Lentiviral vector-mediated expression of OCT4 and SOX2 efficiently reprogram cord blood (CB) CD34⁺ cells into induced pluripotent stem cells (iPSCs). (a) Schematic of the self-inactivating (SIN) lentiviral vector backbones for expression of the human reprogramming factor OCT4, SOX2, KLF4. Δ indicates the SIN design with partially deleted U3 of the 3′ long-terminal repeat. cPPT, central polypurine tract; RRE, rev-responsive element; SFFV, spleen focus-forming virus U3 promoter; Wpre, woodchuck post-transcriptional regulatory element; ψ, packaging signal; 2a, a self-cleavage site derived from equine rhinitis A virus. (b) Experimental strategy for reprogramming human CB CD34⁺ cells using lentiviral vectors. (c) Representative alkaline phosphatase (ALP) staining of iPSC colonies 16 days after lentiviral transduction of 1 × 10⁴ CB CD34⁺ cells. O, OCT4; S, SOX2; K, KLF4. (d) Numbers of induced pluripotent stem cells (iPSCs) generated from 1 × 10⁴ CB CD34⁺ cells. n = 3. O+S vs. OS: P < 0.05. OS vs. OS+K: no significant difference. Data shown are presented as mean ± SEM. (e) Representative fluorescence-activated cell sorting (FACS) diagram of TRA-1-60 expression in cells undergoing reprogramming. Cells at day 16 after transduction were harvested and analyzed. (f) Percentages of TRA-1-60 positive cells in reprogramming cultures. O+S vs. OS: P < 0.05; OS vs. OS+K: no significant difference. Data shown are presented as mean ± s.e.m. (n = 3).

Efficiency of OCT4 and SOX2-mediated reprogramming depends on gene expression levels. (a) Schematic of the self-inactivating (SIN) lentiviral vector backbones for expression of green fluorescent protein (GFP). Δ indicates the SIN design with partially deleted U3 of the 3′ long-terminal repeat. cPPT, central polypurine tract; EF1, elongation factor-1α promoter; PGK, phosphoglycerokinase promoter; RRE, rev-responsive element; SFFV, spleen focus-forming virus U3 promoter; Wpre, post-transcriptional regulatory element; ψ, packaging signal. (b) Representative levels of GFP expression driven by three different promoters in cord blood (CB) CD34⁺ cells. Fluorescence-activated cell sorting (FACS) analysis was conducted at 3 days post-transduction. (c) Distinct GFP expression levels driven by three different promoters in CB CD34⁺ cells. n = 3. PGK-GFP vs. EF-GFP: P = 0.05; EF-GFP vs. SFFV-GFP: P < 0.05. (d) Increased expression of OCT4GFP fusion gene driven by SFFV promoter compared to PGK and EF1 in CB CD34⁺ cells. FACS analysis was conducted at 3 days post-transduction. n = 3. PGK-OCT4GFP vs. EF1-OCT4GFP: P = 0.06; EF1-OCT4GFP vs. SFFV-OCT4GFP: P < 0.01. (e) Alkaline phosphatase (ALP) staining for iPSC cultures from CB cells transduced with PGK-OS, EF1-OS, and SFFV-OS. Note that no colonies were generated in PGK-OS, EF1-OS conditions. (f) Expression of MYC and KLF4 rescues failure of low level OS expression driven by EF1 promoter in generating induced pluripotent stem cells (iPSCs) from CB CD34⁺ cells. Graphed data are presented as mean ± SEM (n = 3).

OCT4 and SOX2-mediated reprogramming using episomal vectors. (a) Schematic of episomal vectors used in this study for conversion of cord blood (CB) CD34⁺ cells into induced pluripotent stem cells (iPSCs). Reprogramming factors were cloned into the pCEP4 backbone; their expression is driven by spleen focus-forming virus U3 promoter (SFFV). 2a is a self-cleavage site derived from equine rhinitis A virus. Wpre, post-transcriptional regulatory element; SV40PolyA, polyadenylation signal from SV40 virus; OriP, EBV origin of replication; EBNA1, Epstein–Barr nuclear antigen 1, which plays essential roles in replication and persistence of episomal plasmid in infected cells. (b) Experimental strategy for reprogramming human CB CD34⁺ cells using EBNA1-based episomal vectors. (c) Representative alkaline phosphatase (ALP) staining shows that inclusion of the Wpre element in the episomal vector pCEP-OS (w/o W) results in successful reprogramming. n = 3. Colonies are from 1 × 10⁵ CB CD34⁺ cells. (d) Inclusion of Wpre element in the CEP episomal vector increases gene expression. 293T cells were infected with same amount of plasmids. 3 days after transfection, OCT4 and SOX2 expression was examined by intracellular staining and fluorescence-activated cell sorting (FACS) analysis. n = 3. pCEP-OS (w/o W) vs. pCEP-OS: P < 0.05. (e) Numbers of ALP positive iPSC colonies at 16 days post-transfection of 1 × 10⁵ CB CD34⁺ cells with pCEP-OS (OS) and pCEP-K (K) or pCEP-MK (MK). n = 3. OS vs. OS+K: P < 0.05; OS+K vs. OS+MK: P < 0.05. Expression of the iPSC markers (f) NANOG and (g) TRA-1-60 in cultures reprogrammed using three different combinations of episomal vectors. Cells were harvested for FACS analysis 20 days after nucleofection.

Characterization of induced pluripotent stem cells (iPSCs) generated with pCEP-OS. (a) Copies of residual episomal vectors after eight passages as indicated by real-time PCR. Data shown are from one pair of primers. Similar results were obtained with second pair of primers. (b) Immunohistochemistry analysis of a representative iPSC line showing expression of indicated pluripotency markers. Images were captured using the Zeiss LSM 710 confocal microscope with a ×10 objective. (c) A representative karyogram of an iPSC clone. All analyzed iPSC clones showed a normal karyotype. (d) Bisulphite genomic sequencing of the OCT4 and NANOG promoters indicates demethylation in three independent clones. Each horizontal row of circles represents an individual sequencing reaction of a given amplicon. Open and filled circles represent unmethylated and methylated CpG dinucleotides, respectively. (e) Hematoxylin and eosin (H&E) staining of representative teratoma from pCEP-OS cord blood (CB) iPSCs shows derivatives of three embryonic germ layers. Cartilage (mesoderm); neurotubules with rosettes (ectoderm); glands (endoderm); retina epithelial cells with pigments (ectoderm). Images were acquired using the Olympus microscope with a ×20 objective.