Acknowledgments This work was supported by the

Acknowledgments This work was supported by the Agency for Science, Technology and Research, Singapore. We would like to thank the Cytogenetics laboratory at Singapore General Hospital for karyotyping the iPSC lines and Luqman Hakim for help in characterizing the iPSC lines. We would like to thank Thong Zhonghui from the DNA profiling laboratory at Health Sciences Authority, Singapore for fingerprinting the DNA of iPSC lines and PBMCs. We would also like to thank Tan Hong Kee for his guidance on PBMC reprogramming.
Resource table. Resource details Primary human dermal fibroblasts (PHDFs) were isolated from the skin of breast cancer diagnosed patient after signing informed consent. The feeder-dependent human induced pluripotent stem cell line (hiPSCs) (GPCCi001-A) was generated with the use of modified EF1a-hSTEMCCA-loxP, a polycistronic vector composed of four reprogramming factors: Oct4, Sox2, Klf4, c-Myc and regulatory element: tet-operator (STEMCCA-tetO) [Sommer et al., 2009; Takahashi et al., 2007] (Fig. 1A). The stem cell cassette of the Stemcca-tetO vector consists of a two fusion cistrons: Oct4/Klf4 and Sox2/cMyc, separated by IRES element, coexpressed from EF-1α promoter. Sequences of reprogramming factors in each cistron are separated by sequences of self-cleaving peptides F2A and E2A. The tetO element enables regulation of transgene expression in target cells. After 18–21days of the reprograming, the colonies with clear boundaries, compact structure and composed of GSK2656157 with high nucleus to cytoplasm ratio were observed. The established colonies were transferred for further culture in feeder-dependent conditions (Fig. 1C). Generated hiPS cells were transduced with pLV-HK lentiviral vector expressing fusion protein tTRKRAB in order to inhibit expression of reprogramming factors. TTRKRAB binding to specific DNA regions recruits chromatin modifying protein complexes through KRAB domain and induces reversible, doxycycline-controllable epigenetic repression of transcription from any promoter placed nearby tetO sequence, in either directions [Wiznerowicz and Trono, 2003]. This protein expressed from regulatory vector pLV-HK binds to the tetO element in reprogramming STEMCCA-tetO vector, which allows for epigenetic repression of exogenous factors, leaving derived hiPS cells deprived of transgene expression (Fig. 1A, C). The use of a lentiviral system has several advantages over other reprogramming methods. The most important are the high reprogramming efficiency and low toxicity. Our method allows for reprogramming of somatic cells towards pluripotent cells with high yield, around 1%, in contrasts to other, less-efficient methods like Sendai virus, episomal plasmids or free mRNA (Stadtfeld et al., 2008; Okita et al., 2013; Warren et al., 2010). Moreover, lentiviral vectors allows for reprograming of variety of cells, including non-dividing cells. In addition, the iPS cell lines generated with this technique are homogenous, and importantly, highly stable, as they remain free of transgene expression in prolonged culture, and during in vitro and in vivo differentiation. A microscopic evaluation of GPCCi001-A cell line indicated, that obtained hiPS colonies are composed of homogenous small cells with high nucleus to cytoplasm ratio, what is characteristic for pluripotent stem cells (Fig. 1B). The hiPS clone GPCCi001-A shows the high expression of genes responsible for maintaining pluripotency state such as NANOG, octamer-binding transcription factor 4 (OCT4), sex determining region Y box 2 (SOX2) and E-cadherin (CDH1) which was confirmed both by RT-qPCR analysis and immunofluorescence staining (Fig. 1D, E). The expression of pluripotency markers in GPCCi001-A cell line is elevated in contrast to PHDF cell line and similar to commercially available iPS cell line ND41658*H (Coriell Cell Repository, NY, USA). Moreover, the flow cytometry analysis of extracellular (TRA-1-81, TRA-1-60) markers confirmed the pluripotency state of obtained GPCCi001-A cell line (Fig. 1F). We considered the possibility that the reprogramming process may cause chromosomal instability in the derived cells. We confirmed that GPCCi001-A clone showed a normal female karyotype, consistent with the parental fibroblast karyotype (Fig. 1G). The cell line is also free of mycoplasma contamination as detected by PCR. To prove the ability of obtained GPCCi001-A cells to differentiate into three germ layers, the spontaneous differentiation through embryoid bodies (EB) was performed. Due to slight possibility of spontaneous transgene reexpression, RT-PCR analysis was performed in iPS-derived EBs, with primers specific for lentiviral mRNA. Primers amplify fragment of transgene Oct4 and Klf4 sequence, including F2A fragment. Since both fusion cistrons, Oct4/Klf4 and Sox2/cMyc are expressed from single promoter, epigenetic repression of transcription by TTRKRAB from this promoter results in lack of all four reprogramming factors in obtained hiPS cells. As we expected, during differentiation there was no detectable expression of exogenous reprogramming factors, and EBs remained transgene-expression free, like initial iPS cells (Fig. 1H). In the two-week EBs (7days suspension culture combined with 7days monolayer culture), the demonstration of three germ layer markers such as endodermal (sex determining region Y box 17, SOX17; forkhead box A2, FOXA2), ectodermal (GATA Binding Protein 4, GATA4; paired box protein 6, PAX6) and mesodermal (Brachyury; alpha smooth muscle actin, α-SMA) was assessed by immunofluorescence staining (Fig. 1I).