, the disease mutations are associated with neurodegenerative diseases, disorders, and / or conditions. In other embodiments, the disease mutations are associated with inflammatory bowel disease, disorders, and / or conditions. In various embodiments, BC-iPSCs have the characteristics of pluripotent stem cells. Some exemplary features of pluripotent stem cells include, among other features understood and recognized by those skilled in the art, differentiation into cells of the three germ layers (ectoderm, endoderm, and mesoderm) either in vitro or in vivo when injected into immunodeficient animals, expression of pluripotency markers such as Oct-4, Sox-2, nanog, TRA-1-60, TRA-1-81, and SSEA4, high levels of alkaline phosphatase ("AP") expression, and unrestricted proliferation in culture.

[0043] Other embodiments include a pharmaceutical composition comprising a certain amount of induced pluripotent stem cells derived from blood cells produced by the method described above and a pharmaceutically acceptable carrier.

[0044] This specification also describes an efficient method for generating induced pluripotent stem cells, comprising preparing a certain amount of cells, delivering a certain amount of reprogramming factors to the cells, and culturing the cells in a reprogramming medium for at least four days, wherein induced pluripotent stem cells are generated by delivering the reprogramming factors and culturing. In certain embodiments, the cells are primary cultured cells. In other embodiments, the cells are blood cells (BCs). In certain embodiments, the blood cells are T cells. In other embodiments, the blood cells are non-T cells. In other embodiments, the cells are mononuclear cells (MNCs), including, for example, peripheral blood mononuclear cells (PBMCs). In other embodiments, the cells are primary granulocytes, monocytes, and B lymphocytes.

[0045] In certain embodiments, the reprogramming factors are Oct-4, Sox-2, Klf-4, c-Myc, Lin-28, SV40 large T antigen ("SV40LT"), and p53-targeting short hairpin RNA ("shRNA-p53"). In other embodiments, these reprogramming factors are encoded in a combination of vectors containing pEP4 E02S ET2K, pCXLE-hOCT3 / 4-shp53-F, pCXLE-hSK, pCXLE-hUL, and pCXWB-EBNA1. This includes, for example, the use of approximately 0.5–1.0 ug of pCXLE-hOCT3 / 4-shp53, 0.5–1.0 ug of pCXLE-hSK, 0.5–1.0 ug of pCXLE-UL, approximately 0.25–0.75 ug of pCXWB-EBNA1, and 0.5–1.0 ug of pEP4 E02S ET2K. This includes, for example, the use of 0.83 ug of pCXLE-hOCT3 / 4-shp53, 0.83 ug of pCXLE-hSK, 0.83 ug of pCXLE-UL, 0.5 ug of pCXWB-EBNA1 and 0.83 ug of pEP4 E02S ET2K, where the stoichiometric ratio of SV40LT (encoded in pEP4 E02S ET2K) to EBNA-1 (encoded in pCXWB-EBNA1) supports the reprogramming of non-T cell components of blood, including peripheral blood mononuclear cells. In various embodiments, the reprogramming medium comprises embryonic stem cell (ESC) medium. In various embodiments, the reprogramming medium comprises bFGF. In various embodiments, the reprogramming medium is E7 medium. In various embodiments, the reprogramming E7 medium comprises L-ascorbic acid, transferrin, sodium bicarbonate, insulin, sodium selenite and / or bFGF. In different embodiments, the reprogramming medium contains at least one chemically derived small molecule. In certain other embodiments, the reprogramming medium contains PD0325901, CHIR99021, HA-100, and A-83-01. In other embodiments, the culture of blood cells in the reprogramming medium is 4 to 30 days.

[0046] In various embodiments, BC-iPSCs can be continuously passaged as a cell line. In various embodiments, BC-iPSCs have genomic stability. Genomic stability can be confirmed by various techniques known in the art. For example, G-band karyotype analysis can identify abnormal cells lacking genomic stability, where the abnormal cells have mosaicism of about 10% or more, or have one or more balanced translocations larger than about 5, 6, 7, 8, 9, 10 or more Mb. Alternatively, genomic stability can be measured using comparative genomic hybridization (aCGH) microarrays, for example, by comparing BC-iPSCs with iPSCs derived from non-blood cell sources such as fibroblasts. Genomic stability may include copy number variations (CNVs), duplication / deletion, and unbalanced translocations. In various embodiments, BC-iPSCs include amplifications and deletions with an average size of about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, or 20 Mb or less. In various embodiments, BC-iPSCs exhibit amplification and deletions with an average size of approximately 20-30 Mb or less. In various embodiments, BC-iPSCs exhibit amplification and deletions with an average size of approximately 30-40 Mb or less. In various embodiments, BC-iPSCs exhibit amplification and deletions with an average size of approximately 40-50 Mb or less. In various embodiments, the average number of new amplifications and deletions acquired in BC-iPSCs is approximately 5, 4, 3, 2, or less than 1. For example, new amplifications and deletions in fib-iPSCs are at least twice as large as in PBMC-iPSCs. In various embodiments, when the cell line is continuously passaged, the method produces iPSC cell lines exhibiting approximately 20%, 15%, 10%, and 5% or less of abnormal karyotypes overall for passages of 4-8, 9-13, 13-17, 17-21, 21-25, or 29 or more.

[0047] In different embodiments, the reprogramming factor may also include one or more of the following: Oct-4, Sox-2, Klf-4, c-Myc, Lin-28, SV40LT, shRNA-p53, nanog, Sall4, Fbx-15, Utf-1, Tert, or Mir-290 cluster microRNAs such as miR□291□3p, miR□294, or miR□295. In different embodiments, the reprogramming factor is encoded by a vector. In different embodiments, the vector may be, for example, a non-integrated episomal vector, a minicircle vector, a plasmid, a retrovirus (integrated and non-integrated), and / or other genetic elements known to those skilled in the art. In different embodiments, the reprogramming factor is encoded by one or more oriP / EBNA1-derived vectors. In different embodiments, a vector may encode one or more reprogramming factors, and a combination of vectors can be used simultaneously to deliver one or more Mir-290 cluster microRNAs such as Oct-4, Sox-2, Klf-4, c-Myc, Lin-28, SV40LT, shRNA-p53, nanog, Sall4, Fbx-15, Utf-1, Tert, or miR□291□3p, miR□294, or miR□295. For example, oriP / EBNA1 is an episomal vector that can encode vector combinations of multiple reprogramming factors such as pCXLE-hUL, pCXLE-hSK, pCXLE-hOCT3 / 4-shp53-F, pEP4 EO2S T2K, and pCXWB-EBNA1.

[0048] In other embodiments, the reprogramming factor is delivered by techniques known in the art, including, among others, nucleofection, transfection, transduction, electrofusion, electroporation, microinjection, and cell fusion. In other embodiments, the reprogramming factor is provided as RNA, linear DNA, peptides or proteins, or as a cell extract of pluripotent stem cells. In certain embodiments, cells are treated with sodium butyrate before delivery of the reprogramming factor. In other embodiments, cells are incubated on a tissue culture surface for 1, 2, 3, or 4 days or more before further culture. This may include, for example, incubation on a Matrigel-coated tissue culture surface. In other embodiments, the reprogramming conditions include the application of normal oxygen conditions, such as 5% O2, less than 21% O2 in air.

[0049] In various embodiments, the reprogramming medium comprises embryonic stem cell (ESC) medium. In various embodiments, the reprogramming medium comprises bFGF. In various embodiments, the reprogramming medium is E7 medium. In various embodiments, the reprogramming E7 medium comprises L-ascorbic acid, transferrin, sodium bicarbonate, insulin, sodium selenite and / or bFGF. In different embodiments, the reprogramming medium comprises at least one chemically derived small molecule. In different embodiments, the at least one chemically derived small molecule includes PD0325901, CHIR99021, HA-100, A-83-01, valproic acid (VPA), SB431542, Y-27632, or thiazovibin ("Tzv"). In different embodiments, BC culture in reprogramming medium is for at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days.

[0050] The efficiency of reprogramming can be readily confirmed by one of many techniques readily understood by those skilled in the art. For example, efficiency can be described by the ratio between the number of donor cells that received a set of reprogramming factors and the number of reprogrammed colonies produced. The number of donor cells that received reprogramming factors can be measured directly if the vector encoding the reprogramming factors contains a reporter gene such as GFP. Alternatively, the indirect measurement of delivery efficiency can be obtained by transfecting a vector encoding a reporter gene as a substitute for evaluating the delivery efficiency in a pair of samples to which the reprogramming factor vector is delivered. Furthermore, the number of reprogrammed colonies produced can be measured by observing the appearance of one or more embryonic stem cell-like pluripotency characteristics, such as alkaline phosphatase (AP)-positive clones, colonies with endogenous expression of the transcription factor Oct or Nanog, or antibody staining of a surface marker such as Tra-1-60. In another example, efficiency can be described by the kinetics of induced pluripotent stem cell generation. For example, efficiency can include the production of cell lines with normal karyotypes, including methods for producing iPSC cell lines exhibiting abnormal karyotypes in approximately 20%, 15%, 10%, and 5% or less overall for 4-8, 9-13, 13-17, 17-21, 21-25, or 29 or more passages, when the cell line is continuously passaged. [Examples]

[0051] Example 1 General iPSC reprogramming protocols for blood Generally, the reprogramming method includes the following steps: Frozen peripheral blood mononuclear cells (PBMCs) or freshly isolated PBMCs can be used. A treated cell culture surface can be used to seed the thawed or freshly isolated PBMCs. This includes, for example, mouse embryonic feeder cells (MEFs) or cell medium surfaces treated with the extracellular matrix laminin L-521A. An expression plasmid mixture is provided, having a plasmid mixture encoding a combination of pluripotency factors. For example, the plasmid is introduced into the PBMCs using nucleofection and then placed on the treated cell culture surface. After 2 days of culture, reprogramming medium is introduced and replenished until colonies are formed.

[0052] Example 2 Peripheral blood collection and minimal processing for reprogramming. The inventors have established a process for isolating lymphocytes from freshly collected or commercially available human or mammalian peripheral blood (PB). A preferred form for collecting and transporting such samples is a CPT tube. This form allows the supplier to centrifuge the vacuum tube to isolate red blood cells from the plasma component before transport. This protocol provides for the isolation of PBMCs from the plasma layer in a CPT tube.

[0053] Optional Testing: Before opening the vacuum container, several tests may be performed, including tests for HIV, hepatitis B and C, and syphilis. The results of the latter tests must be available within 1-3 business days from the date of collection. If the result is positive, the sample(s) will be disposed of appropriately, and all laboratory equipment used to process the samples will be decontaminated.

[0054] Example 3 Collection procedure (1) Red blood cells and platelets are reduced using a container. BD Vacutainer® CPT® tubes containing sodium citrate should be brought to room temperature (18-25°C) and properly labeled for patient identification. Spray the CPT Vacutainer with 70% isopropanol / ethanol and wipe off the alcohol. (2) Blood samples were collected using the standard method with BD Vacutainer® vacuum blood collection tubes, into three 8 ml CPT tubes per sample. After blood collection, the tubes were stored upright at room temperature until centrifugation. Blood samples must be centrifuged within two hours of collection for good results. (3) The tubes / blood samples are centrifuged in a horizontal rotor (swing-out head) at room temperature (18-25°C) for a minimum of 20 minutes (maximum of 30 minutes) at 1500-1800 RCF (relative centrifugal force). (4) Blood transported in CPT tubes should be reprogrammed within 24 hours. After centrifugation, mononuclear cells and platelets are located in a whitish layer just below the plasma layer (see Figure 1). Another procedure for recovering separated mononuclear cells is to resuspend the cells in plasma by gently inverting the unopened BD Vacutainer® CPT® tube 5 to 10 times. This is a preferred method for storing or transporting separated samples up to 24 hours after centrifugation.

[0055] Example 4 Important parameters Temperature: The separation principle depends on the density gradient, and since the density of the components changes with temperature, the system temperature must be controlled to 18-25°C during the separation process. Centrifugation: Since the principle of separation depends on the movement of formed elements in the blood as it passes through the separation medium, the RCF should be controlled between 1500 RCF and 1800 RCF. The centrifugation time should be at least 20 minutes. (As explained in the troubleshooting section, some samples may take up to 30 minutes for optimal separation.) Centrifugation of BD Vacutainer® CPT® tubes for up to 30 minutes is effective in reducing red blood cell contamination of the mononuclear cell fraction. Centrifugation for more than 30 minutes has little additional benefit. BD Vacutainer® CPT® tubes may be recentrifuged if the mononuclear cell "bands" or layers are not disturbed. Time: Blood samples must be centrifuged or separated within 2 hours of collection. Red blood cell contamination in the separated mononuclear cell fraction increases with longer delays in sample separation. Mononuclear cell recovery decreases with increasing time delay before centrifugation, dropping to approximately 40% mononuclear cell recovery after 24 hours. Pour the contents (plasma layer) into a properly labeled 50 ml tube.

[0056] Example 5 Materials and suppliers for the reprogramming procedure Procedures for reprogramming isolated whole blood samples (peripheral blood mononuclear cells, PBMCs) for iPSC production are described herein. Materials used include sterile 1.5 ml Eppendorf tubes, sterile pipette tips (1000 μl, 200 μl, 10 μl), Amaxa Nucleofector® 2b device (Lonza), Human T cell Nucleofector kit (Lonza, catalog no. VPA-1002), MEF 6-well TC-treated plate or L-521 coated plate, 0.22 μm SteriFlip (optional), and primate ESC medium or E7 medium, as listed in Tables 1-4 below.

[0057] The reprogramming plasmids used include (1) pCXLE-hOCT3 / 4-shp53, (2) pCXLE-hSK, (3) pCXLE-UL, (4) pCXWB-EBNA1, and (5) pEP4 E02S ET2K.

[0058] (Table 1) αβ T cell medium TIFF0007943782000001.tif45136

[0059] (Table 2) Non-T cell medium TIFF0007943782000002.tif53135

[0060] (Table 3) MEF medium (optional) TIFF0007943782000003.tif38136

[0061] (Table 4) Prepared primate ESC medium (optional) TIFF0007943782000004.tif23136

[0062] (Table 5) Prepared E7 medium (optional) TIFF0007943782000005.tif61135

[0063] Example 6 Reprogramming procedure [Day 1 - Plate Preparation] [MEF Plate] 1. Coat each well of a 6-well plate with 1 ml of 0.1% gelatin. 2. Place the plate at 37 o °C and incubate for at least 1 hour. 3. Retrieve a vial of frozen MEFs from the LN2 tank. 4. Thaw the vial in a water bath by slowly moving the frozen vial in water in a figure-8 motion. 5. Collect the thawed cells into a 15 ml conical tube. 6. Slowly add MEF medium to a final volume of 10 ml. 7. Centrifuge the tube / MEFs at room temperature (18~25°C) at 1000~1200 RPM (relative centrifugal force) for 5 minutes. 8. While the cells are spinning, aspirate the gelatin from the 6-well plate and add 1 ml of MEF medium to each well. 9. After centrifugation has stopped, remove the conical tube and aspirate the supernatant without disturbing the cell pellet. 10. Resuspend the cell pellet in sufficient MEF medium to a concentration of approximately 50,000 cells / ml. Add 11.1 ml of the cell / MEF medium mixture to each well. 12. Rotate the plate to distribute the MEF evenly, then place it in the incubator overnight. Thaw one 1 ml vial of 13,100 ug / ml L-521, add its contents to 9 ml of sterile PBS, and prepare 10 ug / ml L-521. 14. Filter the mixture through a 0.22 μm filter or SteriFlip. Add 1 ml of 10 ug / ml L-521 mixture to each well of a 15.6-well plate. 16. Wrap the plate in Parafilm and place it in a 37°C incubator for 2 hours. 17. Remove the plate from the incubator and place it in a refrigerator at 4°C. [Day 0 - Cell preparation of frozen PBMCs] 18. Thaw two frozen vials of PBMCs, each containing 5e6 cells, in a tank of water by moving them in a figure-eight pattern until small ice chunks remain. 19. Collect the contents of each tube into two separate sterile 15 ml conical tubes and label tubes 1 and 2. 20. Add sterile PBS to each tube to make a total volume of 10 ml. Mix the cells by inverting the tubes five times. Centrifuge at 21.1000 RPM for 15 minutes. 22. Proceed to step 25. [Cell preparation using newly isolated PBMCs] 23. Place the freshly isolated 3e6 PBMCs into two sterile 15 ml conical tubes, one for each. 24. Complete steps 19-22. [Reprogramming] [When using L-521 coated plates, allow the plates to equilibrate at room temperature for at least one hour.] 25. Prepare a 1.5 ml Eppendorf tube of Lonza Nucleofection solution according to the protocol (82 μl of Nucleofection solution + 18 μl of supplement per reaction). Label the tube with NS. [Note: Each reaction should be performed twice, once under T-cell conditions and once under non-T-cell conditions. A master mix of NS may be prepared for all reactions.] Prepare 3.83 ug of expression plasmid mixture for each reaction in a 26.1.5 ml Eppendorf tube as follows. i.pCXLE-hOCT3 / 4-shp53:0.83ug ii.pCXLE-hSK:0.83ug iii.pCXLE-UL:0.83ug iv.pCXWB-EBNA1:0.5ug v.pEP4 E02S ET2K:0.83ug Prepare a 15 ml conical tube containing 27.6 ml of T cell medium. Prepare a 15 ml conical tube containing 28.6 ml of non-T cell medium. 29. Aspirate the supernatant from one of the 15 ml conical tubes containing the cells. 30. Gently tap the tube to loosen the cell pellet. 31. Add 100 µl of NS mixture to the cell pellet and gently pipette 3-4 times. 32. The total volume of the NS / cell mixture is transferred to a 1.5 ml Eppendorf tube containing the plasmid mixture and mixed by gently pipetting 3-4 times. 33. Transfer the entire volume of the NS / cell / plasmid mixture to the glass cuvette provided in Lonza's T cell kit. 34. Place the cuvette into Amaxa nucleofector and run program V-024. 35. Using the plastic pipette tip included with the Lonza T cell kit, transfer a small amount of T cell medium to a glass cuvette, then transfer the entire contents of the cuvette to a 15 ml conical tube containing 6 ml of T cell medium. 36. Repeat steps 29-34 for the second 15 ml conical tube containing the cells, then proceed to step 37. 37. Using the plastic pipette tip included with the Lonza T cell kit, transfer a small amount of non-T cell medium to a glass cuvette, then transfer the entire contents of the cuvette to a 15 ml conical tube containing 6 ml of non-T cell medium. [Seeding] [MEF / ESC conditions for primates] 38. Remove the MEF medium from each well. 39. Rinse each well with 1 ml of sterile PBS. 40. Transfer the entire contents of the 15ml conical tube from step 35 by adding 2ml to each of the three wells at the top. 41. Add 5 µl of Dynabeads Human T-activator CD3 / CD28 to each well. 42. Transfer the entire contents of the 15ml conical tube from step 37 by adding 2ml to each of the three wells at the bottom. 43. Plate 37 o Place it in incubator C. [L-521 / E7 conditions] 44. Aspirate L-521 from each well. 45. Transfer the entire contents of the 15ml conical tube from step 5.35 by adding 2ml to each of the three wells at the top. 46. ​​Add 5 µl of Dynabeads Human T-activator CD3 / CD28 to each well. 47. Transfer the entire contents of the 15ml conical tube from step 37 by adding 2ml to each of the three wells at the bottom. 48. Plate 37 oPlace it in incubator C. [Day 2] 49. On day 2 after nucleofection, add 2 ml of appropriate reprogramming medium (Primate ESC + bFGF or E7) to each well. Do not aspirate anything from the wells. [Days 4-25+] From day 50.4 onwards, gently aspirate the culture medium from each well and add 2 ml of fresh Replagram medium to each well. Repeat this every other day. [Day 25+] 51. Use a stretched glass pipette to isolate individual colonies and transfer each colony to a single well in a 12-well dish containing appropriate culture medium.

[0064] Example 7 Various quality control assays can be performed to confirm the proper generation of pluripotent stem cells. Table 1 shows examples of such tests illustrating desirable characteristics of pSC generation.

[0065] (Table 1) iPSC QC assay TIFF0007943782000006.tif185170TIFF0007943782000007.tif76170

[0066] Example 8 Alkaline phosphatase For characterizing BC-iPSCs, alkaline phosphatase staining can be performed using Alkaline Phosphatase Staining Kit II (Stemgent, catalog number 00-0055) according to the manufacturer's instructions.

[0067] Example 9 Immunohistochemistry / cytochemistry For further characterization of BC-iPSCs, BC-iPSCs or differentiated cells can be placed on coverslips or optical-bottom 96-well plates (Thermo, #165305) and subsequently fixed with 4% paraformaldehyde. Intestinal organoids can be fixed with 4% paraformaldehyde, transferred to 30% sucrose, embedded in HistoPrep (Thermo Fisher Scientific), and cut into 20 μm sections. All cells are blocked with 510% goat or donkey serum containing 0.1% Triton X-100 and incubated with primary antibody for either 3 hours at room temperature or overnight at 4°C. Cells are then rinsed and incubated with species-specific AF488 or AF594 conjugated secondary antibody, followed by Hoechst 33258 (0.5 μg / ml, Sigma) for counterstaining the nuclei.

[0068] Antibodies suitable for immunocytochemistry and immunoblotting include (as listed by antigen, dilution, catalog number, isotype, and manufacturer): SSEA4, 1:250, MAB4304, mIgG3, Millipore; TRA-1-60, 1:250, 09-0010, IgM, Stemgent; TRA-1-81, 1:250, 09-0011, mIgM, Stemgent; OCT4, 1:250, 09-002 3. Rabbit IgG, Stemgent; NANOG, 1:250, 09-0020; Rabbit IgG, Stemgent; SOX2, 1:500, AB5603; Rabbit IgG, Millipore; TuJ1 (β3-tubulin), 1:1000, T8535; mIgG2b, Sigma; CDX2, 1:500, NBP1-40553; IgG, Novus; FABP2, 1:500, AF3078; IgG, R&D systems; Type I collagen, 1:500, 600-401-103-0.1, Rabbit Rockland; CD73, 1:500, 550257, mIgG1, BD Pharmingen; NKX6.1, 1:100 F55A10, mIgG1, DSHB Iowa; HB9, 1:25, 81.5C10, mIgG1, DSHB Iowa; ISELT1, 1:250, AF1837, goat IgG, R&D systems; SMI32, 1:1000, SMI-32R, mIgG1, Covance; CHAT, 1:250, AB144P, goat IgG, Millipore; SMN, 1:250, 610647, mIgG1, BD Biosciences; Cox-IV, 1:1000, 4850s, Rabbit Cell Signaling includes GAPDH, 1:1000, ab9484, mIgG2b, and Abcam.

[0069] Example 10 Flow cytometry Further characterization may involve flow cytometry. Specifically, BC-iPSCs are dissociated into a single cell suspension using Accutase (Millipore, #SCR005). Surface staining of the IPSCs is performed using SSEA4 (R&D Systems, FAB1435A).

[0070] Next, the cells were fixed, permeabilized, and intracellularly stained with Oct3 / 4 (BD Pharmingen, 560186). The recommended isotypes were used according to the antibody recommendations (R&D Systems, FABIC007 and BD Pharmingen, 562547). All samples were analyzed using a BD LSRFortessa flow cytometer with BD FACSDiva software. All images were generated using FloJo software.

[0071] Example 11 Karyotype Karyotyping can be performed as follows: Human BC-iPSCs are incubated with colsemid (100 ng / mL, Life Technologies) at 37°C for 30 minutes and dissociated using TrypLE for 10 minutes. These can then be washed with phosphate-buffered saline (PBS) and incubated in 5 mL of hypotonic solution (1 g KCl, 1 g sodium citrate in 400 mL of water) at 37°C for 30 minutes. The cells are centrifuged at 1500 RPM for 2.5 minutes and resuspended in fixative (methanol:acetic acid 3:1) at room temperature for 5 minutes. This process is repeated twice, and finally the cells are resuspended in 500 μl of fixative and submitted to the Cedars-Sinai Clinical Cytogenetics Core for G-band karyotyping.

[0072] Example 12 PluriTest Pluritest provides a robust pluripotency measurement. Total RNA can be isolated using the RNeasy Mini Kit (Qiagen) and subsequently measured with the Human HT-12 v4 Expression BeadChip Kit (Illumina). The raw data file (idat file) was then uploaded to the Pluritest widget online (www.pluritest.org).

[0073] Example 13 Quantitative RT-PCR Total RNA was isolated using the RNeasy Mini Kit (Qiagen), and cDNA was synthesized using 1 µg of RNA with the Promega transcription system. qRT-PCR was performed under standard conditions using specific primer sequences (Table 2). "CDS" refers to primers designed as coding sequences to measure the expression of endogenous genes only, while "Pla" refers to primers designed to measure the expression of plasmid-transfer genes only. Data are shown as mean ± SEM values.

[0074] (Table 2) qRT-PCR primer sequences TIFF0007943782000008.tif129148TIFF0007943782000009.tif237148

[0075] Example 14 Cytogenetic stability of PBMC-derived iPSC lines versus fibroblast-derived iPSC lines

[0076] (Table 3A) Cytogenetic stability of PBMCs TIFF0007943782000010.tif41170

[0077] (Table 3B) Cytogenetic stability of PBMCs TIFF0007943782000011.tif42170

[0078] Example 15 Cytogenetic stability of array CGH between PBMC-derived iPSC lines and fibroblast-derived iPSC lines

[0079] (Table 4A) Cytogenetic stability of PBMCs TIFF0007943782000012.tif58128

[0080] (Table 4B) Cytogenetic stability of PBMCs TIFF0007943782000013.tif52128 *Novels or deletions are amplifications or deletions newly acquired during reprogramming and / or during the growth of each iPSC strain, compared to the aCGH profile of the parent donor tissue.

[0081] Various results demonstrating the superior properties of BC-iPSCs, including those of specific cell lines, are shown. Table 5 shows the high success rate of episomal reprogramming based on peripheral blood mononuclear cells (PBMCs) in MEF. The chromosomal frequency of different types of karyotype abnormalities in individuals is observed by G-band karyotype analysis in fibroblast-derived iPSCs. "Rearrangement," "inversion," and "derivation" are shown in Table 6. Table 7 shows the success rate of blood-based reprogramming with human recombinant laminin 521 in standard E7 reprogramming medium. Table 8 shows the lack of success of blood-based episomal reprogramming on Matrigel substrate and limited E7 reprogramming medium.

[0082] (Table 5) High success rate of episomal reprogramming based on peripheral blood mononuclear cells (PBMCs) on MEFs TIFF0007943782000014.tif228139TIFF0007943782000015.tif228149TIFF0007943782000016.tif229126NP: Not implemented T: T-cell reprogramming method NT: Non-T cell reprogramming method 4P / 5P: Reprogramming factors on four or five plasmids

[0083] (Table 6) Frequency of different types of karyotype abnormalities TIFF0007943782000017.tif229146TIFF0007943782000018.tif22928

[0084] (Table 7) Success rate of blood-based reprogramming with human recombinant laminin 521 TIFF0007943782000019.tif229149TIFF0007943782000020.tif22937NP: Not implemented T: T-cell reprogramming method NT: Non-T cell reprogramming method 4P / 5P: Reprogramming factors on four or five plasmids

[0085] (Table 8) Relative lack of success in blood-based episomal reprogramming on Matrigel substrates TIFF0007943782000021.tif23059NP: Not implemented T: T-cell reprogramming method NT: Non-T cell reprogramming method 4P / 5P: Reprogramming factors on four or five plasmids

[0086] Example 16 PBMCs isolated by blood sampling are reliably reprogrammed into iPSCs. Dermal fibroblasts obtained from skin biopsies have been the most common source of iPSC reprogramming material due to their low cost and relatively easy handling. However, compared to blood sampling, skin punch biopsies can be 3-4 mm in size and are often painful procedures, requiring local anesthesia. Compared to fibroblasts derived from skin biopsies, blood is a more readily available outpatient tissue source and is therefore a preferred source tissue choice for iPSC reprogramming by both patients and clinicians. Various human blood reprogramming methods involve recruiting CD34 cells from peripheral blood. + CD34 is reported to be contaminated with cells and other admixtures (see reference). Unless isolated from umbilical cord blood, CD34 + Cells are typically isolated from the peripheral blood or bone marrow of patients undergoing G-CSF mobilization for several days. To produce a sufficient number of cells for reprogramming, isolated CD34 cells are used. + Progenitor cells need to be enriched and proliferated in culture using complex and expensive protocols. The inventors have developed a method for pre-cell proliferation using episomal reprogramming plasmids or CD34 +We successfully reprogrammed PBMCs (Primary Bone Microorganisms) immediately isolated from collected peripheral blood without cell isolation. This approach provides the fastest and most cost-effective procedure for obtaining reprogrammed iPSCs from a patient, from the time of biological sample collection to the generation of strictly characterized iPSCs.

[0087] Simultaneous reprogramming of large cohorts of PBMCs from multiple subjects and generation of iPSC lines considerably after patient blood collection makes it possible to "use iPSCs for the future," which requires a PBMC cryopreservation step to ensure the recovery of viable cells. Furthermore, if genotypic-phenotypic information from more patients is available, good cryopreserved PBMCs from a selective cohort of representative subjects can be converted to iPSCs, thus offering flexibility to the process. However, when cryopreserved PBMCs from multiple individuals were reprogrammed into iPSCs with episomal plasmids expressing POU5F1, SOX2, KLF4, LIN28, L-MYC, and p53shRNA, the inventors observed significant variability in isolating identifiable iPSC clones even after 35-40 days, regardless of PBMC cell type, ECM base, and culture medium. No clone iPSC lines were obtained using the non-T cell method with cryopreserved PBMCs, while the T cell reprogramming method was successful in only 33% of PBMC samples (data not shown).

[0088] Crucially, the reliability of PBMC reprogramming in our technique was significantly improved when we utilized: (a) two additional episomal plasmids containing SV40 large T antigen (SV40LT) and EBNA-1 in specific stoichiometric proportions to minimize PBMC cell death and maintain reprogramming factor plasmid expression; and (b) a prescribed reprogramming medium to promote high surface adhesion of nucleofection PBMCs (Figure 8A). This novel protocol resulted in the efficient generation of multiple adherent PBMC-iPSC clones that can be mechanically isolated and scaled up for proliferation 25–35 days after nucleofection (Figure 8A). Importantly, the success rate in reprogramming multiple individual cryopreserved PBMCs from non-disease controls or diseased patients was 90% with the T-cell method and 83% with the non-T-cell method in mouse embryonic fibroblasts (MEFs) (Table 5). Considering the limitations imposed by T cell-iPSCs and the low efficiency of non-T cell reprogramming from PBMCs, this method provides reliable reprogramming of non-T cell-derived BC-iPSCs.

[0089] The inventors tested whether this novel protocol was still applicable when using clinically more suitable reprogramming methods. Using recombinant human laminin 521 substrate and chemically defined reprogramming media, favorable PBMC reprogramming results were obtained, despite slightly lower success rates of 75% and 58%, respectively. The average reprogramming efficiencies for T cells and non-T cells were 2.2 × 10⁻⁶, respectively. -4 % and 1.6 × 10 -6The percentage was % (Table 5). All PBMC-iPSC lines exhibited typical PSC characteristics, including tightly packed colonies, a high nucleus-to-cytoplasmic ratio, robust alkaline phosphatase activity, and expression of pluripotent antigens (Figure 8B). BC-iPSCs passed the pluripotency quality control metric determined by the PluriTest assay, and the PBMC-iPSC transcription profile was demonstrated to be similar to well-established hESCs and fib-iPSCs, but not to differentiated fibroblasts and neural progenitor cells (Figure 8C). BC-iPSCs maintained a normal G-band karyotype (Figure 8D) and were confirmed to be either T cell or non-T cell clonal derivatives from PBMCs using T cell receptor (TCR)-β and -γ, and gene rearrangement / clonality assays (Figure 8E). The triseries differentiation potential of BC-iPSCs was demonstrated by spontaneous embryoid body formation and measurement of germ layer-specific gene expression profiles by the TaqMan Scorecard assay (Figure 8F). The "trace-free" state of BC-iPSCs was confirmed by demonstrating endogenous pluripotency gene expression and the absence of exogenous reprogramming transgenes using RT-qPCR (Figure 11A). Latent infection elements associated with the EBNA plasmid were ultimately eliminated from the established BC-iPSCs (Figure 11B).

[0090] Example 17 BC-iPSCs possess neuronal differentiation equivalent to that of fib-iPSCs. It has been reported that iPSCs, regardless of their source tissue, ultimately lose most of the gene expression and epigenetic profile associated with their original cell source. However, it remains unclear whether blood-derived iPSCs can differentiate into various other cell types with the same efficiency as fibroblast-derived iPSCs, likely due to the strong retention of epigenetic memory in blood-derived iPSCs. We addressed this by orienting mesoderm-derived blood or fibroblast-derived iPSCs into different germ layers, such as the ectoderm. Specifically, we performed neuroectoderm differentiation from a large number of fibroblasts (n=26) and PBMC lines (n=8) derived from both healthy volunteers and diseased patients. Based on immunocytochemistry and subsequent cell counts against the neuroectoderm markers TUBB3 (β3-tubulin) and NKX6.1, we showed that neuronal differentiation occurred with similar efficiency between fibroblast-iPSCs and BC-iPSCs (Figure 8G), further demonstrating the usefulness of BC-iPSCs.

[0091] Example 18 BC-iPSCs maintain a much more stable karyotype compared to fib-iPSCs. Recurrent chromosomal abnormalities have been previously described for an excessive number of hESC strains. While some reports document common chromosomal abnormalities in hiPSC strains, these do not systematically explain the variability in source tissue, reprogramming methods, and cell culture methods. To the best of our knowledge, there have been no systematic studies describing cytogenetic analyses comparing the frequencies of iPSCs derived from fibroblasts and blood. Over the past six years, we have performed conventional cytogenetic analyses on 104 iPSC strains derived from intrinsic fibroblasts or PBMC donors, including 339 independent clonal iPSC strains. All fibroblast and blood-derived iPSCs evaluated were reprogrammed using similar non-integrated episomal reprogramming and standard feeder-free Matrigel / mTeSR cell culture methods.

[0092] While fib-iPSCs and BC-iPSCs derived from iPSC Cores are similar to each other in terms of their reprogramming methods, pluripotency, and differentiation, the early observation of significant differences in cytogenetic stability between the two source cell types prompted the need for large-scale comparative analysis. Many laboratories and stem cell repositories routinely perform quality control assays for cytogenetic changes in PSCs using G-band karyotype analysis and Giemsa staining obtained at metaphase of chromosomes. This method, which easily achieves reliable identification of 5% abnormal cells, allows for the easy identification of small subpopulations of abnormal cells, enabling the detection of mosaicism (>10%) and balanced translocations at low resolution (>5-10 Mb).

[0093] The inventors performed karyotype analysis on 364 human iPSC cultures, from which source fibroblasts and PBMCs were collected from multiple laboratories or public storage facilities. The inventors' data reveal a significant difference in the incidence of chromosomal abnormalities between fib-iPSCs and BC-iPSCs. Of 258 cultures derived from clonally independent human fib-iPSC lines from 78 unique donors, 59 (22.9%) showed abnormal karyotypes with clonal abnormalities (Figure 6). In stark contrast, only 4 (3.8%) of 106 cultures from cloned human PBMC-iPSC lines from 32 unique donors showed low-frequency abnormalities. The more pronounced recurrent abnormalities in fib-iPSCs compared to BC-iPSCs are evident from the schematic chromosome diagrams in the ideograms (Figure 6). In fact, this remarkable cytogenetic stability is unique to PBMCs because iPSCs reprogrammed from other cell origins, including lymphoblastoid cell lineages (25%) and primary epithelial cells (27%), exhibit similarly high rates of karyotype abnormalities (Figure 12).

[0094] The most frequent karyotype abnormalities in fib-iPSCs were observed on chromosome 13 (13.2%), chromosome 1 (11.8%), chromosome 12 (11.1%), and chromosome X (10.4%) (Table 6). Less frequent karyotype changes were also observed on chromosome 21, chromosome 11, and chromosome 6, in descending order of frequency. Among aneuploidy, chromosome expansion (trisomy or duplication) was most commonly observed on chromosome 12 (21.1%), chromosome 1 (15.8%), chromosome 11 (13.2%), and chromosome 6 (10.5%), while chromosome reduction was repeatedly confirmed on chromosome X (38.5%) and chromosome 21 (23.1%). Translocation rearrangements were observed on chromosome 1, chromosome 13 (13.9%), and chromosome 14 (11.1%) in fib-iPSCs. One BC-iPSC strain exhibited chromosome augmentation at either chromosome 14 (trisomy) or chromosome 18 (duplication). The other two PBMC-iPSC strains showed rare abnormalities (translocation 46, XY, t(7;14)(q34;q11.2) and mosaic deletion 46, X, del(X)(q22q26) in 19% of cells (Table 6).

[0095] Example 19 The karyotype distribution of Fib-iPSCs and BC-iPSCs is related to the passage number, not the donor's age. Unlike blood, skin is routinely exposed to external environmental factors such as the possibility of sunburn, and biopsy-derived fibroblasts require a certain level of proliferation before reprogramming. This is similar to further aging in culture. Considering this, the inventors hypothesized that donor age may be a contributing factor influencing the cytogenetic instability of fib-iPSCs. However, the inventors found no significant trend or correlation between donor age and the frequency of cytogenetic abnormalities in fib-iPSCs (Figure 9A). In fact, based on G-band karyotype analysis, the highest percentage of karyotype abnormalities (31.9%) was observed in fibroblast-iPSCs from the 21-40 year old group.

[0096] Pluripotent stem cells possess high proliferative properties. Therefore, the inventors then attempted to analyze how fib-iPSCs and BC-iPSCs changed over time in cell culture and whether the tendency for cytogenetic abnormality accumulation increased with increasing passage number. Approximately 21.6% of fibroblast-derived iPSCs were observed to have abnormal karyotypes (more than 1 / 20 cells with clonal abnormalities) in the first G-band karyotype evaluation after iPSC generation, typically between passage numbers 4 and 23. In stark contrast, only 2.8% of BC-iPSCs had abnormal karyotypes in the initial evaluation. iPSC lines determined to have abnormal karyotypes were not cultured or evaluated further. Therefore, karyotypes in the 2nd to 4th repeats were evaluated only for iPSC lines that were initially identified as cytogenetically normal in their initial karyotypes. This analysis allowed us to determine whether fib-iPSCs possess a unique level of cytogenetic instability compared to BC-iPSCs during the duration of culture and passage, even after being established after normal iPSC generation (at the time of initial G-band karyotype evaluation). In G-band karyotype analysis, a larger proportion of fib-iPSCs (27.8%) acquired abnormal karyotypes in culture during repeated karyotype analyses compared to the proportion in the initial karyotype (P<0.001, two-way ANOVA with Bonferroni post-hoc test), while the proportion of abnormal karyotypes in BC-iPSCs did not show a significant difference between the initial and repeated karyotypes (Figure 9B). In analyzing the origin of fib-iPSC cytogenetic instability, the highest proportion of abnormal karyotypes (initial or repeated) appeared to occur between passages 10-23 of the iPSC line's lifespan (Figures 9C-D), which typically coincides with the time when the maximum amount of iPSC proliferation occurs for any given cell line. The significant increase in iPSC proliferation between passages 10 and 23 is largely due to characterization and cell banking occurring simultaneously around these passages. Nevertheless, although this is a similar process for both fib-iPS and BC-iPSCs, PBMC-derived cells do not show an increasing trend toward abnormal karyotypes during long-term culture or proliferation.

[0097] Example 20 BC-iPSCs acquire only slight amplification and virtually no loss. G-banding karyotypes have the advantage of being able to detect chromosomal locations of balanced translocations, inversions, culture mosaics of less than 20%, and genomic amplification or deletion commonly observed in fib-iPSC cultures (Figure 6 and Table 5). However, since G-banding karyotypes cannot detect very small genomic abnormalities (<5Mb), we decided to perform comparative genomic hybridization (aCGH) microarrays on subsets of fib-iPSC and PBMC-iPSC strains. Although aCGH cannot detect balanced translocations, inversions, and culture mosaics of less than 20%, it is an excellent auxiliary method for detecting smaller genomic amplification and deletion, copy number variations (CNVs), duplication / deletion, and unbalanced translocations in iPSC strains. We analyzed and recorded novel amplifications or deletions acquired in iPSCs in comparison with biological samples from parental fibroblast or PBMC sources. In this analysis, only amplifications and deletions newly acquired in iPSCs were considered if they were not normal CNVs in cross-reference with the Database of Genomic Variants (DGV), which includes genomic mutations observed in healthy individuals.

[0098] Including iPSC strains with abnormal and normal G-band karyotypes, the average size of amplifications and deletions detected by aCGH was significantly larger in fib-iPSC strains (44 Mb) compared to 2.1 Mb in PBMC-iPSC strains (Figure 10A), with the dominance attributed to fib-iPSC strains with abnormal karyotypes (Figure 10B). When size analysis comparisons were separated among iPSC strains with normal karyotypes, the average number of new amplifications / deletions was 2.31 Mb in fib-iPSC strains and 2 Mb in PBMC-iPSC strains. Supporting this data, the average number of new amplifications / deletions obtained overall in fib-iPSCs (3.7) was at least twice as high as in BC-iPSCs (1.8). Even in iPSC strains determined to have normal G-band karyotypes, the number of new amplifications / deletions was 1.8 in BC-iPSCs compared to 3.3 in fib-iPSCs. The most commonly acquired minute (0.8–1.5 Mb) new amplifications or deletions detected by aCGH in fib-iPSC or PBMC-iPSC strains were amplifications of chromosome 7q31.32 or deletions of chromosomes 10q15.2–q25.1, 16p11.2, and 21p11.2–p11.1 (Figure 10E).

[0099] Here, we report a novel and more reliable method for reprogramming the non-T cell component of blood using an episomal plasmid expressing a pluripotency factor. Using the described reprogramming protocol, non-T cells can be consistently reprogrammed from non-T cell or non-B cell raw materials with a near 100% success rate. Further advantages include the use of a specified reprogramming medium E7 and recombinant human L-521, a substrate with specified clinical compatibility. Blood cell-derived iPSCs ("BC-iPSCs") exhibit the same characteristics as fibroblast-derived iPSCs ("fib-iPSCs"), retaining their genotype, showing a normal pluripotency profile, and readily differentiating into all three germ layer cell types. This method for reliably inducing iPSCs from patient blood samples opens the way for the rapid generation of novel human iPSC lines, including patient-specific lineages, providing a vast bioresource for disease modeling, drug discovery, and regenerative medicine applications.

[0100] Example 21 Additional characterization tests G-band nucleotype Human iPSCs were incubated in colsemid (100 ng / mL, Life Technologies) at 37°C for 30 minutes, followed by dissociation using TrypLE for 10 minutes. These were then washed with phosphate-buffered saline (PBS) and incubated in 5 mL of hypotonic solution (1 g KCl, 1 g sodium citrate in 400 mL of water) at 37°C for 30 minutes. The cells were centrifuged at 1500 RPM for 2.5 minutes and resuspended in fixative (methanol:acetic acid 3:1) at room temperature for 5 minutes. This was repeated twice, and finally the cells were resuspended in 500 μl of fixative and submitted to the Cedars-Sinai Clinical Cytogenetics Core for G-band karyotype analysis. Analysis included chromosome testing of at least 20 cells per culture. Dataset 4 in this specification includes only abnormalities that conform to the ISCN 2013 definition of clonality. All normal and abnormal karyotypes are presented in tabular form. Next, ideograms of chromosomes were generated by importing a table into R Bioconductor (a programming language) using the IdeoViz package, which plots data related to arbitrary genomic intervals along chromosomal ideograms. This package is available on the open-source Bioconductor website. To accurately reflect the frequency of chromosomal rearrangements, the width of the bars in each ideogram was increased according to the number of samples examined. For example, the ideogram of fibroblasts showing the number of abnormalities observed in the sample set was 2.16 times larger than that of the PBMC set, so the bars showing abnormalities in PBMC-iPSC strains are 2.16 times wider than the bars showing abnormalities in fib-iPSC strains. The letters above the chromosomal translocation (dark blue bars) category are coded to correspond between the chromosomal locations where the translocation abnormalities occurred.

[0101] Copy number variations by array comparative genomic hybridization (aCGH) Array CGH is a high-resolution karyotype analysis solution for structural and numerical chromosomal variations due to its unbalanced detection capabilities and high throughput. High-quality genomic DNA was isolated from primary dermal fibroblasts or isolated PBMCs and their reprogrammed iPSCs. Genomic DNA quality was determined by UV spectroscopy (NanoVue), fluoroscopy (Qubit), and agarose gel analysis. Samples were analyzed for CNVs using the Agilent 60K Standard aCGH platform suitable for human stem cell lines and cancer cell lines by Cell Line Genetics (Madison, WI). Comparison of iPSC lines to source parental donor cells revealed novel copy number variations (increase / amplification or decrease / deletion) at the indicated loci.

[0102] TCRG Chain Rearrangement Assay Genomic DNA (350 ng) was recovered from all cell lines using the MasterPure DNA Purification Kit (Epicenter Biotechnologies). Embryonic stem cell line (H9) was used as a negative control. A primer set recognizing three framework regions at the heavy chain locus of the IgH gene was obtained from InVivoScribe Technologies (Cat No. 11010010, San Diego, CA), and PCR was performed according to the manufacturer's protocol. Genomic DNA (400 ng) for episomal plasmid-related gene analysis was recovered from all cell lines, and embryonic stem cell line (H9) was used as a negative control. In addition to GAPDH, which was used as a housekeeping gene, primers recognizing EBNA-1 were also included in this study. PCR was performed for 35 cycles of 30 seconds at 95°C, 30 seconds at 60°C, and 30 seconds at 72°C.

[0103] Neural differentiation from iPSCs Human fibroblast-iPSCs and PBMC-iPSCs were seeded in 6-well Matrigel-coated plates. The iPSCs were grown to near confluence under normal maintenance conditions before differentiation initiation. The following day, neuronal differentiation was initiated by double inhibition of SMAD and GSK-3beta using LDN193189 (0.2 μM, Cayman), SB431542 (10 μM, Cayman), and CHIR99021 (3 μM, Cayman) to induce neuroectoderm differentiation. This treatment was carried out for 6 days in Iskov-modified Dulbecco's medium (IMDM) / F12 (1:1) containing non-essential amino acids (NEAA; 1%), B27 (2%), and N2 (1%). After 6 days, differentiated cells were then gently detached by Accutase treatment at 37°C for 5 minutes. Next, 7.5 × 10⁶ units were added to the above-mentioned neuronal differentiation medium, which had been supplemented with 0.1 μM all-trans retinoic acid and 1 μM sonic hedgehog agonist (SAG). 5 Cells were seeded at a density in 6-well plates or 1 × 10⁴ cells in 96-well plates. The medium was changed to fresh medium every other day. On day 12, the differentiation medium was changed to terminal differentiation (IMDM) / F12 (1:1) medium supplemented with NEAA (1%), B27 (2%), N2 (1%), compound E (0.1 μM, Calbiochem), DAPT (2.5 μM, Cayman), retinoic acid (0.5 μM) all-trans, SAG (0.1 μM), ascorbic acid (200 ng / ml), dibutyryl cyclic adenosine monophosphate (1 μM), brain-derived neurotrophic factor (10 ng / ml), and glial cell line-derived neurotrophic factor (10 ng / ml). Differentiated neurons were fixed and analyzed on day 18 of differentiation.

[0104] Example 22 Cryopreserved PBMCs isolated from blood are reliably reprogrammed into iPSCs. Human iPSCs can be generated from non-proliferating PBMCs (Primary Birth Cells) that have just been isolated using episomal plasmids. However, there is no documentation on reprogramming frozen PBMCs into iPSCs. When the inventors reprogrammed frozen PBMCs from multiple individuals into iPSCs using episomal plasmids expressing POU5F1, SOX2, KLF4, LIN28, L-MYC, and TP53shRNA (referred to as the 4p method), they found that even after 35-40 days, the isolation of identifiable iPSC clones was either unsuccessful or showed significant mutability. Other attempts to generate iPSCs from whole blood have shown that isolated CD34+ progenitor cells need to be enriched and grown in culture using complex and expensive protocols to generate a sufficient number of cells for reprogramming. This highlights the need to simplify this process and reprogram while avoiding the proliferation of somatic cells. Because this somatic cell source provides the fastest and most cost-effective procedure from multiple target cohorts, we instead isolated and cryopreserved non-proliferating PBMCs for reprogramming into iPSCs. While no clonal iPSC lines were obtained using the non-T cell method with cryopreserved PBMCs, T cell reprogramming using the 4p method was successful in only 14.3% of the PBMC samples.

[0105] To enhance the efficiency of PBMC reprogramming, the inventors used: (a) an additional episomal plasmid (referred to as the 5p method) containing SV40 large T antigen (SV40LT) in a specific stoichiometric ratio to minimize PBMC cell death; and (b) a specified E7 reprogramming medium to promote high surface adhesion of nucleofected PBMCs. This novel protocol yielded favorable and efficient generation of multiple adherent PBMC-iPSC clones that can be mechanically isolated and scaled up for proliferation within 25–35 days after nucleofection. Importantly, the success and efficiency of reprogramming multiple cryopreserved PBMCs from non-disease controls or diseased patients were significantly higher with the 5p method (85% with the T-cell method and 92% with the non-T-cell method), in contrast to 0–15% with the 4p method. Reprogramming of PBMCs on mouse embryonic fibroblasts (MEFs) in commercially available culture media (primate ESC medium, PESC) was nearly 89–95% successful in both T-cell and non-T-cell methods, with good reprogramming efficiency. Because this may be required for clinically suitable iPSC-based cell therapies, the inventors then tested whether this 5p reprogramming protocol could also be adapted with xenobiotic-free components. Using recombinant human laminin 521 substrate and chemically defined reprogramming medium 1 (E7)21, T-cell and non-T-cell reprogramming was successful at 80% and 64%, respectively. In summary, these results demonstrate that the 5p method significantly enhances the success and efficiency of PBMC (both T-cell and non-T-cell) reprogramming compared to the 4p method, and provides a valuable protocol for reliably reprogramming cryopreserved PBMCs. Each PBMC-iPSC strain exhibited typical PSC characteristics, including densely packed colonies, a high nucleus-to-cytoplasmic ratio, robust alkaline phosphatase activity, and expression of multiple pluripotent antigens, all of which were evident in representative reprogrammed iPSC colonies derived from PBMCs of two healthy volunteers (03iCTR10 NTn1 from a non-T cell source and 80iCTR-Tn1 from a T cell source).PBMC-iPSCs passed the pluripotency quality control criteria determined by the PluriTest assay, and their transcriptional profiles were demonstrated to be similar to well-established hESCs and fib-iPSCs, but not to differentiated fibroblasts and neural progenitor cells. PBMC-iPSCs maintained a normal G-band karyotype and were confirmed to be either T-cell or non-T-cell clonal derivatives from PBMCs using T-cell receptor (TCR)-β and -γ and gene rearrangement / clonality assays. The triseries differentiation potential of PBMC-iPSCs was demonstrated by spontaneous embryoid body formation and measurement of germ layer-specific gene expression profiles using the TaqMan Scorecard assay. The absence of transgenes in the genome of PBMC-iPSCs is confirmed by (1) evidence that latent elements related to the EBNA plasmid were ultimately eliminated from the established PBMC-iPSCs and were not detected in the genomic DNA, and (2) the absence of any exogenous reprogramming transgenes when endogenous pluripotency genes are expressed and RT-qPCR is used. Furthermore, the SV40LT factor, a component of the 5p reprogramming mixture, was not detected at the genomic DNA level and was not expressed in the reprogrammed PBMC-iPSC strains.

[0106] Example 23 PBMC-iPSCs possess neuronal differentiation equivalent to that of Fib-iPSCs. Many early reports suggested that newly reprogrammed iPSCs retain epigenetic memory of their parental donor cell type, but recent reports suggest that hiPSCs, regardless of the source tissue, ultimately lose most of the gene expression and epigenetic profile associated with their original cell source after proliferation. However, it remains unclear whether blood-derived iPSCs can differentiate into various other cell types with the same efficiency as fibroblast-derived iPSCs, perhaps because blood-derived iPSCs retain a stronger epigenetic memory compared to other donor cells. We addressed this by orienting iPSCs derived from blood or fibroblasts, both of which originate from the mesoderm, to a different germ layer, namely the neuroectoderm. Specifically, we performed neuroectoderm differentiation from iPSC lines derived from numerous fibroblasts (n=26) and PBMC lines (n=20) from both healthy subjects and patients with diseases. Immunocytochemistry and subsequent quantification of the neuroectoderm markers TUBB3 (β3-tubulin) and NKX6.1 demonstrated similar efficiency in neuronal differentiation between fib-iPSCs and PBMC-iPSCs, further demonstrating the usefulness of PBMC iPSCs. It has been previously described for hESC strains that PBMC-iPSCs reprogrammed at 5p maintain significantly more stable karyotype recurrent chromosomal abnormalities. While some reports also document common chromosomal abnormalities for hiPSC strains, these do not systematically explain the variability in source tissue, reprogramming methods, and cell culture methods. To our knowledge, there have been no systematic studies describing cytogenetic analyses comparing the frequencies of iPSCs derived from fibroblasts and blood. We performed detailed cytogenetic analyses of iPSC strains derived from 171 unique donors, consisting of 16 fibroblasts (77) or PBMCs (94). Since each donor had multiple clonal lines, this included karyotypes derived from 348 iPSC lines of independent clonal fibroblasts (205) or PBMCs (143). All evaluated fibroblasts, lymphoblastoid cell lines, epithelial and hematopoietic iPSCs were reprogrammed using a similar non-integrated episomal reprogramming method.The main difference between the fibroblast and PBMC reprogramming reported here is the number of reprogramming factor plasmids used: 3p31 for fibroblasts and 4p or 5p for PBMCs. After successful reprogramming, all iPSC lines analyzed and reported here were maintained using a feeder-free Matrigel® / mTeSR1® cell culture method and passaged using the mechanically operated StemPro® EZPassage® tool. To establish the genetic stability of these lineages, the inventors first performed G-band karyotype analysis on 552 human iPSC cultures derived from fibroblasts (260), PBMCs (206), epithelial cells (26), and lymphoblastic cell lines (60) collected from multiple laboratories or public storage. The inventors' data show a remarkably low incidence of chromosomal abnormalities in PBMC iPSCs. Of 260 cultures derived from clonally independent human fib-iPSC lines from 77 unique donors, 61 (23.5%) exhibited clonal abnormalities and abnormal karyotypes. Typically, the fibroblasts had proliferated for 9–10 passages before reprogramming. In stark contrast, of 206 cultures from clonal human PBMC-iPSC lines derived from 94 unique donors, only 4 (1.9%) showed any arbitrary karyotype abnormalities. The four abnormalities observed were also infrequent. Recurrent abnormalities are more pronounced in fib-iPSCs compared to PBMC-iPSCs, as is evident from schematic diagrams of chromosomal ideograms. Indeed, this remarkable cytogenetic stability is unique to PBMCs reprogrammed with the novel 5p method because iPSCs reprogrammed from other cell origins, including lymphoblastoid cell lineage (25%, 15 abnormalities out of 60) and primary epithelial cells (27%, 5 abnormalities out of 26), exhibit high karyotypic abnormality rates similar to those seen in fibroblasts. The most frequent karyotypic abnormalities in fib-iPSCs were observed on chromosome 13 (14.9%), chromosome 1 (11.7%), chromosome 12 (9.6%), and chromosome X (8.5%). Less severe karyotypic changes were also observed on chromosomes 21, 11, and 6.Interestingly, karyotype abnormalities were never detected in chromosomes 2, 5, and 7 of fib-iPSCs. Of the aneuploidy, chromosome increase (trisomy or duplication) was most commonly observed in chromosome 12 (20.0%), chromosome 1 (15.0%), and chromosome 13 (12.5%), while chromosome decrease was repeatedly confirmed in chromosome X (38.5%) and chromosome 21 (23.1%). Structural rearrangements, including translocations, inversions, and derived chromosomes, recurred in chromosome 13 (22.0%), chromosome 1 (12.2%), and chromosome 14 (9.8%) in fib-iPSCs. In stark contrast, only two PBMC-iPSC strains derived from the 178 iPSC cultures analyzed showed chromosome increase in either chromosome 14 (trisomy) or chromosome 18 (duplication). The other two PBMC-iPSC strains showed unusual abnormalities (translocation 46,XY,t(7;14)(q34;q11.2) and mosaic deletion 46,X,del(X)(q22q26) in 19% of cells).

[0107] Example 24 The enhanced karyotype stability of PBMC-iPSCs is independent of donor age and passage number. Unlike blood, skin is routinely exposed to external environmental factors such as the possibility of sunburn, and biopsy-derived fibroblasts require a certain level of proliferation before reprogramming. This is similar to further aging in culture. Considering this, the inventors hypothesized that donor age may be a contributing factor influencing the cytogenetic instability of fib-iPSCs. G27 band karyotype analysis was used to assess this possibility. There was no clear trend or correlation between increasing donor age and the frequency of cytogenetic abnormalities in fib-iPSCs. However, the results confirmed that PBMC-iPSCs have much higher karyotype stability than fib-iPSCs in all donor age groups. Except for the 0-20 year old donor group, PBMC-iPSCs were significantly more stable than fib-iPSCs in all donor groups between 21 and 90 years old. To establish how iPSC proliferation in culture may affect genetic stability, the inventors then analyzed fib-iPSCs and PBMC-iPSCs over time. Approximately 22.4% of fibroblast-derived iPSCs were observed to have abnormal karyotypes (more than 1 / 20 cells with clonal abnormalities) in the first G-band karyotype assessment after iPSC generation, typically between passages 1 and 23. In stark contrast, only 1.4% of PBMC-derived iPSCs had abnormal karyotypes in the first assessment (p<0.0001). iPSC lines identified as having abnormal karyotypes in the first karyotype assessment were not cultured or evaluated further. Therefore, karyotypes in the 2nd to 4th repeats were evaluated only for iPSC lines that were initially identified as cytogenetically normal in those first karyotypes. Compared to the proportion of initial karyotypes, the proportion of fib-iPSCs (27.3%) that acquired abnormal karyotypes in culture tended to increase with repeated karyotype analyses and was significantly higher than that of PBMC-iPSCs (p<0.001). Surprisingly, the rate of abnormal karyotypes in PBMC-iPSCs did not show a significant difference between the first and repeat karyotypes. Analysis of the causes of cytogenetic instability in fib-iPSCs suggests that a high incidence of abnormal karyotypes (first or repeat) occurred between passages 1 and 23 during the survival period of the iPSC strain.This passage range is typically when the maximum amount of iPSC proliferation occurs for any given cell line, and is largely due to characterization and cell banking that occur simultaneously around these passage numbers. Nevertheless, this is a similar process for both fib-iPSCs and PBMC-iPSCs, and more importantly, PBMC-derived cells reprogrammed by our novel method do not show an increasing trend toward abnormal karyotypes during long-term culture or proliferation. Since G-band karyotypes cannot detect minute genomic abnormalities (<5Mb), PBMC-iPSCs that acquire sub-ultramicroscopic amplification and deletions were then subjected to array comparative genomic hybridization (aCGH) microarrays for subsets of fib-iPSC and PBMC-iPSC strains. While aCGH cannot detect balanced translocations, inversions, and culture mosaics in less than 20% of cases, it is an excellent auxiliary method for detecting smaller-sized genome augments and decrements, copy number variations (CNVs), duplications / deletions, and unbalanced translocations in iPSC strains 11, 30, 32, and 33. The inventors analyzed and recorded novel amplifications or deletions acquired in iPSCs compared to biological samples from parental fibroblast or PBMC sources. This analysis considered only those amplifications and deletions newly acquired in iPSCs if they were not normal CNVs in cross-reference to a Database of Genomic Variants containing genomic mutations observed in healthy individuals.

[0108] Including iPSC strains with abnormal and normal G-band karyotypes, the average size of amplifications and deletions (amplification / deletion) detected by aCGH was significantly larger in fib-iPSC strains (44 Mb) compared to 2.1 Mb in PBMC-iPSC strains, with the dominance attributed to fib-iPSC strains with abnormal karyotypes. When size analysis comparisons were separated between iPSC strains with normal karyotypes, the average number of new amplifications / deletions was 2.31 Mb in fib-iPSC strains and 2 Mb in PBMC-iPSC strains. Supporting this data, the average number of new amplifications / deletions obtained overall in fib-iPSCs (3.7) was significantly higher (twice as high) than in PBMC-iPSCs (1.8). Even in iPSC strains determined to have normal G-band karyotypes, the number of new amplifications / deletions was 1.8 in PBMC-iPSCs compared to 3.3 in fib-iPSCs. The most commonly acquired minute (0.8–1.5 Mb) new amplifications or deletions detected by aCGH were amplifications of chromosome 7q31.32 or deletions of chromosomes 10q15.2–q25.1, 16p11.2, and 21p11.2–p11.1. In summary, these data demonstrate that this novel method of reprogramming PBMCs yields more stable human iPSCs in terms of the acquisition of chromosomal abnormalities compared to any other published method.

[0109] Example 25 Additional characterization of the 5p method

[0110] (Table 9) More PBMCs are being reprogrammed into iPSCs. TIFF0007943782000022.tif47128

[0111] The inventors performed further analysis to confirm whether the p53 (TP53) of the PBMC-iPSC strains was reprogrammed using the inventors' method. These strains were reprogrammed on the L521 substrate in a specified E7 medium.

[0112] The inventors conducted extensive analyses of multiple iPSC strains. Based on whole-genome sequencing (of another manuscript in preparation), the inventors identified no variants (SNPs or indels) in the exons (coding regions) of the TP53 gene at the genomic level in any of the iPSC strains.

[0113] Whole-genome sequencing was performed using 750 ng of genomic DNA according to the Illumina TruSeq PCR-free library preparation kit (Illumina, San Diego, CA). Library quality was verified using BioAnalyzer (Agilent Technologies, Santa Clara, CA), and the libraries were sequenced to at least 40x read count using Illumina Hi-Seq10X at Fulgent Therapeutics (Temple City, CA). An average of over 460 million reads were generated per sample. The Fastq files were aligned to the reference genome when constructing hg38 using the Burroughs-Wheeler Aligner BWA-mem algorithm with over 400 million reads, resulting in good alignment for each sample, which generated an average of 43x read count across the entire genome.

[0114] Using this pipeline, the inventors did not identify any variants (SNPs or indels) in the coding region (exon) of TP53 that occur only in the iPSC strain or its clones. Among the multiple iPSC strains and their clone derivatives analyzed, a novel single-intron SNP was detected in two of the three iPSC clones derived solely from one donor iPSC strain, rs12940247 (hg38 chr17:7,664,385 G / A), located in intron 1 of a single (and rare) isoform of TP53. This common SNP was found at a minor allele (G) frequency of 0.403 in the reference population of Caucasian samples (the donor lineage iPSC strains were derived from Caucasian donors) and in at least 36% of the entire population assayed. This common variant is not involved in any function or is annotated in typical clinical annotation databases used to assign potential effects of cancer variants (CiVIC, ClinVar, COSMIC Cancer Consensus). There is no evidence that this variant acts as a regulator of TP53 expression levels in healthy tissues in the Gene Tissue Expression (GTEx) dataset. The variant rs12940247 does not cross-reference with any regulatory function in Tier 1 cell lines (including GM12878, a lymphoblast cell line derived from PBMCs). We also used the Illumina Omni 2.5M genotyping array to characterize these samples. Consistent with whole-genome sequencing results, no variants were detected in iPSC clones that did not match the genotype of germline (blood) samples, and no copy number variations were identified across this locus.

[0115] (Table 10) The non-coded common variant rs12940247 is observed as a new phenomenon in two clones derived from donor CS-002. TIFF0007943782000023.tif59165

[0116] The location of the SNP in intron 6 of one isoform of TP53 is shown by a gray line. TP53 isoforms are shown in black (corresponding to function in the Protein Data Bank) or dark blue (transcripts investigated or validated by either RefSeq or SwissProt) in GENCODE v24 Comprehensive. Below the gene annotation are four tracks showing transcription, active promoter (neither observed in the plotted genomic intervals), and active enhancer (tracks 3 and 4, again, neither observed in the plotted genomic intervals). Several stable regulatory elements are observed within the gene body of TP53 in HUVEC cells (represented as blue peaks, indicating enrichment of the H3K4me1 signal by ChIP-Seq). There is no evidence for the observed untranslated region variants that arise in the two clones from donor iPSCs with any regulatory function of TP53.

[0117] A simplified method: Germline variant detection was performed using the Genome Analysis Toolkit (GATK) with Best Practices for Germline Variant Detection. Briefly, aligned sequences were pre-processed with Picard, and duplicate reads (derived from a single fragment of DNA, which can be generated during sample preparation or when using cluster amplification during the sequencing process) were identified by the sequence of the 5' end of the reads. Before identifying variants, the base quality score was recalibrated after local genomic rearrangements around in-dels to improve genotyping accuracy (a process heavily dependent on the base quality score). Then, reliable variant detection was achieved by recalibrating variant detection with the joint genotyping module using the detected single nucleotide polymorphisms (SNPs) and in-dels.

[0118] In conclusion, based on a very detailed and extensive analysis of multiple iPSC strains and clones at the SNP and WGS levels, we have not detected any mutations in the coding region of the TP53 gene that would disrupt its normal regulation.

[0119] (Table 11) The non-coded common variant rs12940247 is observed as a new phenomenon in two clones derived from donor CS-002. TIFF0007943782000024.tif218119

[0120] Example 26 Consideration In this study, the inventors demonstrated that human iPSCs (PBMC-iPSCs) induced by a novel method from non-proliferating and cryopreserved PBMCs were significantly more cytogenetically stable than iPSCs generated from other somatic cells (including dermal fibroblast cultures, LCL, epithelial cells, and adipocytes) that require cell proliferation before reprogramming. These PBMC-iPSCs reprogrammed using the 5p method also exhibited a higher stability ratio compared to other pluripotent stem cell lines, such as hESCs and iPSCs, previously reported in other laboratories. All of this research, involving over 200 unique donors, identical stem cell maintenance procedures, pluripotency characterization, and episomal plasmid repranging of various cell types by karyotype analysis, was conducted in the inventors' laboratory and at the Cytogenetics Core. The inventors systematically analyzed over 200 individual clonal PBMC-iPSC lines generated in the inventors' laboratory using both lymphoid T cell and myeloid non-T cell populations that reliably reprogram regardless of substrate and medium. Furthermore, the inventors demonstrated the stable production of PBMC-iPSCs using xeno-free recombinant human L521 substrate and a defined Reprograng medium. Most importantly, PBMC-iPSCs reprogrammed by the 5p method maintain karyotype stability during long-term culture. In summary, this improved technique, combined with non-proliferating cell sources such as PBMCs, provides both a clinically suitable reprogramming method and easy availability for a large number of patient samples.

[0121] In our laboratory, aneuploidy and rearrangement of chromosomes 1, 12, 13, and X were the most frequent abnormalities in fib-iPSCs generated by episomal reprogramming. In contrast, these abnormalities were never detected in any PBMC-iPSCs generated and analyzed by us. Some of these changes observed in fib-iPSCs are similar to recurrent abnormalities seen in human ESCs, which involve an increase or decrease in the short arm of chromosome 12 and an increase or decrease in chromosomes 1, 17, and X, and are similar to their malignant human embryonic carcinoma (EC) stem cell counterparts. Using aCGH microarrays, PBMC-iPSCs showed half as many novel CNVs compared to fib-iPSCs. These novel CNVs were not shared between hiPSCs and their respective parent fibroblasts or PBMCs and are likely acquired during reprogramming or iPSC proliferation. The average base pair size of chromosomal changes in CNVs in fib-iPSCs is also much larger compared to PBMC-iPSC strains, which further supports the stability of this novel reprogramming method.

[0122] The increased karyotype abnormalities, coding mutations, and minor genomic alterations previously reported in iPSCs likely reflect the mutagenic effects of the reprogramming procedures themselves. However, most of these reported genomic abnormalities have been tested in iPSC lines generated from dermal fibroblasts using embedded retroviral and lentiviral reprogramming methods. Aneuploidy rates and CNVs comparing different non-embedded and lentiviral reprogramming methods for low-passage iPSCs have been described. While aneuploidy rates of approximately 12–13% have been reported in low-passage iPSCs using episomal and embedded reprogramming methods, this data mainly includes abnormality rates in iPSCs derived from proliferating fibroblast cultures and is not separated by donor cell origin or obtained from non-proliferating cells such as PBMCs. Various factors, including oxygen partial pressure, growth adjuvants, growth factors, passaging techniques, cryopreservation, and extracellular matrix for cell proliferation, can affect the genetic stability of PSCs during long-term culture and proliferation.36 Considering these aforementioned factors, while there is often substantial laboratory variability in PSC culture techniques, all of these factors were strictly controlled in the inventors' research. The Progenitor Cell Biology Consortium (www.synapse.org) confirmed the correlation between gene expression and CNVs between major developmental and oncogenic regulators using donor lineage stability, reprogramming techniques, and origin cell results. However, karyotype aberrations and line stability were not distinguished in this study by origin cells and their previous cell proliferation history, and only a small number of abnormal fib-iPSCs were analyzed.

[0123] The methylation and transcriptional profiles of iPSCs were somewhat differentiated based on their originating blood and fibroblast cells, but these were not directly attributable to the somatic cell epigenetic memory profiles. In this group as well, a tendency for higher levels of CNVs in lineages generated using embedded programming methods was observed. More recently, another group has shown that blood cells and induced iPSCs are much closer to true hiPSCs and ESCs in their methylation profiles (higher methylation state) compared to any other parental tissue or their iPSCs with more abnormal DNA methylation. This supports our hypothesis that cell populations not previously grown in tissue culture, such as adult PBMCs or umbilical cord blood, may be preferred choices for reprogramming and maintaining cytogenetic stability at the iPSC stage.

[0124] Compared to iPSCs generated from proliferating somatic cell types such as dermal fibroblasts, LCLs, epithelial cells, and adipose stem cells, the underlying mechanisms of genetic stability in iPSCs derived from non-proliferating PBMCs are likely multifactorial. Firstly, the bone marrow niche allows for blood turnover more frequently than almost any other cell type in the body (1-5 days for most cells in PBMC strains). Therefore, host blood may be considered far less "aged" than endogenous dermal fibroblasts, epithelial cells, or adipocytes, which regenerate over months to years. In particular, reprogramming donor-derived dermal fibroblasts, LCLs, epithelial cells, and adipocytes requires prolonged ex vivo somatic cell proliferation in tissue culture. These factors lead to an initial pooling of cells accompanied by increased cellular stress and aging associated with tissue culture, and thus a reduced DNA repair capacity. Therefore, cultures of previously grown somatic cells may begin with overall heterogeneity and inherent genotypic instability, and low levels of mosaicism may remain undetectable in the conventional G-band karyotype in bulk xenocellular cultures before reprogramming. Subsequently, fib-iPSC, LCL-iPSC, or epithelial-iPSC strains may be isolated in which low levels of karyotypic abnormalities become significant during clonal growth and expansion. In our method, this risk is avoided because the isolated PBMCs have no prior period of cell growth and reprogramming begins immediately after isolation. In conclusion, the 5p reprogramming protocol for PBMCs includes an additional episomal plasmid, and one of the reprogramming factors, SV40LT, can also enhance the stability of PBMCs during the stressful reprogramming period by minimizing PBMC cell death and promoting surface adhesion. The use of SV40LT as a component of the reprogramming factor mixture has also been shown in the past to be non-transformative because the transgene is not incorporated into the genome of the iPSC strain induced by episomal plasmid reprogramming, and this is also supported by our data.While somatic cell types such as pre-grown fibroblasts may exhibit superior genetic stability when reprogrammed with the 5p protocol, we believe that this single difference in reprogramming between pre-grown and non-grown PMBCs does not fully explain the significant difference in karyotype stability. Importantly, apart from additional factors, all iPSCs from pre-grown and non-grown blood cell types were reprogrammed using the same non-integrated episomal reprogramming method and maintained using the same feeder-free Matrigel® / mTeSR1® cell medium and manual StemPro® EZPassage® tools. Nevertheless, if our novel 5p protocol facilitates the reprogramming of additional cell types other than non-grown PBMCs, it could represent a significant advance to the field, and ongoing research is evaluating fibroblast reprogramming using the optimized protocol. A first-of-its-kind human iPSC clinical trial organized by RIKEN using iPSCs from patients with age-related macular degeneration was discontinued because single nucleotide variants (SNVs) and cellular nucleotide variants (CNVs) were detected in the patient's iPSC lines that were not found in the patient's original fibroblasts. All CNVs were single-gene deletions, but one SNV had been previously identified in cancer-related somatic mutation databases.6,16 This highlights the crucial importance of a stable karyotype for the successful clinical application of human iPSCs. Furthermore, scaling up iPSCs and derivatives for predictive toxicology and high-throughput drug screening for therapeutic drug discovery require a source of iPSCs with substantial cellular stability. This optimal reprogramming method in non-proliferating PBMCs minimizes the impact of acquired genomic abnormalities. These results demonstrate that optimized reprogramming techniques, as well as the quality and duration of pre-culture before reprogramming, are crucial for long-term iPSC line stability. This data will help improve the safety of iPSC-derived cell products for clinical and drug discovery research applications, and enhance reproducibility in iPSC disease modeling studies.

[0125] The various methods and techniques described above provide many means for carrying out the present invention. Naturally, it should be understood that not all objectives or benefits described may necessarily be achieved according to any particular embodiment described herein. Therefore, for example, a person skilled in the art will recognize that it may be possible to carry out the invention in a manner that achieves or optimizes one or more benefits as taught herein, without necessarily achieving other objectives or benefits as taught or suggested herein. Various advantageous and disadvantageous alternatives are referred to herein. It will be understood that some preferred embodiments include one, another, or several advantageous features, while others particularly mitigate the current disadvantages by specifically eliminating one, another, or several disadvantages, while simultaneously incorporating one, another, or several advantageous features.

[0126] Furthermore, those skilled in the art will understand the applicability of various features from various embodiments. Similarly, the various elements, features, and steps described above, as well as other known equivalents of each such element, feature, or step, can be combined and adapted by those skilled in the art to carry out methods according to the principles described herein. Some of the various elements, features, and steps may be included in particular in various embodiments, while others may be excluded in particular.

[0127] While the present invention is disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that embodiments of the present invention extend beyond the scope of the specifically disclosed embodiments to other alternative embodiments, and / or their uses, modifications, and equivalents.

[0128] Many variations and alternatives are disclosed in embodiments of the present invention. Further variations and alternatives will be apparent to those skilled in the art. Among these variations are, but are not limited to, sources of blood cells, cellular components of blood, pluripotent stem cells derived therefrom, techniques and compositions relating to induced pluripotent stem cells derived from blood cells, techniques and compositions relating to cell differentiation, biomarkers relating to such cells, and specific uses of products produced by teachings of the present invention. Various embodiments of the present invention may specifically include or exclude any of these variations or elements.

[0129] In some embodiments, numerical values ​​representing characteristics such as the amount, concentration, and reaction conditions of components, used to describe and claim certain embodiments of the present invention, should be understood to be modified in some cases by the term "approximately." Therefore, in some embodiments, the numerical parameters described in the description and appended claims are approximations that may vary depending on the desired characteristics to be obtained by the particular embodiment. In some embodiments, numerical parameters should be interpreted in light of the number of significant figures reported and by the application of ordinary rounding methods. Despite the numerical ranges and parameters representing a wide range of embodiments of the present invention being approximations, the numerical values ​​shown in a particular example are reported as accurately as possible. In some embodiments of the present invention, the presented numerical values ​​may include certain errors that inevitably arise from the standard deviation observed in each test measurement.

[0130] In some embodiments, the terms “a,” “an,” and “the” and similar references used in the context of describing specific embodiments of the Invention (particularly in the context of the following claims) may be interpreted as covering both singular and plural forms. Details of value ranges in this Specified are intended solely as abbreviations to refer individually to each individual value that falls within the range. Unless otherwise indicated herein, each individual value is incorporated into the Specified as if it were individually detailed herein. All methods described herein may be performed in any suitable order unless otherwise indicated herein or unless it is clearly inconsistent with the context. Any and all examples or illustrative words (e.g., “etc.”) provided relating to a particular embodiment of this Specified are merely for the purpose of better illustrating the Invention and do not limit the scope of the Invention as otherwise claimed. No word herein should be interpreted as indicating any unclaimed element essential to the practice of the Invention.

[0131] The grouping of alternative elements or embodiments of the present invention disclosed herein should not be construed as limiting. Members of each group may be referenced and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group may be included in or removed from a group for convenience and / or patentability reasons. In the event of such inclusion or omission, this specification shall be deemed to include the modified group and thus satisfy the description of all Markush groups used in the appended claims.

[0132] Preferred embodiments of the Invention, including the best methods known to the inventors for carrying out the Invention, are described herein. Variations of these preferred embodiments will become apparent to those skilled in the art by reading the preceding description. Those skilled in the art will find that such variations can be used as needed, and that the Invention can be carried out in other ways other than those specifically described herein. Accordingly, many embodiments of the Invention include all modifications and equivalents of the subject matter described in the appended claims, as permitted by applicable law. Furthermore, unless otherwise indicated herein or clearly rejected by context, any combination of the above elements in all possible variations is incorporated into the Invention.

[0133] Furthermore, numerous references are made throughout this specification to patents and publications. Each of the above cited references and publications is incorporated into this specification in its entirety by reference.

[0134] Finally, it should be understood that the embodiments of the present invention disclosed herein are illustrative of the principles of the present invention. Other modifications that can be used may also be within the scope of the invention. Therefore, alternative forms of the invention can be used in accordance with the teachings herein, not as examples but as examples. Thus, embodiments of the present invention are not limited to those illustrated and described in detail.

[0135] Sequence information SEQUENCE LISTING <110> CEDARS-SINAI MEDICAL CENTER <120> A NOVEL AND EFFICIENT METHOD FOR REPROGRAMMING BLOOD TO INDUCED PLURIPOTENT STEM CELLS <150> US 15 / 184,241 <151> 2016-06-16 <160> 48 <170> PatentIn version 3.5 <210> 1 <211> 23 <212> DNA <213> Homo sapiens <400> 1 ccccagggcc ccatttttggt acc 23 <210> 2 <211> 26 <212> DNA <213> Homo sapiens <400> 2 acctcagttt gaatgcatgg gagagc 26 <210> 3 <211> 20 <212> DNA <213> Homo sapiens <400> 3 cattcaaact gaggtaaggg 20 <210> 4 <211> 22 <212> DNA <213> Homo sapiens <400> 4 tagcgtaaaa ggagcaacat ag 22 <210> 5 <211> 24 <212> DNA <213> Homo sapiens <400> 5 ttcacatgtc ccagcactac caga 24 <210> 6 <211> 26 <212> DNA <213> Homo sapiens <400> 6 tcacatgtgt gagaggggca gtgtgc 26 <210> 7 <211> 24 <212> DNA <213> Homo sapiens <400> 7 ttcacatgtc ccagcactac caga 24 <210> 8 <211> 22 <212> DNA <213> Homo sapiens <400> 8 tttgtttgac aggagcgaca at 22 <210> 9 <211> 24 <212> DNA <213> Homo sapiens <400> 9 accatcctt cctgcccgat caga 24 <210> 10 <211> 24 <212> DNA <213> Homo sapiens <400> 10 ttggtaatgg agcggcggga cttg 24 <210> 11 <211> 23 <212> DNA <213> Homo sapiens <400> 11 ccacctcgcc ttacacatga aga 23 <210> 12 <211> 22 <212> DNA <213> Homo sapiens <400> 12 tagcgtaaaa ggagcaacat ag <210> 13 <211> 26 <212> DNA <213> Homo sapiens <400> 13 gcgaaccccaa gcccaggcc tgctcc <210> 14 <211> 25 <212> DNA <213> Homo sapiens <400> 14 cagggggtct gctcgcaccg tgatg <210> 15 <211> 21 <212> DNA <213> Homo sapiens <400> 15 ggctgagaag aggatggcta c <210> 16 <211> 22 <212> DNA <213> Homo sapiens <400> 16 tttgtttgac you are at <210> 17 <211> 25 <212> DNA <213> Homo sapiens <400> 17 agccatatgg tagcctcatg tccgc <210> 18 <211> 30 <212> DNA <213> Homo sapiens <400> 18 tcaattctgt gcctccggga gcagggtagg 30 <210> 19 <211> 25 <212> DNA <213> Homo sapiens <400> 19 agccatatgg tagcctcatg tccgc 25 <210> 20 <211> 22 <212> DNA <213> Homo sapiens <400> 20 tagcgtaaaa ggagcaacat ag 22 <210> 21 <211> 21 <212> DNA <213> Homo sapiens <400> 21 cctggaggag aagaggaaag a 21 <210> 22 <211> 21 <212> DNA <213> Homo sapiens <400> 22 ttgaggacct ctgtgtattt in the 21 <210> 23 <211> 21 <212> DNA <213> Homo sapiens <400> 23 tgctgtctcc atgtttgatg t 21 <210> 24 <211> 21 <212> DNA <213> Homo sapiens <400> 24 tctctgctcc ccacctctaa g 21 <210> 25 <211> 21 <212> DNA <213> Homo sapiens <400> 25 atcagggcca agacatagag a 21 <210> 26 <211> 20 <212> DNA <213> Homo sapiens <400> 26 gccaatgcaa cttggacgtt 20 <210> 27 <211> 24 <212> DNA <213> Homo sapiens <400> 27 catagaagaa gaagaggatg aaga 24 <210> 28 <211> 24 <212> DNA <213> Homo sapiens <400> 28 gtagggattc gagggaatta ctga 24 <210> 29 <211> 19 <212> DNA <213> Homo sapiens <400> 29 atggaacacg accttgaga 19 <210> 30 <211> 20 <212> DNA <213> Homo sapiens <400> 30 tgagcaggat gaggtctagg 20 <210> 31 <211> 20 <212> DNA <213> Homo sapiens <400> 31 cacctcaacc tggagacaat 20 <210> 32 <211> 20 <212> DNA <213> Homo sapiens <400> 32 tgaagcaggc gtggtttcaa 20 <210> 33 <211> 20 <212> DNA <213> Homo sapiens <400> 33 tcgggggtgt tagagacaac 20 <210> 34 <211> 20 <212> DNA <213> Homo sapiens <400> 34 ttccacgagg gtagtgaacc 20 <210> 35 <211> 20 <212> DNA <213> Homo sapiens <400> 35 accacagtcc atgccatcac 20 <210> 36 <211> 20 <212> DNA <213> Homo sapiens <400> 36 tccaccaccc tgttgctgta <210> 37 <211> 20 <212> DNA <213> Homo sapiens <400> 37 tccttctacg gacggaactg <210> 38 <211> 20 <212> DNA <213> Homo sapiens <400> 38 agaaatgcct 20 years <210> 39 <211> 20 <212> DNA <213> Homo sapiens <400> 39 20. gattcctcct ccaccctcac <210> 40 <211> 23 <212> DNA <213> Homo sapiens <400> 40 caatattctg cctggcctgg atg <210> 41 <211> 21 <212> DNA <213> Homo sapiens <400> 41 21. ccacacccac tcagagccat <210> 42 <211> 20 <212> DNA <213> Homo sapiens <400> 42 caccccacca ccaaaacctt 20 <210> 43 <211> 24 <212> DNA <213> Homo sapiens <400> 43 cgagaggacc ccgtggatgc agag 24 <210> 44 <211> 24 <212> DNA <213> Homo sapiens <400> 44 ggcggccatc ttcagcttct ccag 24 <210> 45 <211> 28 <212> DNA <213> Homo sapiens <400> 45 gaatgctgca aactgaccac gctggaac 28 <210> 46 <211> 28 <212> DNA <213> Homo sapiens <400> 46 tggcattcaa gagggttttc agtctgga 28 <210> 47 <211> 18 <212> DNA <213> Homo sapiens <400> 47 ctatcatgct ggctgcct 18 <210> 48 <211> 20 <212> DNA <213> Homo sapiens <400> 48 ctacaacacc cttctcacag 20

Claims

1. A blood cell-derived induced pluripotent stem cell (BC-iPSC) line, The BC-iPSC strain is a T cell and is derived from non-proliferating peripheral blood mononuclear cells (PBMCs) containing a T cell receptor gene rearrangement, or the BC-iPSC strain is a non-T cell and is derived from non-proliferating peripheral blood mononuclear cells that do not contain a T cell receptor gene rearrangement. The aforementioned BC-iPSC strain, To deliver a reprogramming factor containing a certain amount of EBNA1 and Oct-4, Sox-2, Klf-4, l-Myc, Lin-28, SV40 large T antigen ("SV40LT"), and p53-targeting short hairpin RNA ("shRNA-p53") to isolated non-proliferating peripheral blood mononuclear cells, and The isolated non-proliferating peripheral blood mononuclear cells are cultured in reprogramming medium for at least four days. This includes, and is generated by the process, By delivering EBNA1 and reprogramming factors and culturing them in reprogramming medium, induced pluripotent stem cells derived from blood cells are generated. The aforementioned reprogramming factor is encoded in one or more oriP / EBNA1-derived plasmid vectors, The BC-iPSC strain produced by the above process has an abnormal karyotype in 5% or less of cases over 4 to 8 passages, or the BC-iPSC strain does not show a statistically significant difference between the initial and repeated G-band karyotype analyses, and the initial or repeated G-band karyotype analysis occurs between 10 and 23 passages. The aforementioned BC-iPSC strain.

2. The BC-iPSC strain according to claim 1, wherein the T cell receptor gene is T cell receptor β (TCRβ), T cell receptor γ (TCRγ), or both.

3. The BC-iPSC strain according to claim 1, which is a T cell and is derived from a non-proliferating PBMC containing a T cell receptor gene rearrangement.

4. The BC-iPSC strain according to claim 3, wherein the T cell receptor gene is T cell receptor β (TCRβ), T cell receptor γ (TCRγ), or both.

5. The BC-iPSC strain according to claim 1, which exhibits a lower incidence of genomic abnormalities compared to iPSC strains produced from fibroblast-derived iPSCs (Fib-iPSCs).

6. The BC-iPSC strain according to claim 1, which exhibits an abnormal karyotype of less than 5% after 4 to 8 passages.

7. The BC-iPSC strain according to claim 1, which exhibits an abnormal karyotype of less than 2.8% after 4 passages.

8. The BC-iPSC strain according to claim 1, wherein there is no statistically significant difference between the initial and repeated G-band karyotype analyses, and the initial or repeated G-band karyotype analysis occurs between passages 10 and 23.

9. A BC-iPSC strain according to claim 1, having a disease mutation.

10. The BC-iPSC strain according to claim 9, wherein the disease mutation is associated with neurodegenerative diseases, disorders, and / or conditions.

11. The BC-iPSC strain according to claim 9, wherein the disease mutation is associated with inflammatory bowel disease, disorder, and / or condition.

12. The BC-iPSC strain according to claim 1, wherein the non-proliferating PBMC is a mononuclear cell that is non-T cell and non-B cell.

13. The BC-iPSC strain according to claim 1, wherein delivering a certain amount of reprogramming factor comprises nucleofection.

14. The BC-iPSC strain according to claim 1, wherein the plasmid vector is pEP4 E02S ET2K, pCXLE-hOCT3 / 4-shp53-F, pCXLE-hSK, pCXLE-hUL, and pCXWB-EBNA1.

15. The BC-iPSC strain according to claim 1, wherein the ratio of SV40LT to EBNA1 is 0.83 μg of pEP4 E02S ET2K vector encoding SV40LT to 0.5 μg of pCXWB-EBNA1 vector encoding EBNA1.

16. The aforementioned process, Prepare a certain amount of non-proliferating peripheral blood cells. The fractionation of a certain amount of non-proliferating peripheral blood cells, and To isolate a layer of fresh, non-proliferating peripheral blood mononuclear cells from the aforementioned fractionated peripheral blood cells. The BC-iPSC strain according to claim 1, further comprising:

17. The aforementioned reprogramming factor is A first vector encoding Oct4, Sox2, SV40LT and Klf4, A second vector encoding Oct4 and shRNA-p53, A third vector encoding Sox2 and Klf4, and A fourth vector encoding l-Myc and Lin-28 The BC-iPSC strain according to claim 1, encoded by four oriP / EBNA1-derived plasmid vectors containing the above.

18. The BC-iPSC strain according to claim 17, wherein the fifth vector encodes EBNA1.

19. The BC-iPSC strain according to claim 18, wherein the first, second, third, fourth, and fifth vectors are pEP4 E02S ET2K, pCXLE-hOCT3 / 4-shp53-F, pCXLE-hSK, pCXLE-hUL, and pCXWB-EBNA1, respectively, and the ratio of SV40LT to EBNA1 that minimizes isolated non-proliferating peripheral blood mononuclear cell (PBMC) death is 0.5–1.0 μg of the first vector encoding SV40LT to 0.25–0.75 μg of the fifth vector encoding EBNA1.

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