Reprogramming of human peripheral blood cells to induced pluripotent stem cells

Abstract

Direct reprogramming of human fibroblasts to induced pluripotent stem cells (iPS) has been achieved by ectopic expression of defined transcription factors. Derivation of human fibroblasts however is a time consuming process and requires punch biopsies or isolation of patient foreskin. Here we use a polycistronic vector encoding Oct4, Klf4, Sox2 and c-Myc to generate iPS cells from from frozen peripheral blood of several donors. Genomic DNA analyses indicated that iPS cells were derived from mature T cells as well as myeloid donor cells. Inducing pluripotency in peripheral blood would allow utilization of easy to get samples from the adult and, more importantly, provide convenient access to numerous patient samples stored in blood banks. The latter is of major interest as frozen blood samples, when reprogrammed to iPS cells, would allow the retrospective molecular analyses of rare diseases.

Figure 1. Characterization of peripheral blood derived iPS cells

A) Schematic drawing demonstrating the strategy to derive iPS cells from human peripheral blood. B) Immunostaining for pluripotency markers Oct4, Nanog and Tra1-81 in one representative T- and M-iPS line. C) Southern blot analyses showing that telomeres are elongated in T- and MiPS cells compared to mononuclear (D1MNC, D3MNC) and myeloid cells (D3myeloid). D) Quantitative RT-PCR assay for expression analyses of mesodermal (Brachyury), endodermal (AFP) and ectodermal (NCAM) lineage markers in differentiated embryoid bodies and iPS cells E) Teratoma analysis from each one representative T-iPS and M-iPS line. F) Methylation analyses of the Oct4 promoter region in human ES, T-iPS, M-iPS and peripheral blood cells. See also Supplemental Table 1 and Figure S1.

Figure 2. Analyses of TCR gene rearrangements in T-iPS cells

A) PCR analyses of TCRB Vβ-Jβ gene rearrangements in 6 iPS lines derived from peripheral blood of donor 4. PCR products within the indicated valid size range were cloned and sequenced. B) PCR analyses of TCRG Vγ-Jγ gene rearrangements in 6 iPS lines derived from donor 4. PCR products within the indicated valid size range were cloned and sequenced. C) PCR analyses showing that all iPS clones from donor 4 tested negative for Vδ-Jδ TCRD gene rearrangements. D) Sequencing analyses of PCR products obtained in 2A and 2B. Shown are productive TCRB and productive (D4 #4) and unproductive (D4 #1) TCRG gene rearrangements in selected T-iPS clones of donor 4. Genomic DNA from human ES cell line BGO2 was used as negative control in all PCR assays; clonal positive controls were purchased from InVivoScribe Technologies. See also Supplemental Table 1, Figure S1 and Figure S2.