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.

Induction of Noonan syndrome-specific human-induced pluripotent stem cells under serum-, feeder-, and integration-free conditions

“…Briefly, DPCs were cultured in a gelatin (Millipore, Billerica, MA)-coated 12-well plate at a density of 1 × 10 5 cells in RD6F serum-free medium (Sato et al 1987) and were infected with SeVdp (KOSM) (Nishimura et al 2011) vector at MOI 6 once at room temperature for 2 h and then at 37°C overnight in a humid atmosphere of 95% air/5% CO 2 in RD6F medium. Then, the infected cells were trypsinized and seeded on fibronectin (2 μg/cm 2 ) (Sigma-Aldrich, St. Louis, MO)-coated 6-well plates at a density of 1.0 × 10 4 cells in hESF9medium (Furue et al 2008;Yamasaki et al 2014;Hamada et al 2020) at 38°C in a humid atmosphere of 95% air/5% CO 2 . The medium was changed every other day.…”

“…Isolation of peripheral blood mononuclear cells (PBMCs) and infection protocol of SeVdp under serum-free conditions Induction of PBMC-hiPSCs was performed as reported previously (Hamada et al 2020). Briefly, PBMCs were prepared by density gradient centrifugation in a Histopaque 1077 (Sigma-Aldrich) and were cultured in RD6F serum-free medium supplemented with IL-2 (CELEUK, Takeda Pharm., Osaka, Japan) for 6 d at 37°C in a humidified atmosphere of 95% air/5% CO 2 .…”

“…Differentiation of NS-hiPSCs into three germ layers in vitro and in vivo The in vitro and in vivo differentiation of WT-DPC-hiPSCs and WT-PBMC-hiPSCs was performed as described previously (Yamasaki et al 2014) Hamada et al 2020. To confirm the in vitro differentiation capacity of NS-hiPSCs, we performed embryoid body (EB) assay.…”

“…Previously, we reported the induction of hiPSCs derived from peripheral blood mononuclear cells (PBMCs) (Hamada et al 2020) without using serum or feeder cells (Sato et al 1987;Takahashi and Yamanaka 2006;Takahashi et al 2007;Yamasaki et al 2013;Yamasaki et al 2014) and also without virus integration (Nishimura et al 2011;Nakanishi and Otsu, Supplementary Information The online version of this article (https:// doi.org/10.1007/s11626-020-00515-9) contains supplementary material, which is available to authorized users. 2012; Yamasaki et al 2016).…”

“…Previously, we reported the induction of hiPSCs derived from peripheral blood mononuclear cells (PBMCs) (Hamada et al . 2020 ) without using serum or feeder cells (Sato et al . 1987 ; Takahashi and Yamanaka 2006 ; Takahashi et al .…”