Induction of integration-free human-induced pluripotent stem cells under serum- and feeder-free conditions

Abstract

Human-induced pluripotent stem cells (hiPSCs) have shown great potential toward practical and scientific applications. We previously reported the generation of human dental pulp stem cells using non-integrating replication-defective Sendai virus (SeVdp) vector in feeder-free culture with serum-free medium hESF9. This study describes the generation of hiPSCs from peripheral blood mononuclear cells to increase the donor population, while reducing biopsy invasiveness. From 6-d-old primary culture of peripheral blood mononuclear cells (PBMCs) with IL-2, hiPSCs were established using SeVdp(KOSM)302L with recombinant Laminin-511 E8 fragments under serum-free condition. The established PBMC-derived hiPSCs showed pluripotency and differentiation ability both in vivo and in vitro. In addition, we evaluated microarray data from PBMC- and dental pulp-derived hiPSCs. These hiPSCs will be beneficial for characterizing the molecular mechanisms of cellular differentiation and may provide useful substrates for developing cellular therapeutics.

Keywords: Human-induced pluripotent stem cells; Peripheral blood mononuclear cells; Reprogramming efficiency; hESF9 serum-free defined media.

Figure 1.

Procedure for hiPSC induction under serum-, feeder-, and integration-free conditions. Time schedule of hiPSC induction. (a) Phase contrast images of WT4-PBMCs on Day 0. (b, c) Phase contrast images of WT4-PBMC on Day 10 (b) and Day 14 (c) after SeVdp transfection. ESC-like colonies were detected approximately 14 d after transduction. Hamada et al.

Figure 2.

ALP-positive colonies and number of colonies after culture with IL-2 supplemented RD6F for 0, 3, and 6 d. (A) Images of ALP staining on Day 25 after transfection with SeVdp(KOSM)302L under serum-free culture conditions, displaying reprograming efficiencies of 0.024% in 0-d primary culture, 0.051% in 3-d primary culture, and 0.0033% in 6-d primary culture. (B) Images of ALP staining of control, conducted by KSR and MEF feeder co-culture under serum-supplemented conditions, displaying efficiencies of 0% in 0-d primary culture, 0.0035% in 3-d primary culture. Hamada et al.

Figure 3.

Phase contrast of DPC-hiPSCs and PBMC-hiPSCs. Phase contrast images of iPSCs derived from DPC or PBMC, WT1-DPC-iPSC clone25 at passage 40, WT2-DPC-iPSC clone6 at passage 26, WT3-PBMC-iPSC clone9 at passage 10, WT4-PBMC-iPSC clone1 at passage 10, WT5-PBMC-iPSC clone7 at passage 40, WT6-PBMC-iPSC clone3 at passage 10, WT7-PBMC-iPSC clone2 at passage 4, WT8-PBMC-iPSC clone9 at passage 5, and WT9-PBMC-iPSC clone5 at passage 16. Each hiPSC showed smooth edges and a high cell ratio in small cells. Bars shown in each figure are 500 μm. Hamada et al.

Figure 4.

RT-qPCR. Gene expression of pluripotent markers by RT-qPCR before and after hiPSC induction of each cell line. Although Oct3/4 was detected before reprogramming, Nanog, Sox2, and Rex1 were expressed after reprogramming. SeVdp was not detected under all conditions. Hamada et al.

Figure 5.

ICC (pluripotency). Immunocytochemistry of pluripotency marker proteins WT-iPSCs. WT-iPSCs were fixed and reacted with antibodies (Oct4, Nanog, SSEA-4, and Tra-1-81). Binding of these antibodies was visualized with Alexa Fluor® 488-conjugated secondary antibodies (green). Nuclei were stained with DAPI (blue). Scale bars represent 100 μm. Hamada et al.

Figure 6.

ICC (in vitro differentiation ability). Immunofluorescence staining of differentiation markers after 3-wk differentiation using embryoid body formation in vitro in each WT-hiPSCs. Immunocytochemistry of βIII-tubulin (ectoderm), α-smooth muscle actin (α-SMA, mesoderm), and α-fetoprotein (AFP, endoderm) are shown. Binding of these antibodies was visualized with Alexa Fluor® 488-conjugated secondary antibodies (green). Nuclei were stained with DAPI. Bar indicates 100 μm. Hamada et al.

Figure 7.

H-E staining (in vivo differentiation ability). WT-hiPSCs formed teratomas in SCID mice (CB17/Icr-Prkdc scid/CrlCrlj). Histological analysis with H-E staining demonstrated that those tumors contained various tissues, including gut-like epithelial tissues (endoderm), cartilage (mesoderm), and neural tissues (ectoderm), and were confirmed as teratomas. Scale bars represent 200 μm. Hamada et al.

Figure 8.

STR (short tandem repeat) analysis. STR analysis showing a match (16 loci out of 16) to verify that hiPSCs are derived from the same individual. Hamada et al.

Figure 9.

Microarray analysis. A comparison of the global gene expression patterns between PBMCs during primary culture on Days 0–6 and hiPSCs. Clustering analysis was performed based on WT4-PBMC (cultured for 6 d). Blue yellow and red indicate low, middle, and high expression level, respectively. Hamada et al.