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.

We describe a reproducible method for growing small intestinal epithelium (derived from the suckling rat intestine) in short-term (primary) cultures. Optimal culture conditions were determined by quantitative assays of proliferation (i.e. changes in cellularity and DNA synthesis). Isolation of the epithelia and, significantly, preservation of its three-dimensional integrity was achieved using a collagenase/dispase digestion technique. Purification of the epithelium was also facilitated by the use of a simple differential sedimentation method. The results presented below support the idea that proliferation of normal gut epithelium ex vivo is initially dependent upon the maintenance of the structural integrity of this tissue and upon factors produced by heterologous mesenchymal cells. Proliferation in vitro was also critically dependent upon the quality of the medium and constituents used. Cultures reached confluence within 10–14 days and consisted of epithelial colonies together with varying amounts of smooth-muscle-like cells. Cultures have been maintained for periods up to one month, but the longer-term potential for growth by sub-culturing has not been examined. Strategies for reducing the proliferation of these non-epithelial cells are also described.

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