Epstein-Barr virus immortalized lymphoblastoid cell lines (LCLs) were obtained from the Coriell Institute. The Coriell cell repository maintains the consent and privacy of the donor LCLs. LCLs were cultured in RPMI 1640 (Gibco) supplemented with 15% fetal bovine serum (FBS; Biochrom) and 2 mM L-glutamine (Sigma-Aldrich). CRL-2097 neonatal skin fibroblasts and HEK293 cells were purchased from ATCC and cultured in Dulbecco’s Modified Eagle’s medium high glucose (DMEM; Welgene) supplemented with 10% FBS, 1×nonessential amino acids (NEAAs; Sigma-Aldrich), 1×GlutaMAX (Gibco), and 1×penicillin/treptomycin (P/S; 100 U/ml each, Sigma-Aldrich). Human iPSCs were cultured on the Matrigel-coated plate with MEF-conditioned medium (MEF-CM). The MEF-CM medium was prepared as described previously (ref). The cells described in this study were cultured at 37℃ and 5% CO₂ in a humidified Heraeus BB15 incubator (Thermo Fisher Scientific).

pLEX307-GFP and pCXLE-mCherry were cloned in house. pMXs-OCT4, pMXs-SOX2, pMXs-KLF4, pMXs-c-MYC, pMXs-GFP and pRRL-hOSKM-tdTomato were previously described (14-16). Other plasmids including pMD2.G (#12259), psPAX2 (#12260), pUMVC (#8449), pCMV-VSV-G (#8454), pCXLE-hOCT3/4 (#27076), pCXLE-hSK (#27078), pCXLE-hUL (#27080), and pCXLE-mp5 3DD (#41859) were purchased from Addgene. Plasmid DNA was isolated from E. coli and purified using Nucleo-Bond Xtra Kit (Macherey-Nagel).

To produce retroviruses, 4.5 μg of pMXs-GFP, 3 μg of pUMVC, and 1.5 μg of pCMV-VSV-G were transfected into HEK293 cells using 27 μl of Polyethylenimine (PEI; Polysciences) in 600 μl Opti-MEM (Invitrogen). At 48 h post-transfection, virus-containing supernatants were collected and filtered through a 0.45-μm PVDF filter (Mil-lipore), concentrated at 23,000 rpm for 2 h using an Optima XL-100 K Ultracentrifuge (Beckman), and resuspended in 1 ml of DMEM. Retroviral suspensions were stored at −80℃ until use. To produce lentiviruses, 1.5 μg of pMD2.G, 3 μg of psPAX2 and 4.5 μg of the pRRL-hOSKM-tdTomato were transfected into HEK293 cells using 27 μl of PEI in 600 μl of Opti-MEM. At 48 h post-tra-nsfection, virus-containing supernatants were collected and filtered through a 0.45-μm PVDF filter, concentrated at 23,000 rpm for 2 h using an Optima XL-100 K Ultrace-ntrifuge and resuspended in 1 ml of DMEM. Lentiviral suspensions were stored at −80℃ until use. The LCLs and CRL2097 fibroblasts were then infected with viruses twice over the course of three days in the presence of 8 μg/ml protamine sulfate (Sigma-Aldrich).

Sodium butyrate (NaB; Sigma-Aldrich) was used at a final concentration of 250 μM. SB431542 (purity: ≥98%; Cayman Chemical) was used at a final concentration of 2 μM. SGC0946 (purity: ≥98%; Cayman Chemical) was used at a final concentration of 3 μM.

1×10⁶ of LCLs were electroporated with total 2 μg of pCXLE-hOCT3/4, pCXLE-hSK, pCXLE-hUL, and pCXLE-mp53DD (0.5 μg each) using the Neon^TM Transfection System 10 μl Kit (MPK10096; Thermo Fisher Scientific). The transfected LCLs were then transferred to 35 mm dishes pre-coated with hESC-qualified Matrigel (BD Bios-ciences) and cultured in MEF-CM supplemented with NaB, SB431542, and SGC0946. The medium was changed every other day. Chemicals were added until colonies reached the size of over 200 μm in diameter.

The cells were mechanically or enzymatically disso-ciated and resuspended in 300 μl of 3% FBS/PBS. The cell suspension was transferred to 5 ml polystyrene round-bottom tubes through a cell strainer cap (Falcon) and subjected to flow cytometry analysis. The cells were then separated from debris and aggregates by forward scatter/side scatter (FSC/SSC) gating. Single cells were identified by plotting FSC area versus FSC width. Dead cells were excluded by staining with DAPI and gating on DAPI^＋ cells. Cells that had not been infected with viruses or had been electroporated with empty plasmids were used as negative controls for gating. Fluorescence was measured using a FACSCanto II (BD Biosciences) and flow cytometry data were analyzed using FlowJo software (Tree Star Inc.).

The cells were fixed with 10% formalin (Cellnest) for 20 mins, incubated with 0.1% Triton X-100/PBS for 30 mins, and blocked in 5% BSA/PBS for 1 h. The cells were incubated with appropriate primary antibodies overnight at 4℃. The primary antibodies used were following: anti-SSEA-4 (1：100, 90231; Millipore), anti-NANOG (1：1,000, 5232S, Cell Signaling Technology), anti-OCT4 (1：1,000, 5677S; Cell Signaling Technology), anti-SALL4 (1：1,000, ab29112; Abcam). The cells were then washed three times with PBS and incubated with appropriate fluorescently labeled Alexa-Fluor secondary antibodies (1：1,000, Invitrogen) for 1 hr. The cells were then washed three times with PBS, incubated with 0.5 μg/ml 4’,6-dia-midino-2-phenylindole (DAPI, Molecular Probes) for 10 mins, and washed once with PBS. The cells were then subjected to immunofluorescence microscopy analysis. Images were acquired using a fluorescence inverted microscope (IX71; Olympus) equipped with the CCD camera (DP30BW; Olympus) and analyzed with the DP-BSW (Olympus) and Fiji software (17).

Retroviral transduction of OSKM has been widely used for generation of iPSCs from different types of human cells (15, 18). Since retroviruses can only infect actively dividing cells (19), our first choice for gene delivery into LCLs was retroviral transduction. We transduced LCLs with retroviruses containing OSKM and cultured in MEF-CM medium supplemented with sodium butyrate (a HDAC inhibitor), SB431542 (a TGFb inhibitor) and SGC0946 (a DOT1L inhibitor) (Fig. 1A). These chemicals positively influence iPSC generation (20). Surprisingly, retroviral transduction of OSKM in LCLs did not yield any iPSC colonies in contrast to concurrently transduced fibroblasts (Fig. 1B). We then hypothesized that the failure of generating iPSCs from LCLs might be due the fact that retrovirus might not effectively transduce LCLs. To test this hypothesis, we transduced LCLs with retroviruses encoding green fluorescent protein (GFP) and evaluated GFP expression by a fluorescence microscope. At five days post-infection, we found that GFP was not expressed in retroviral GFP-transduced LCLs (Fig. 1C). Neither increased titers of GFP virus or longer culturing periods (days 7 and 9) changed GFP negativity (Fig. 1C). In contrast, GFP was expressed in retroviral GFP-transduced fibroblasts (Fig. 1D). Flow cytometry analysis further confirmed that GFP was not expressed in retrovirally GFP-transduced LCLs, in contrast to concurrently transduced fibroblasts which yield over 20% GFP^＋ cells at day 5 post-infection (Fig. 1E). Together, these data demonstrate a decisive difference in retroviral transduction between two cell types and show that retrovirus-mediated gene delivery is not effective for LCLs.

Lentiviruses can infect a wider range of cell types than retroviruses (21). We thus postulated that lentiviral transduction might be in fact an effective way to deliver genes into LCLs. To test this, we produced lentiviruses with a polycistronic lentiviral vector encoding OCT4, SOX2, KLF4, c-MYC, and tdTomato (hereafter referred as OSKMT) and transduced LCLs (Fig. 2A). In this system, tdTomato was linked to OSKM by an internal ribosomal entry site (IRES) and thus transgene expression can be monitored by tdTomato expression. Surprisingly, at forty-eight hours post-infection, we found that lentiviral OSKMT-transduced LCLs did not yield any tdTomato^＋ cells, in contrast to concurrently transduced fibroblasts which yielded over 70% tdTomato^＋ cells (Fig. 2B∼D). Consequently, le-ntiviral OSKMT-transduced LCLs failed to produce iPSC colonies, whereas concurrently transduced fibroblasts produced iPSC colonies (Fig. 2E). Together these data demonstrate lentiviral transduction is also not an effective way to deliver genes into LCLs and is not suitable for generating iPSCs from LCLs.

Beside retroviral and lentiviral transduction, electroporation is an efficient gene delivery method which can introduce genes into a wide range of cell types, especially hard-to-transfect cells, like primary cells and cells that are cultured in suspension (22, 23). Electroporation uses an electrical pulse to create temporary pores in cell membranes through which substances like plasmids can enter into the cells. Different cell types require distinct electroporation settings (i.e. pulse time, voltage strength) to achieve high viability and reproducible transfection efficiency (22, 23). To identify an optimal electroporation condition for LCLs, we electroporated LCLs with episomal vector encoding mCherry with different ranges of voltage stren-gth and pulse width (Fig. 3A). We selected an OriP/EBNA1-based episomal vector particularly as a vehicle to deliver mCherry into LCLs, since two elements (oriP and EBNA-1) allow for replication and long-term retainment of vectors in mammalian cells (24). At forty-eight hours post-transfection, mCherry expression was analyzed by a fluorescence microscope. Intriguingly, of six condition we tested, two conditions (20 ms/1,400 v, 30 ms/1,100 v) yie-lded over 20% mCherry^＋ cells (Fig. 3B and 3C). Of note, cell death rates increased with higher voltage conditions (20 width: 1,700 v, 30 width: 1,400 v, 40 width: 1,200 v), indicating that voltage strengths influence the cell viability (Fig. 3C). Together these data reveal defined electroporation conditions that enable efficient gene delivery into LCLs.

Having established that electroporation is a powerful and versatile method for delivery genes into LCLs, we next attempted to use this method to reprogram LCLs into iPSCs. For this, we electroporated LCLs, which were derived from a PDM patient, with episomal vectors encoding repro-graming factors [OCT4, SOX2, KLF4, L-MYC, LIN28 and p53DD (a dominant-negative inhibitor of p53 function)] and subsequently cultured them in MEF-CM medium supplemented with sodium butyrate, SB431542, and SGC0946 (Fig. 4A). At one week after transfection, we observed a number of small iPSC colonies firmly attached to the bottom of the culture plate (Fig. 4B). After the colonies reached over 800 μm in diameter (Fig. 4C), they were mechanically isolated and cultured independently. To confirm if the LCLs had transformed into iPSCs, we stained them with the number of pluripotency makers (Fig. 4D). We found that the LCL-derived iPSCs indeed expressed OCT4, SSEA4, DPPA4, and SALL4 (Fig. 4D). Further-more, they could form embryoid bodies when the cells were cultured in ultra-low attachment culture plates (Fig. 4E). Together these data demonstrate that electroporation of episomal vectors enables iPSC generation from LCLs.