Somatic cell nuclear transfer allows trans-acting factors present in the mammalian oocyte to reprogram somatic cell nuclei to an undifferentiated state. We show that four factors (OCT4, SOX2, NANOG, and LIN28) are sufficient to reprogram human somatic cells to pluripotent stem cells that exhibit the essential characteristics of embryonic stem (ES) cells. These induced pluripotent human stem cells have normal karyotypes, express telomerase activity, express cell surface markers and genes that characterize human ES cells, and maintain the developmental potential to differentiate into advanced derivatives of all three primary germ layers. Such induced pluripotent human cell lines should be useful in the production of new disease models and in drug development, as well as for applications in transplantation medicine, once technical limitations (for example, mutation through viral integration) are eliminated.

Reprogramming differentiated human cells to induced pluripotent stem (iPS) cells has applications in basic biology, drug development, and transplantation. Human iPS cell derivation previously required vectors that integrate into the genome, which can create mutations and limit the utility of the cells in both research and clinical applications. Here we describe the derivation of human iPS cells using non-integrating episomal vectors. After removal of the episome, iPS cells completely free of vector and transgene sequences are derived that are similar to human embryonic stem (ES) cells in proliferative and developmental potential. These results demonstrate that reprogramming human somatic cells does not require genomic integration or the continued presence of exogenous reprogramming factors, and removes one obstacle to the clinical application of human iPS cells.

Spinal muscular atrophy (SMA) is one of the most common inherited forms of neurological disease leading to infant mortality. Patients exhibit selective loss of lower motor neurons resulting in muscle weakness, paralysis, and often death. Although patient fibroblasts have been used extensively to study SMA, motor neurons have a unique anatomy and physiology which may underlie their vulnerability to the disease process. Here we report the generation of induced pluripotent stem (iPS) cells from skin fibroblast samples taken from a child with SMA. These cells expanded robustly in culture, maintained the disease genotype, and generated motor neurons that showed selective deficits compared to those derived from the child's unaffected mother. This is the first study to show human iPS cells can be used to model the specific pathology seen in a genetically inherited disease. As such, it represents a promising resource to study disease mechanisms, screen novel drug compounds, and develop new therapies.Spinal muscular atrophy (SMA) is an autosomal recessive genetic disorder caused by mutations in the survival motor neuron 1 gene (SMN1) significantly reducing SMN protein expression 1, 2 and resulting in the selective degeneration of lower α-motor neurons 3 . Clinically, patients with SMA 1 typically show symptoms at 6 months of age and die by age 2 4 . The SMN2 gene is an almost identical copy of SMN1 except that SMN2 has a single nucleotide difference that results in only 10% of full-length protein being produced and high levels of a truncated, unstable protein lacking exon 7 (SMNΔ7) 5 . However, patients with multiple copies of SMN2 produce more full-length protein and have a less severe phenotype 6 . While current model systems using worms, flies, or mice have provided invaluable data concerning the genetic cause of SMA, mechanisms of motor neuron death, and potential drug therapies 7 , they have Correspondence: Reprints and permissions information is available at npg.nature.com/reprintsandpermissions Correspondence should be addressed to ADE (ebert@waisman.wisc.edu) or CNS (cnsvendsen@wisc.edu). Requests for material should be addressed to CNS. Contributions: ADE participated in all aspects and prepared the manuscript; JY generated and aided in characterization of iPS-SMA and iPS-WT clones; FR, VBM, and CLL performed SMN analysis and manuscript preparation; JAT participated in the generation of the iPS clones; CNS conceived the project and participated in planning, data analysis, and manuscript preparation. The authors declare no competing financial interest.Supplementary Information accompanies the paper on www.nature.com/nature. A schematic outlining the main results is included as Supplementary Figure 1.

Abstract-Human

Defined transcription factors can induce epigenetic reprogramming of adult mammalian cells into induced pluripotent stem cells. Although DNA factors are integrated during some reprogramming methods, it is unknown whether the genome remains unchanged at the single nucleotide level. Here we show that 22 human induced pluripotent stem (hiPS) cell lines reprogrammed using five different methods each contained an average of five protein-coding point mutations in the regions sampled (an estimated six protein coding point mutations per exome). The majority of these mutations were non-synonymous, nonsense, or splice variants, and were enriched in genes mutated or having causative effects in cancers. At least half of these reprogramming-associated mutations pre-existed in fibroblast progenitors at low frequencies, while the rest were newly occurring during or after reprogramming. Thus, hiPS cells acquire genetic modifications in addition to epigenetic modifications. Extensive genetic screening should become a standard procedure to ensure hiPS safety before clinical use.

No abstract

For the promise of human induced pluripotent stem cells (iPSCs) to be realized, it is necessary to ask if and how efficiently they may be differentiated to functional cells of various lineages. Here, we have directly compared the neural-differentiation capacity of human iPSCs and embryonic stem cells (ESCs). We have shown that human iPSCs use the same transcriptional network to generate neuroepithelia and functionally appropriate neuronal types over the same developmental time course as hESCs in response to the same set of morphogens; however, they do it with significantly reduced efficiency and increased variability. These results were consistent across iPSC lines and independent of the set of reprogramming transgenes used to derive iPSCs as well as the presence or absence of reprogramming transgenes in iPSCs. These findings, which show a need for improving differentiation potency of iPSCs, suggest the possibility of employing human iPSCs in pathological studies, therapeutic screening, and autologous cell transplantation.

No abstract