We have attempted to generate iPSCs from T lymphocytes in peripheral blood of rhesus monkeys via reprogramming with Sendai virus (SeV) vectors carrying OCT3/4, KLF4, SOX2, c-MYC, LIN28, and SV40 large T antigen as we have previously reported (11, 17). As expected, the combinatorial addition of an ERK1/2 inhibitor, PD0325901, and a GSK3β inhibitor, CHIR99021, during the reprogramming phase resulted in the generation of iPSCs from T cells obtained from independent monkeys (Figure 1A). Pluripotency of rhesus T-iPSCs (rhT-iPSCs) was confirmed in vitro by their morphology (Figure 1B), alkaline phosphatase staining (Figure 1C), and immunohistochemical staining for NANOG (Figure 1D). Following subcutaneous injection into immunodeficient mice, rhesus T-iPSCs formed a teratoma containing all three germ layers (Figures 1E, G). A total of eight SeV transgene-free clones derived from two animals (R496 and R557) were selected for further experiments (Table 1). Finally, these Rh-iPSCs were confirmed to be the normal karyotype (Figures 1H, I).

In order to generate T cells, rhT-iPSCs were induced to differentiate into the hematopoietic lineage by coculturing with a murine cell line, C3H10T1/2, supplemented with VEGF and BMP4 for the first 7 days, and SCF and FLT3L for the next 7 days as shown in Figure 2A. On the 14th day after induction, differentiating cells containing CD34⁺CD45⁺ cells were harvested and seeded onto another murine cell line, OP9-DLL1, in the presence of FLT3L and IL-7 to induce T-cell differentiation (Figure 2B). Flow cytometric analysis of day 21 after T-cell differentiation demonstrated that majority of the differentiating cells expressed CD3 and approximately 10% of the cells expressed both CD4 and CD8β (Figure 2C), indicating that rhT-iPSCs successfully differentiated into T cells with the protocol similar to T-cell differentiation from human iPSCs (11, 18). As summarized in Table 1, we observed some interclonal variations in T-cell differentiation efficiency. After differentiating the eight transgene-free iPSCs, we selected R557–1 and R496–14 for further analysis.

Resulting cells from day 21 after T-cell differentiation were activated to induce proliferation by coculturing them with inactivated human peripheral blood mononuclear cells (PBMCs) with added phytohemagglutinin (PHA) (Figure 2A). RhiPSC-T cells derived from R496–14 and R557–1 clones proliferated on averages of 59-fold (R496-14) and 38-fold (R557-1), respectively, in 14 days after activation and could be activated again in the next round of activation (Figure 2D). At the end of two rounds of activation and proliferation, rhiPSC-T cells from the R496–14 clone expressed CD3, but lost CD4 and CD8β expressions (Figure 2E). Nonetheless, nearly half of the rhiPSC-T cells from the R557–1 clones were CD3⁻ at this stage (Figure 2E). RhiPSC-T cells from both clones showed a CCR7⁻CD62L⁻ phenotype similar to human effector T cells (Figure 2F). In contrast to human iPSC-derived T cells, they expressed high levels of CD103 and CD69 (Figure 2G). These findings collectively indicate that regenerated rhiPSC-T cells exhibit an immunophenotype similar to innate-like tissue-resident T cells, which is inconsistent with iPSC-T cells differentiated from human T-iPSCs (11, 12, 19).

To evaluate the antigen-dependent in vitro effector functions of rhiPSC-T cells, we transduced them with CD20 CAR and performed cytotoxic assays using PBMCs obtained from autologous monkeys (Supplementary Figure S1A). As shown in Supplementary Figure S1B, we confirmed that CD20 CAR rhiPSC-T cells recognized and eliminated autologous B cells within PBMCs (Supplementary Figure S1B). Cytotoxicity of CD20 CAR rhiPSC-T cells was lower than that of rhesus primary CAR-T (Supplementary Figure S1C). Moreover, rhiPSC-T cells killed K562 cells, demonstrating their TCR-independent NK-cell-like cytotoxicity similar to human iPSC-T cells (Figure 2H).

Finally, we conducted a series of in vivo experiments to understand safety and in vivo cellular kinetics of regenerated T cells using the two original monkeys (Table 2) (Figure 3A). In order to distinguish infused cells from recipient cells, we retrovirally transduced rhiPSC-T cells with EGFP. We evaluated EGFP expressions during in vitro culture and found their stable expression (Figure 3B). All transplantation studies were carried out with over 8.4 × 10⁷ cells/injection. Approximately 1% of EGFP⁺ cells, which represents infused rhiPSC-derived T cells, were detected in PBMCs at 6 h after transplantation and they subsequently disappeared from the blood by day 7 (Figure 3C). Flow cytometric analysis of bone marrow and lymph nodes of the recipients did not show the presence of EGFP⁺ cells (Supplementary Figure S2). Given that rhiPSC-T cells expressed tissue-resident markers CD103 and CD69, we examined if rhiPSC-T cells could be detected in non-lymphoid tissues. Semi-quantitative PCR using genomic DNA obtained from R496 monkey tissues showed the presence of EGFP⁺ cells in the lung (Figure 3D). To further confirm the presence of iPSC-T cells in the lung, paraffin-embedded lung sections and cytospin samples of bronchoalveolar lavage fluid were prepared and stained with hematoxylin–eosin (HE) and an anti-GFP antibody (Figures 3E, F). Microscopic analysis revealed that GFP-positive cells were present in alveolar spaces of the recipients (Figure 3F). Transduction of CD20 CAR and preconditioning of recipient R557 prior to infusion did not improve peripheral blood cellular kinetics (Table 2 and Figure 3C). CD20 CAR rhiPSC-T-cell administration did not induce B-cell reduction in blood samples (Supplementary Figures S3A, B). These results indicate that rhiPSC-T cells had short in vivo persistence and actively migrated to alveoli.

To assess the side effect of rhiPSC-T cell infusion, we monitored body weight and body temperature (Figure 4). Hematological and biochemical analyses were also performed (Figure 5 and Supplementary Table S1). Body weight did not change during the study, except for the third infusion to R557, which included preconditioning (Figure 4A). Body temperature remained the normal range of between 39°C and 37.5°C, except in one measurement where the R557 monkey showed transient decrease around day 195 (Figure 4B). No remarkable changes in complete blood counts that are categorized to grade III to IV adverse effects were observed. We did not notice notable changes in white blood cell counts, except for the first infusion to R557 (Figure 5A). Red blood cell counts and hemoglobin concentrations remained constant (Figures 5B, C). A sudden rise in platelet count was observed after 2 weeks of the third infusion to R557 (Figure 5D). All changes were transient and recovered by 8 weeks after infusion. Biochemical tests with plasma samples indicated no signs of abnormal elevations or drops in reference to rhesus reference values (20) (Supplementary Tables S1–S4). Resected organs (brain, lung, heart, thymus, liver, spleen, pancreas, kidney mesentery, and intestine) were histologically assessed for tumorigenesis. Although lymphocyte aggregates that were GFP⁻ were found in hepatic sinusoid, there was no evidence for tumorigenesis in all tissues examined by rhiPSC-T cells (Supplementary Figures S4A, B).

Two rhesus macaques (Macaca mulatta) were used in this study: one is female (R496, 10 years old) and the other is male (R557, 6 years old). Experiments were carried out at the Institute for Frontier Life and Medical Sciences, Kyoto University after approval by the Committee on the Ethics of Animal Experiments in Kyoto University (permission numbers: B12-1–3 and B12-1-4). Cell transfusion, blood collection, tissue biopsy, and necropsy were performed under ketamine anesthesia. Euthanasia of the rhesus macaques was performed by administering sodium pentobarbital (Kyoritsu Seiyaku) at a dose of 4.212 mg/kg, followed by exsanguination via cardiac puncture.

We used 4- to 8-week-old immunodeficient NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ (NSG). Mice experiments were performed in accordance with the institutional regulations approved by Kyoto University. For euthanasia of mice, CO₂ was administered at a displacement rate of 30%–70% of the chamber volume per minute.

Collected blood was centrifuged at 1,700 rpm for 25 min. The buffy coat was collected and diluted with D-PBS (−) (Nacalai, Japan). Diluted buffy coats were put onto Ficoll-Paque PLUS (GE healthcare) and centrifuged at 540g for 25 min. The middle layer was collected as PBMCs.

Mouse embryonic feeder cells (MEFs) were seeded 4 × 10⁶ cells on a 10-cm dish in MEF medium [Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum (FBS) and 1% L-glutamine–penicillin–streptomycin (PSG) (Sigma)]. The next day, MEF culture medium was added with mitomycin C (Wako) (fc 10 μg/mL). MEFs were incubated for 90 min at 37°C in a 5% CO₂ incubator. Medium was removed. Inactivated MEFs were washed with D-PBS (−) three times to remove Mitomycin C. MEF medium was added to MEFs and incubated for 2 h at 37°C in a 5% CO₂ incubator. MEF medium was removed and MEFs were washed with D-PBS (−). 0.05% Trypsin-EDTA (Sigma) was added to MEFs and incubated for 5 min at 37°C in a CO₂ incubator. MEF medium was added into MEFs to stop reaction. MEFs were corrected into a centrifugation tube and centrifuged at 1,200 rpm for 5 min. Supernatant was discarded. MEFs were resuspended with DMEM medium. MEFs were seeded at 3 × 10⁵ cells/dish to a 0.1% gelatin (Sigma)-coated six-well plate.

Rhesus iPSCs were established from T cells as previously described (17). T cells were stimulated by α-NHP-CD3/2/28 antibody-coated beads (Miltenyi Biotec) at a 1:1.5 ratio in R-Medium [RPMI-1640 (Sigma) with 10% FBS and 1% PSG]. Mixture was rotated at approximately 6–10 rpm at room temperature for 45 min. Bead-conjugated cells were separated by a magnet. Separated T cells were suspended with T-cell medium [RPMI-1640 supplemented with 10% FBS, 1% PSG, 0.01 mM 2-mercaptoethanol (Invitrogen), and 100 U/mL recombinant human IL-2 (PeproTech)]. T cells were seeded at 5 × 10⁵ cells/mL in 2 mL of T-cell medium on six-well plates. After 4 days of stimulation, 2 × 10⁶ activated cells were collected in a 15-mL tube and centrifuged at 1,500 rpm for 5 min. Supernatant was removed. T-cell pellets were dissociated by tapping. T-cell suspension was supplemented with 3 MOI of reprogramming factors (Klf4, Oct3/4, Sox4, and c-Myc) and SV40 large T antigen expression SeV vectors. T cells were incubated for 2 h at 37°C. Infected T cells were washed with R-medium and centrifuged at 1,500 rpm for 5 min. Supernatant was removed. Infected cells were suspended in T-cell medium and seeded on inactivated MEFs. Medium was gradually replaced with 2i medium {Dulbecco’s modified Eagle’s medium/F12 (Sigma) supplemented with 20% knockout serum replacement (KSR) (Thermo Fisher Scientific), 1% PSG, 1% nonessential amino acid (Thermo Fisher Scientific), 100 μM 2-mercaptoethanol (Thermo Fisher Scientific), 5 ng/mL basic fibroblast growth factor [bFGF] (Wako; Japan), 1 μM PD0325901 [ERK1/2 inhibitor] (Wako; Japan), and 3μM CHIR99021 [GSK3b inhibitor]} (Tocris). The established iPSC clones were transduced with small interfering RNA L527 (11) and L1913 using Lipofectamine RNAiMAX Reagent (Invitrogen).

Rh-iPSCs were cultured using a 6-cm dish in 3 mL of 2i medium. Medium was replaced every day and passaging was performed 1 day per week.

2i medium was removed from the dish. Rh-iPSCs were incubated with 1 mL/dish dissociation solution [D-PBS (−) (Nacalai; Japan) with 0.25% Trypsin (Invitrogen), 20%KSR, 10 mM CaCl₂] at 37°C in a 5% CO₂ incubator for 5 min. After removing the dissociation solution, 1 mL/dish 2i medium was added into the dish. Rh-iPSC clusters were broken up into small cell clumps. Small clumps were seeded onto inactivated MEFs in 2i medium.

Alkaline phosphatase (AP) staining was performed with Alkaline Phosphatase Substrate Kit II (Vector Laboratories, Inc.).

Manufacturer’s protocol was used for AP staining. Stained samples were exposed by a microscope.

Rh-iPSCs were washed with D-PBS (−). Fixative solution was added and incubated at room temperature for 30 s. Fixative solution was removed. Fixed Rh-iPSCs were washed two times with rinse buffer. Staining solution was added and incubated at room temperature for 15 min in the dark. Samples were washed two times with rinse buffer. Samples were filled with D-PBS (−).

To fix the rh-iPSCs, half volume of medium was removed from the iPSC culturing well and the same volume of 4% paraformaldehyde was added. iPSCs were fixed for 30 min at 4°C. Fixing solution was removed. Blocking buffer (0.1% Triton X-100 and 10% Donkey serum) was added and incubated for 30 min at room temperature. Diluted goat anti-NANOG IgG (R&D:AF1997) was added after removing the blocking buffer. Samples were incubated overnight at 4°C. Primary antibody was removed and Donkey anti-goat IgG (H+L) conjugated with Alexa Fluor 488 (Thermo Fisher; A11055) was added. Samples were incubated for 1.5 h at room temperature. After staining, samples were washed with D-PBS (−) and exposed by a fluorescence microscope.

2i medium was removed from the dish. One milliliter of 0.05% Trypsin-EDTA was added to the dish and incubated at 37°C for 5 min. Trypsin-EDTA was removed from the dish. D-PBS (−) (1 mL) with 2% FBS was added to the dish. Rh-iPSCs were dissociated by pipetting. Rh-iPSCs were collected into a centrifugation tube and centrifuged at 1,200 rpm for 5 min. Supernatant was removed. iPSCs were suspended with cold D-PBS (−) to 2 × 10⁷ cells/mL. Suspended Rh-iPSCs were mixed with Matrigel in a 1:1 ratio. Cell suspension (200 μL) was injected into 4- to 8-week-old immunodeficient NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ (NSG) subcutaneously under isoflurane anesthesia by a 27G needle-connected syringe. At approximately 2 cm of teratoma diameter or after 11 weeks, teratomas were excised and fixed with neutral buffered formalin at 4°C overnight. The next day, neutral buffered formalin was exchanged with 70% ethanol.

Samples were cut and embedded with paraffin. Paraffin-embedded samples were sectioned and stained by HE staining.

Embedding and HE staining were performed at the Center for Anatomical, Pathological and Forensic Medical Researches (Kyoto University).

To check residual SeV genome, RNAs were eluted from Rh-iPSCs clones using an RNeasy mini kit (QUIAGEN). cDNAs were synthesized from eluted RNA by a High-Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific). For amplification of the SeV genome-specific sequence, PCR was performed using primers Fw: 5&-AGACCCTAAGAGGACGAAGA-3& and Rv: 5&-ACTCCCATGGCGTAACTCCATAGTG-3& and ExTaq HS (TAKARA). The PCR condition has three steps (dissociation step: 94°C at 2 min, amplification step: 20 s at 94°C; 40 cycles and elongation step: 5 min at 72°C). The PCR products were electrophoresed in agarose gel.

To arrest Rh-iPSCs at metaphase, Colcemid (Invitrogen) was added to 2i medium at a final concentration of 250 ng/mL. Rh-iPSCs were incubated at 37°C in a 5% CO₂ incubator for 2 to 3 h. Rh-iPSCs were dissociated with the same method for the passage. The dissociated Rh-iPSCs were collected into a centrifugation tube and centrifuged at 200g for 5 min. Supernatant was removed. The iPSCs pellet was suspended with 7 mL of 0.075 M KCl and incubated at room temperature for 20 min. Carnoy’s fixative (7 mL; mixture of methanol and glacial acetic acid in a 3:1 ratio) was slowly added to the cell suspension, followed by gentle mixing. The suspension was centrifuged at 200g for 5 min, and the supernatant was removed. The cells were washed twice with 10 mL of Carnoy’s fixative and then centrifuged at 200g for 5 min. After the final centrifugation and removal of the supernatant, the cell pellet was resuspended in 5 mL of Carnoy’s fixative and stored at −20°C. Fixed sample was analyzed by researchers at the Tottori Bioscience Promotion Foundation, Japan.

RNA extractions were performed with an RNeasy mini kit. A total of 1 × 10⁶ cells were harvested from Rh-iPSCs. Rh-iPSCs were suspended with 350 μL of Buffer RLT and the same volume of 70% ethanol was added. Samples were put to the RNeasy mini column. Columns were centrifuged at 10,000 rpm for 15 s. Columns were added with 350 μL of RW1 and centrifuged at 10,000 rpm for 15 s. DNase mixture was applied on columns and incubated for 15 min. Columns were washed with 350 μL of RW1 and centrifuged at 10,000 rpm for 15 s. Columns were added with 500 μL of RPE supplemented with ethanol and centrifuged at 10,000 rpm for 15 s. Columns were added with 500 μL of RPE supplemented with ethanol and centrifuged at 10,000 rpm for 2 min. Collection tube was exchanged for a new one. Columns were centrifuged at 15,000 rpm for 1 min. RNase-free water was added to the column and centrifuged at 10,000 rpm for 1 min. Eluted RNA solutions were stored at −80 °C.

cDNAs were synthesized from eluted RNA by a High-Capacity cDNA reverse Transcription kit. RNA (13.2 μL) was mixed with 2 μL of 10× RT buffer, 0.8 μL of dNTP Mix, 2 μL of RT Random Primers, 1 μL of MultiScribe Reverse Transcriptase, and 1 μL of RNase Inhibitor. Mixed samples were incubated at 25°C for 10 min, at 37°C for 120 min, and at 85°C for 5 min. Samples were stored at −30°C.

DLL1 overexpressing OP9 were cultured in Minimum Essential Medium α (α-MEM) (Invitrogen) supplemented with 15% FBS and 1% PSG. C3H10T1/2 were cultured in Basal Medium Eagle (Invitrogen) supplemented with 10% FBS and 1% PSG. 293GP, MEFs, and FLY-RD18 were cultured in MEF medium. These cells were cultured at 37°C in an atmosphere having a 5% CO₂ incubator.

To dissociate iPSCs, iPSCs were incubated with dissociation solution at 37 °C in a 5% CO₂ incubator for 5 min. After removing the dissociation solution and MEFs, the remaining iPSCs clusters were broken up into large cell clumps. iPSC clumps were seeded on C3H10T1/2 feeder cells in HSPC differentiation medium composed of Iscove’s modified Dulbecco’s medium (Sigma) supplemented with 20% FBS, 1% PSG (Sigma), 1% Insulin-Transferrin-Selenium solution (Thermo Fisher Scientific), 450 μM monothioglycerol (Nacalai), 50 μg/mL ascorbic acid 2-phosphate (Nacalai), and 20 ng/mL VEGF (R&D Systems) under 5% O₂ atmosphere. BMP4 (20 ng/mL; R&D Systems) was added until day 4. After day 7, HSPC differentiation medium was supplemented with 20 ng/mL SCF (R&D Systems) and 10 ng/mL Flt3L (PeproTech). HSPCs were harvested on day 14. HSPCs were seeded on OP9DL1 feeder cells with T-cell differentiation medium [α-MEM with 15% FBS, 1 ng/mL IL7 (PeproTech), and 10 ng/mL Flt3L]. T-cell lineage redifferentiated cells were harvested on day 35.

iPSC-T cells were cocultured with irradiated human PBMCs at a 1:20 ratio in expansion medium (RPMI-1640 supplemented with 0.01 mM 2-ME, 10 ng/mL IL-7 and 10 ng/mL IL-15) with 2 μg/mL PHA-P (Wako). The next day, iPSC-T cells were washed two times with D-PBS (−) to exclude PHA-P. iPSC-T cells were maintained with the expansion medium. Half the medium was changed every 2–3 days with re-plating as needed. Stimulation was repeated after the almost 2 weeks of stimulation.

All flow cytometry staining was performed with 2% FBS containing D-PBS (−) buffer on ice. PI was added to all samples before analysis. LSR II and FACS Aria II (BD Biosciences) were used for flow cytometry analysis. The following monoclonal antibodies were used for flow cytometry analysis: APC-CD34 (clone 563; BD Biosciences), BV510-rhesus CD45 (clone D058-1283; BD Biosciences), APC-CD3 (clone SP34-2; BD Biosciences), BV421-CD4 (clone OKT4; BioLegend), PerCPcy5.5-CD8α (clone SK1; BioLegend), PEcy7-CD8β (clone SIDI8BEE; eBioscience), PE-CD62L (clone SK11; BD Biosciences), APC-CCR7 (clone G043H7; BioLegend), PEcy7-CD56 (clone HCD56; BioLegend), APC-CD103 (clone 2G5; BECKMAN), APCcy7-CD69 (clone FN50; BioLegend), BV421-CD20 (clone 2H7; BD Biosciences), APC-CD161 (clone HP-3G10; BioLegend), and BV421-PD-1 (clone EH12.2H7; BioLegend). Data were analyzed with FlowJo (Tree Star).

293GP cells were seeded on a poly-L-Lysine (Sigma)-coated 10-cm dish. The next day, 3 μg of VSV-G vector and 3 or 6 μg of retrovirus vector were diluted with 500 μL of HBSS (−). Twenty-four microliters of 1 mg/mL polyethylenimine (PEI) (Polysciences Inc.) was added to the vector solution. The solution was mixed vigorously by voltex and incubated for 15 min at room temperature. The mixed solution was added to a 293GP culturing dish and incubated for 16 h in a 5% CO₂ incubator. After incubation, medium was exchanged with MEF medium with 10 μM Forskolin (Sigma). After 48 h, the supernatant was collected as viral supernatant.

At first, VSV-G envelope retrovirus was produced from plasmid-transduced 293GP using PEI. VSV-G retrovirus supernatant was passed through a 0.45-μmφ PVDF syringe filter. FLY-RD18 retrovirus packaging cells were cultured with VSV-G retrovirus supernatant. Retrovirus-infected FLY-RD18 constitutively produce retrovirus having an RD114 envelope (RD virus). Infected FLYRD18 cells were sorted with FACS Aria II. RD virus supernatant was passed through a 0.45-μmφ PVDF syringe filter. RD virus was attached on a retronectin (TAKARA)-coated well. Activated iPSC-T cells were cultured on an RD virus-coated well to infect retrovirus.

To check the cross-reactivity of anti-human CD19 CAR and anti-human CD20 CAR, each CAR-expressing iPSC-T cell was cocultured with purified rhesus B cells at a ratio of 1:10 in RPMI-1640 supplemented with 10% FBS, 5 ng/mL IL-7, and 20 U/mL IL-2 for 13 h at 37°C in a 5% CO₂ incubator. Activated rhiPSC-T cells make clusters. Activated iPSC-T cells were exposed by a fluorescence microscope.

PBMCs were centrifuged at 1,200 rpm for 5 min. Supernatant was discarded. PBMCs were incubated for 10 min in HBSS (−) (Nacalai) with 1× CytoTell Red 650 (AAT Bioquest). PBMCs were centrifuged at 1,200 rpm for 5 min and washed with RPMI medium. Stained PBMCs were used to distinguish between rhiPSC-T cells and PBMCs. Anti-CD20 CAR or blank vector (only EGFP) expressing rhiPSC-T cells was harvested after stimulation day 17. iPSC-T cells were washed two times with D-PBS (−) with 2% FBS at 1,200 rpm for 5 min. iPSC-T cells were seeded 1 × 10⁵ cells/well onto a 96-well round-bottom plate. Coculture with stained auto-PBMCs was performed at effector-to-target (E:T) ratios ranging from 1:10 to 1:1 for 24 h at 37°C in a 5% CO₂ incubator. After coculture, cells were collected and stained with BV421-anti-CD20 antibody and analyzed by FACS Aria II.

Expanded iPSC-T cells were harvested 12 days after stimulation and were passed through a 40-μmφ filter. rhiPSC-T cells were washed two times with D-PBS (−) and resuspended in 20 mL of saline solution containing 2% auto-serum. iPSC-T cells were intravenously administered. Administered rhiPSC-T cell numbers displayed in Table 2 were calculated as EGFP-positive cells.

To promote the engraftment of rhiPSC-T cells, we administered the following agents according to the schedule. rhiPSC-T cells were administered on day 0. Cyclophosphamide (30 mg/kg) was administered on day −4. Fludarabine (25 mg/m²) was administered on days −6, −5, and −4. Uromitexan (12 mg/kg) was administered two times on day −4. Palonosetron (20 μg/kg) was administered on day −6. Dexamethasone (3.3 mg/body) was administered on days −6, −5, and −4.

As shown in Figure 3C, organs were isolated from euthanized rhesus at R496 second administration on day 2. Approximately 5-mm square samples were collected from these organs. Samples were incubated in Lysis buffer containing 10 mM Tris-HCl, 0.1 M EDTA, 0.5% (w/v) SDS, and 1 mg/mL Protease K overnight at 55°C. The same volume of phenol–chloroform mixture [Phenol/Chloroform/Isoamyl Alcohol (25:24:1)] was added. Samples were rotated slowly for 10 min. Samples were centrifuged at 15,000 rpm for 5 min, and the upper layer was collected. Sodium acetate (3 M) and ethanol were added. Samples were then rotated slowly for 30 min. Samples were centrifuged at 15,000 rpm for 30 min, and the DNA pellet was washed two times with 70% ethanol solution at 15,000 rpm for 5 min. Dried pellet was resuspended with TE buffer. The concentration of DNA was determined by nanodrop (Thermo Fisher Scientific).

DNA (200 ng) was used for PCR template. For the detection of EGFP, genomic PCR was performed using the primers Fw: 5&-CGAGCTGGACGGCGACGTAAAC-3& and Rv: 5&-GCGCTTCTCGTTGGGGTCTTTG-3& (29) and ExTaq HS (TAKARA). For the detection of GAPDH, genomic PCR was performed using the primers Fw: 5&-TGGAAAAACCTGCCAAGTACG-3& and Rv: 5&-ACCTAGAAGATGAAAAGAGTCGT-3& and ExTaq HS. The PCR condition had three steps (dissociation step: 98°C at 1 min; amplification step 10 s at 98°C, 40 cycles; and elongation step: 5 min at 72°C).

Excluded lung sample was fixed with 4% paraformaldehyde for 42 h at 4°C. Fixation buffer was exchanged with neutral buffered formalin. Fixed sample was analyzed by outsourcing (shinsoshikikagakukenkyujo, Japan).

Collected blood samples were analyzed by outsourcing (FALCO Biosystems). RBC; Red Blood Cells. WBC; White Blood Cells. HGB; Hemoglobin. HCT; Hematocrit. MCV; Mean Cell volume. MCH; Mean Cell Hemoglobin Concentration. MCHC; Mean Cell Hemoglobin Concentration. PLT; platelet. A/G; albumin/globulin ratio. AST(GOT); asparate aminotransferase. ALT(GPT); alanine aminotransferase. ALP; alkaline phosphatase. LD(LDH); lactate dehydrogenase. γ-GT(γGTP); gamma-glutamyl transpeptidase. CK(CPK); creatine kinase. LDL cholesterol; low density lipoprotein cholesterol. HDL cholesterol; high density lipoprotein cholesterol. CRP; C-reactive protein, quantitative. MCV =Ht (%)/RBC(×10⁴/μL) × 1,000. MCH=HB (g/dL) /RBC(×10⁴/μL) × 1,000. MCHC = Hb(g/dL)/Ht (%) × 100.