The utility of induced pluripotent stem (iPS) cells for investigating the molecular logic of pluripotency and for eventual clinical application is limited by the low efficiency of current methods for reprogramming. Here we show that reprogramming of juvenile human primary keratinocytes by retroviral transduction with OCT4, SOX2, KLF4 and c-MYC is at least 100-fold more efficient and twofold faster compared with reprogramming of human fibroblasts. Keratinocyte-derived iPS (KiPS) cells appear indistinguishable from human embryonic stem cells in colony morphology, growth properties, expression of pluripotency-associated transcription factors and surface markers, global gene expression profiles and differentiation potential in vitro and in vivo. To underscore the efficiency and practicability of this technology, we generated KiPS cells from single adult human hairs. Our findings provide an experimental model for investigating the bases of cellular reprogramming and highlight potential advantages of using keratinocytes to generate patient-specific iPS cells.

Lentiviral vectors offer unique versatility and robustness as vehicles for gene delivery. They can transduce a wide range of cell types and integrate into the host genome in both dividing and post-mitotic cells, resulting in long-term expression of the transgene both in vitro and in vivo. This protocol describes how lentiviral vectors can be produced, purified and titrated. High titer suspensions can be routinely prepared with relative ease: a low-titer (10(6) viral particles/ml) unpurified preparation can be obtained 3 d after transfecting cells with lentiviral vector and packaging plasmids; a high-titer (10(9) viral particles/ml) purified preparation requires 2 more days.

The generation of induced pluripotent stem (iPS) cells has enabled the derivation of patient-specific pluripotent cells and provided valuable experimental platforms to model human disease. Patientspecific iPS cells are also thought to hold great therapeutic potential, although direct evidence for this is still lacking. Here we show that, on correction of the genetic defect, somatic cells from Fanconi anaemia patients can be reprogrammed to pluripotency to generate patient-specific iPS cells. These cell lines appear indistinguishable from human embryonic stem cells and iPS cells from healthy individuals. Most importantly, we show that corrected Fanconi-anaemia-specific iPS cells can give rise to haematopoietic progenitors of the myeloid and erythroid lineages that are phenotypically normal, that is, disease-free. These data offer proof-of-concept that iPS cell technology can be used for the generation of disease-corrected, patient-specific cells with potential value for cell therapy applications.

We describe the use of lentiviral vectors expressing small interfering RNAs (siRNAs) to knock down the expression of specific genes in vitro and in vivo. A lentiviral vector capable of generating siRNA specific for GFP after transduction of 293T-GFP cell lines showed no GFP fluorescence. Furthermore, no GFP-specific RNA could be detected. When eggs from GFP-positive transgenic mice were transduced with lentivirus-expressing siGFP virus, reduced fluorescence could be seen in blastocysts. More interestingly, pups from F1 progeny, which expressed siGFP, showed considerably diminished fluorescence and decreased GFP. We propose that an approach of combining transgenesis by lentiviral vectors expressing siRNAs can be used successfully to generate a large number of mice in which the expression of a specific gene(s) can be down-regulated substantially. We believe that this approach of generating ''knockdown'' mice will aid in functional genomics.gene silencing ͉ transgenesis ͉ functional genomics

Induced pluripotent stem (iPS) cells have generated keen interest due to their potential use in regenerative medicine. They have been obtained from various cell types of both mice and humans by exogenous delivery of different combinations of Oct4, Sox2, Klf4, c-Myc, Nanog, and Lin28. The delivery of these transcription factors has mostly entailed the use of integrating viral vectors (retroviruses or lentiviruses), carrying the risk of both insertional mutagenesis and oncogenesis due to misexpression of these exogenous factors. Therefore, obtaining iPS cells that do not carry integrated transgene sequences is an important prerequisite for their eventual therapeutic use. Here we report the generation of iPS cell lines from mouse embryonic fibroblasts with no evidence of integration of the reprogramming vector in their genome, achieved by nucleofection of a polycistronic construct coexpressing Oct4, Sox2, Klf4, and c-Myc.iPS cells ͉ nonintegrative technique ͉ reprogramming ͉ pluripotency T he reprogramming of somatic cells to induced pluripotent stem (iPS) cells by delivery of exogenous factors was first achieved in mouse embryonic fibroblasts (MEFs) by retroviral transduction of the transcription factors Oct4, Sox2, Klf4, and c-Myc (OSKM) (1). This result was confirmed and expanded in a series of reports which established that iPS cells are similar to ES cells in morphology, expression of pluripotency markers, and ability to differentiate into the 3 germ layers in vitro and in vivo, including the ability to produce germline-competent chimeras (2-4). The technique was quickly reproduced with human fibroblasts (5-7), providing a source of patient-specific pluripotent cells with potential for regenerative medicine. Reprogramming has been achieved in a range of cell types, including embryonic and adult fibroblasts, keratinocytes, stomach cells, liver cells, pancreatic ␤ cells, lymphocytes, and neural progenitor cells (5-13). Moreover, reprogramming can be attained using different combinations of exogenous factors: human fibroblasts have been reprogrammed with Oct4, Sox2, Nanog, and Lin28 (14), and c-Myc is not strictly required for reprogramming, although it enhances its efficiency and speed (15, 16). Certain cell types have been reprogrammed with only 2 factors, like neural progenitors with Oct4 and c-Myc or Oct4 and Klf4 (10), and both mouse and human fibroblasts with Oct4 and either Sox2 or Klf4 in combination with small molecules (17,18).Although different reprogramming protocols have been reported, the delivery of the original OSKM transcription factor set remains the most commonly used method. Reprogramming requires the delivery of all of the factors to the cell and their adequate expression for a period of Ϸ8-12 days for fibroblasts and keratinocytes (13,19,20). The retroviral and (both constitutive and inducible) lentiviral (14,19,21,22) vectors commonly used meet these requirements, but their permanent integration into the genome limits their use for eventual therapeutic applications because of the risk of bo...