Generation of Transgene-Free Lung Disease-Specific Human Induced Pluripotent Stem Cells Using a Single Excisable Lentiviral Stem Cell Cassette  

Abstract

The development of methods to achieve efficient reprogramming of human cells while avoiding the permanent presence of reprogramming transgenes represents a critical step towards the use of induced pluripotent stem cells (iPSC) for clinical purposes, such as disease modeling or reconstituting therapies. While several methods exist for generating iPSC free of reprogramming transgenes from mouse cells or neonatal normal human tissues, a sufficiently efficient reprogramming system is still needed in order to achieve the widespread derivation of disease-specific iPSC from humans with inherited or degenerative diseases. Here we report the use of a humanized version of a single lentiviral ‘stem cell cassette’ vector in order to accomplish efficient reprogramming of normal or diseased skin fibroblasts obtained from humans of virtually any age. Simultaneous transfer of either 3 or 4 reprogramming factors into human target cells using this single vector allows derivation of human iPSC containing a single excisable viral integration, that upon removal generates human iPSC free of integrated transgenes. As a proof of principle, here we apply this strategy to generate >100 lung disease-specific iPSC lines from individuals with a variety of diseases affecting the epithelial, endothelial, or interstitial compartments of the lung, including cystic fibrosis, alpha-1 antitrypsin deficiency-related emphysema, scleroderma (SSc), and sickle cell disease. Moreover, we demonstrate that human iPSC generated with this approach have the ability to robustly differentiate into definitive endoderm in vitro, the developmental precursor tissue of lung epithelia.

Brief Report: Impaired Cell Reprogramming in Nonhomologous End Joining Deficient Cells

“…Passage 2 MEFs were either transduced or transfected using newly developed reprogramming lentiviral vectors (LV) [12,13] or Sleeping Beauty transposon (SB) [14,15] in conjunction with hyperactive transposase mutant SB100X [14] and then cultured for iPSC derivation. Further details can be found in Supporting Information Material and Methods.…”

Scalable stirred suspension culture for the generation of billions of human induced pluripotent stem cells using single‐use bioreactors

“…FSiPS cells were generated from normal human dermal fibroblasts (NHDF; Promocell, Heidelberg, Germany) by reprogramming using hSTEMCCA-lentiviral construct as described by Somers and colleagues with minor changes (Somers et al, 2010). Briefly, 1 × 10 5 cells were plated on a well of a six-well plate pre-coated with 0.1% gelatin (Sigma-Aldrich, St Louis, MO, USA) in fibroblast medium consisting of Dulbecco's modified Eagle's medium (DMEM; Gibco®, Waltham, MA, USA), 10% fetal calf serum (FCS; Biochrom, Berlin, Germany), 1% non-essential amino acids (NEAA; Gibco), and 100 μM β-mercaptoethanol (Gibco).…”

Induced pluripotent stem cells used to reveal drug actions in a long QT syndrome family with complex genetics

“…Human fibroblast reprogramming and characterization of iPSCs Reprogramming was performed with the hSTEMCCA-loxPlentiviral vector, encoding the four human factors, OCT4, KLF4, SOX2, and cMYC, as previously described (Sommer et al, 2009;Somers et al, 2010;Wang et al, 2011). In brief, 10 5 human dermal fibroblasts were plated in Dulbecco's modified Eagle's medium (DMEM) with 10% FBS on a gelatin-coated 35-mm plastic tissue culture dish.…”

“…To understand the limitations of this therapeutic approach and the complications of high dose mexiletine therapy, we studied the effects of the drug in iPSC-CMs derived from members of this LQTS family (Sommer et al, 2009;Somers et al, 2010). This approach enabled us to determine channel activity and pharmacology within a genetically correct cardiac background of the patient.…”