No Evidence for Clonal Selection Due to Lentiviral Integration Sites in Human Induced Pluripotent Stem Cells (original) (raw)
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Derivation of Induced Pluripotent Stem Cells by Retroviral Gene Transduction in Mammalian Species
Methods in Molecular Biology, 2012
This chapter provides a method for reprogramming human dermal fibroblasts into induced pluripotent stem cells (iPSCs) using three lentiviruses containing cDNAs for OCT4 and SOX2, KLF4 and C-MYC, and NANOG and LIN28, respectively. Lentiviral vectors are based on the human immunodeficiency virus (HIV) and provide an effective means for the delivery, integration, and expression of exogenous genes in mammalian cells. Lentiviruses are attractive gene delivery vehicles as they are able to infect both proliferating and nonproliferating cells. Lentiviruses stably integrate into the genome without incurring cellular toxicity and can maintain sustained transgene expression during prolonged host cell proliferation and differentiation. In this protocol, we describe how to prepare lentiviruses, stably transduce human fibroblasts, and identify bona fide iPSC colonies based on morphological similarity to human embryonic stem cell (ESC) colonies and live-cell immunological staining using cell-surface markers of human PSCs such as Tra-1-60 and Tra-1-81.
Induced pluripotent stem cells: opportunities and challenges
Philosophical Transactions of the Royal Society B: Biological Sciences, 2011
Somatic cells have been reprogrammed into pluripotent stem cells by introducing a combination of several transcription factors, such as Oct3/4, Sox2, Klf4, and c-Myc.
Induced Pluripotent Stem Cell Generation Using a Single Lentiviral Stem Cell Cassette
Stem Cells, 2009
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 postnatal 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.
Induced pluripotent Stem Cells: Where we are currently?
Halo 194, 2020
Induced Pluripotent Stem Cells (iPSCs) are a type of pluripotent stem cells generated by reprogramming an adult somatic cell genome to the stage of a pluripotent stem cell in vitro by inducing a forced expression of specific transcription factors that are important for the maintenance of pluripotency. The iPSCs seem to be very similar to Embryonic Stem Cells (ESCs) in terms of morphology, cell surface markers and gene expression levels, but recent studies have demonstrated some differences between the two cell types. However, iPSCs might have potential application in regenerative medicine, transplantation, drug testing, disease modelling, and avoidance of tissue rejection and with less ethical concern than ESCs. This paper aims to present the most important characteristics of iPSCs which have therapeutic significance.
Induced pluripotent stem cells generated without viral integration
Hepatology, 2009
Pluripotent stem cells have been generated from mouse and human somatic cells by viral expression of the transcription factors Oct4, Sox2, Klf4, and c-Myc. A major limitation of this technology is the use of potentially harmful genome-integrating viruses. Here, we generate mouse induced pluripotent stem cells (iPS) from fibroblasts and liver cells by using non-integrating adenoviruses transiently expressing Oct4, Sox2, Klf4, and c-Myc. These adenoviral iPS (Adeno-iPS) cells show DNA demethylation characteristic of reprogrammed cells, express endogenous pluripotency genes, form teratomas, and contribute to multiple tissues, including the germline, in chimeric mice. Our results show that insertional mutagenesis is not required for in vitro reprogramming. Adenoviral reprogramming may provide an improved method for generating and studying patient-specific stem cells and for comparing embryonic stem (ES) cells and iPS cells.
Journal of Biomedicine, 2016
Background: Human somatic cells are reprogrammed to induced pluripotent stem (iPS) cells when four transcription factors (Oct4, Sox2, Klf4, and Myc) are ectopically expressed in them. These iPS cells, which evade immunological rejection, are a valuable source for patient-specific cell therapy. Lentiviral systems have been proved to be powerful tools for cellular reprogramming, an example of which is the induction of iPS cells from somatic cells that requires a high transduction efficiency of lentiviruses harboring the four reprogramming factors. Objectives: The purpose of this study was to define an optimized calcium phosphate transfection method to produce high-titer lentiviral vectors for the generation of human iPS cells. Materials and Methods: In this study, the calcium phosphate transfection method was used to generate lentiviruses. The virus supernatant was concentrated using Amicon Ultra-4 column. Results: This method resulted in 80% GFP-positive cells and viral preparations of 2.4 × 10 8 viral particles/mL. Conclusions: This method is both cost effective and simple to adopt.
Human Induced Pluripotent Stem Cells: The Past, Present, and Future
Clinical Pharmacology & Therapeutics
In 2006, Yamanaka and Takahashi electrified the scientific community by discovering that mouse somatic cells can be converted into embryonic stem cell-like cells by retroviral transduction of four transcription factors: Oct4, Sox2, Klf4, and c-Myc (OSKM). The first generation of mouse induced pluripotent stem (iPS) cells was incompletely reprogrammed, and failed to contribute to germline transmission. Nearly one year later, three groups, including Yamanaka's, improved the reprogramming methodology and generated iPS cells that were in many respects, indistinguishable from ES cells, and also contributed to chimera formation and germline transmission. Shortly thereafter, the successful reprogramming of human somatic cells opened the gate for the development of patient-specific iPS cells for biomedical research and clinical application. Though human iPS cells resemble human ES cells in many aspects, the current iPS cell technologies showed several limitations for clinical usage. First, the efficiency of iPS cell generation is still low and the reprogramming process takes at least two weeks. Second, the virus-delivery of reprogramming factors introduces inconceivable risks of insertional mutagenesis in the genome. Third, given the various strategies for direct reprogramming, it remains difficult to assess the quality of iPS cells generated in each lab and for each patient. These issues should be addressed properly before any iPS cells could be translated into clinic. Here, we review recent progress in human iPS cell technologies, with a focus on the virus-free and integration-free iPS cell generation, which may lead towards the eventual goal of clinical applications.
Gene Transfer into Pluripotent Stem Cells via Lentiviral Transduction
Recombinant lentiviral vectors are powerful tools to stably manipulate human pluripotent stem cells. They can be used to deliver ectopic genes, shRNAs, miRNAs, or any possible genetic DNA sequence into diving and nondividing cells. Here we describe a general protocol for the production of self-inactivating lentiviral vector particles and their purification to high titers by either ultracentrifugation or ultrafiltration. Next we provide a basic procedure to transduce human pluripotent stem cells and propagate clonal cell lines.