Generation of induced pluripotent stem cells from a small amount of human peripheral blood using a combination of activated T cells and Sendai virus

Department of Cardiology, Keio University School of Medicine, Tokyo, Japan.
Nature Protocol (Impact Factor: 9.67). 03/2012; 7(4):718-28. DOI: 10.1038/nprot.2012.015
Source: PubMed


Induced pluripotent stem cells (iPSCs) have become important cell sources for genetic disease models, and they have the potential to be cell sources for future clinical therapies. However, invasive tissue sampling reduces the number of candidates who consent to donate cells for iPSC generation. In addition, integrated transgenes can potentially insert at inappropriate points in the genome, and in turn have a direct oncogenic effect. Technical modifications using a combination of activated T cells and a temperature-sensitive mutant of Sendai virus (SeV) can avoid invasive tissue sampling and residual transgene issues in generating iPSCs. Such advances may increase the number of consenting patients for cell donations. Here we present a detailed protocol for the generation of iPSCs from a small amount of human peripheral blood using a combination of activated T cells and mutant SeV encoding human OCT3/4, SOX2, KLF4 and c-MYC; T cell-derived iPSCs can be generated within 1 month of blood sampling.

Full-text preview

Available from:
  • Source
    • "Most of the commonly-used source tissues or cells for iPSC production are skin biopsies and blood cells, and there are multiple ways of delivering the pluripotent re-programming factors (Oct3/4, Klf4, Sox2, and c-Myc), which have been reviewed extensively elsewhere [94] . The optimal way to generate iPSC lines would be free of virus integration, e.g., by Sendai virus [95] , that has been used by the NIMH stem cell center ( to establish a resource of iPSC lines for psychiatric research. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Schizophrenia (SZ) is a devastating mental disorder afflicting 1% of the population. Recent genome-wide association studies (GWASs) of SZ have identified >100 risk loci. However, the causal variants/genes and the causal mechanisms remain largely unknown, which hinders the translation of GWAS findings into disease biology and drug targets. Most risk variants are noncoding, thus likely regulate gene expression. A major mechanism of transcriptional regulation is chromatin remodeling, and open chromatin is a versatile predictor of regulatory sequences. MicroRNA-mediated post-transcriptional regulation plays an important role in SZ pathogenesis. Neurons differentiated from patient-specific induced pluripotent stem cells (iPSCs) provide an experimental model to characterize the genetic perturbation of regulatory variants that are often specific to cell type and/or developmental stage. The emerging genome-editing technology enables the creation of isogenic iPSCs and neurons to efficiently characterize the effects of SZ-associated regulatory variants on SZ-relevant molecular and cellular phenotypes involving dopaminergic, glutamatergic, and GABAergic neurotransmissions. SZ GWAS findings equipped with the emerging functional genomics approaches provide an unprecedented opportunity for understanding new disease biology and identifying novel drug targets.
    Full-text · Article · Jan 2015 · Neuroscience Bulletin
  • Source
    • "However, obtaining a number of suitable RPE cells for in vitro study is still a problem. Although fibroblasts from skin biopsy or other sources (such as dental pulp cells) were used in many studies for the generation of iPSCs, peripheral blood mononuclear cells (PBMCs) have been widely accepted as a more convenient and an almost unlimited resource for cell reprogramming (Staerk et al., 2010; Seki et al., 2012). In this study, we used EBNA1-based episomal vectors, a non-viral system that can reprogram somatic cells into iPSCs in both feeder-dependent and feeder-free conditions, to generated iPSCs from T cells via electroporation (Figure 2A). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Age-related macular degeneration (AMD) is one retinal aging process that may lead to irreversible vision loss in the elderly. Its pathogenesis remains unclear, but oxidative stress inducing retinal pigment epithelial (RPE) cells damage is perhaps responsible for the aging sequence of retina and may play an important role in macular degeneration. In this study, we have reprogrammed T cells from patients with dry type AMD into induced pluripotent stem cells (iPSCs) via integration-free episomal vectors and differentiated them into RPE cells that were used as an expandable platform for investigating pathogenesis of the AMD and in-vitro drug screening. These patient-derived RPEs with the AMD-associated background (AMD-RPEs) exhibited reduced antioxidant ability, compared with normal RPE cells. Among several screened candidate drugs, curcumin caused most significant reduction of ROS in AMD-RPEs. Pre-treatment of curcumin protected these AMD-RPEs from H2O2-induced cell death and also increased the cytoprotective effect against the oxidative stress of H2O2 through the reduction of ROS levels. In addition, curcumin with its versatile activities modulated the expression of many oxidative stress-regulating genes such as PDGF, VEGF, IGFBP-2, HO1, SOD2, and GPX1. Our findings indicated that the RPE cells derived from AMD patients have decreased antioxidative defense, making RPE cells more susceptible to oxidative damage and thereby leading to AMD formation. Curcumin represented an ideal drug that can effectively restore the neuronal functions in AMD patient-derived RPE cells, rendering this drug an effective option for macular degeneration therapy and an agent against aging-associated oxidative stress.
    Full-text · Article · Aug 2014 · Frontiers in Aging Neuroscience
  • Source
    • "Thus, using this approach in human fibroblasts, transgene-free iPSCs have been generated with efficiencies over 1.5 % [39]. It should be noted that different cell types including fibroblasts, CD34+ cord blood cells [85], and activated T-lymphocytes [86, 87] have been successfully reprogrammed using SeV F protein-deficient and termosensitive (ΔF/TS) vectors to date. Fully reprogrammed iPSCs with Yamanaka factors Oct4, Sox2, Klf, and c-Myc are able to passively eliminate viral RNA through successive cell passages [39]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The therapeutic potential of induced pluripotent stem cells (iPSCs) is well established. Safety concerns remain, however, and these have driven considerable efforts aimed at avoiding host genome alteration during the reprogramming process. At present, the tools used to generate human iPSCs include (1) DNA-based integrative and non-integrative methods and (2) DNA-free reprogramming technologies, including RNA-based approaches. Because of their combined efficiency and safety characteristics, RNA-based methods have emerged as the most promising tool for future iPSC-based regenerative medicine applications. Here, I will discuss novel recent advances in reprogramming technology, especially those utilizing the Sendai virus (SeV) and synthetic modified mRNA. In the future, these technologies may find utility in iPSC reprogramming for cellular lineage-conversion, and its subsequent use in cell-based therapies.
    Full-text · Article · Jul 2013 · Journal of Cardiovascular Translational Research
Show more