br Experimental Procedures br Author

Experimental Procedures
Author Contributions
Acknowledgments B.Z. was supported by an NYSTEM post-doctoral training fellowship, The SKI Stem Cell Research Facility is supported by NYSTEM grants C029153 and C024175 and The Starr Foundation. The work was further supported in part by NIH/NCI grants R21CA176700-02 and P30CA008748. The authors thank M. Navare for technical support and A.F. Parlow and the National Hormone & Peptide program for the antisera against pituitary hormones, The PAX3 antibody developed by C.P. Ordahl, the TFAP2A antibody (3B5) developed by T.J. Williams, and the LHX3 antibody (Lim3, 67.4E12) developed by T.M. Jessell and S. Brenner-Morton were obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at the Department of Biology, University of Iowa, Iowa City.
Introduction One decade ago, Takahashi and Yamanaka (2006) made a stunning discovery that mouse somatic transferases can be reprogrammed into a pluripotent state after forced expression of defined factors composed of OCT4 (also known as POU5F1), SOX2, KLF4, and MYC (also termed c-MYC). The finding in mouse cells was soon reproduced with human fibroblasts (Takahashi et al., 2007; Yu et al., 2007). This breakthrough has changed the landscape of personalized cell therapy, disease modeling, and drug screening. Fibroblasts are the widely used cellular source for many reprogramming studies reported in the last decade but with noticeable limitations (Zhang, 2013). A major drawback is that the derivation of a sufficient amount of fibroblasts for reprogramming requires a lengthy 2–3 weeks of in vitro culture. Human fibroblasts are often obtained by skin biopsy, which is an invasive and non-sterile procedure. Of more concern, skin cells bear more mutations due to environmental insults than cells from inside the body (Abyzov et al., 2012). In contrast to dermal fibroblasts, peripheral blood (PB) has been widely used in medical diagnostics and is the most accessible resource to procure large quantities of cells. Compared with human fibroblasts, PB can be obtained from freshly drawn samples or existing blood stocks. After drawing blood, gradient centrifugation separates red blood cells and granulocytes from mononuclear cells (MNCs) with lower density (Zhang, 2013). The original protocol using retroviral vectors expressing Yamanaka factors (OCT4, SOX2, KLF4, and MYC) has been found to be successful in many cell types, including hematopoietic cells (Aasen et al., 2008; Broxmeyer et al., 2011; Loh et al., 2009; Mali et al., 2008, 2010; Park et al., 2008). Reprogramming of T cells, a major subpopulation of MNCs, into pluripotency has been achieved by many laboratories using different approaches (Loh et al., 2010; Okita et al., 2013; Staerk et al., 2010) and T cell reprogramming has the potential to rejuvenate aged T cells for immunotherapy (Nishimura et al., 2013; Wakao et al., 2013). However, induced pluripotent stem cells (iPSCs) from non-lymphoid cells may be more useful, since mature T cells harbor a single T cell receptor (TCR) after somatic recombination and are unable to regenerate the T cell repertoire with unlimited possibilities. In contrast to mature T cells, hematopoietic progenitors contain an intact genome and are readily reprogrammable after ex vivo expansion in conditions that favor the proliferation of myeloid cells or erythroid cells (Agu et al., 2015; Chou et al., 2011, 2015; Diecke et al., 2015; Dowey et al., 2012; Hu et al., 2011; Liu et al., 2014; Loh et al., 2009; Mack et al., 2011; Meng et al., 2012; Merling et al., 2013; Okita et al., 2013). For cell replacement therapies, the use of integration-free iPSCs that bear no exogenous genetic elements is required. We and other groups have demonstrated that episomal vectors (EV) are capable of reprogramming human somatic cells, including MNCs, into integration-free iPSCs (Chou et al., 2011, 2015; Dowey et al., 2012; Meraviglia et al., 2015; Okita et al., 2013; Su et al., 2013a, 2016; Yu et al., 2009, 2011). The most commonly used EV is a plasmid incorporated with two elements from the Epstein-Barr (EB) virus, origin of viral replication (oriP) and EB nuclear antigen 1 (EBNA1) (Dorigo et al., 2004). One transfection of the EV is sufficient for iPSC generation due to oriP/EBNA1-mediated plasmid retention in mammalian cells, while a gradual loss of EV during each cell division eventually renders the iPSC lines to become void of ectopic factors (Chou et al., 2011; Okita et al., 2013). However, EV-mediated reprogramming was very inefficient, thus potentially risky factors such as SV40 large T antigen and p53 shRNA were used in some studies to achieve appreciable efficiency (Okita et al., 2011; Yu et al., 2009). For reprogramming of human PB MNCs, the success rate was frustratingly low without SV40 large T antigen and p53 suppression (Chou et al., 2011; Dowey et al., 2012). With the use of spleen focus-forming virus U3 (SFFV), a strong promoter in hematopoietic cells, and an additional pro-survival factor BCL-XL, the reprogramming efficiency of PB MNCs was increased by 10- to 100-fold (Meng et al., 2012; Su et al., 2013a, 2016).