No abstract

Induced Pluripotent Stem (iPS) cells can be generated using retroviral vectors expressing Oct4, Klf4, Sox2 and cMyc. Most prior studies have required multiple retroviral vectors for reprogramming, resulting in high numbers of genomic integrations in iPS cells and limiting their use for therapeutic applications. Here we describe the use of a single lentiviral vector expressing a ‘stem cell cassette’ comprised of the four transcription factors and a combination of 2A peptide and IRES technology, generating iPS cells from post-natal fibroblasts. iPS cells generated in this manner display ES cell-like morphology, express stem cell markers and exhibit in vivo pluripotency as evidenced by their ability to differentiate in teratoma assays and their robust contribution to mouse chimeras. Combining all factors into a single transcript achieves the most efficient reprogramming system to date, and allows derivation of iPS cells with a single viral integration. The use of a single lentiviral vector for reprogramming represents a powerful laboratory tool and a significant step toward the application of iPS technology for clinical purposes.

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.

The residual presence of integrated transgenes following the derivation of induced pluripotent stem (iPS) cells is highly undesirable. Here we demonstrate efficient derivation of iPS cells free of exogenous reprogramming transgenes using an excisable polycistronic lentiviral vector. A novel version of this vector containing a reporter fluorochrome allows direct visualization of vector excision in living iPS cells in real time. We find that removal of the reprogramming vector markedly improves the developmental potential of iPS cells and significantly augments their capacity to undergo directed differentiation in vitro. We further propose that methods to efficiently excise reprogramming transgenes with minimal culture passaging, such as those demonstrated here, are critical since we find that iPS cells may acquire chromosomal abnormalities, such as trisomy of chromosome 8, similar to ESC after expansion in culture. Our findings illustrate an efficient method for the generation of transgene-free iPS cells and emphasize the potential beneficial effects that may result from elimination of integrated reprogramming factors. In addition, our results underscore the consequences of long-term culture that will need to be taken into account for the clinical application of iPS cells.

Transcripts from five cell cycle related genes accumulate in isolated cells dispersed throughout the actively dividing regions of plant meristems. We propose that this pattern reflects gene expression during particular phases of the cell division cycle. The high proportion of isolated cells suggests that synchrony between daughter cells is rapidly lost following mitosis. This is the first time that such a cell specific expression pattern has been described in a higher organism. Counterstaining with a DNA specific dye revealed that transcripts from three genes (two mitotic cyclins and a cdc2‐like gene) accumulate during part of interphase and early mitosis whereas transcripts from a histone H4 gene are preferentially detected only in interphase cells. Double labelling for cyclin and histone H4 transcripts confirms that these genes are expressed in different cells, and therefore at different phases of the cell cycle. The results suggest that transcriptional regulation of cell cycle related genes may be important in controlling cell division in plants, and that these genes are useful markers for identifying cells at specific phases of the cell cycle within plant meristems.