Reprogramming Of Human Adipose Mesenchymal Stem Cells To Induced Pluripotent Stem Cells Using A Polycistronic Plasmid

Induced pluripotent stem cells (iPSCs) are derived from somatic cells by ectopic expression of a few transcription factors that have the ability of self-renew indefinitely and to differentiate into all cell types of an orgaism. The generation of patient-specific iPSCs provies an invaluable resource for regenerative medicine that can circumvent both the practical and ethical concerns. Therefore, iPSCs have received widespread attention in basic and clinical research and holds tremendous potential for pharmacologic and medical applications.Although the generation of iPSCs has been proved as a robust technology, recently, the safety remains to be addressed for iPSCs in the clinical applications. In order to reproducibly obtain iPSCs, several variables must be considered which included:effective methods to deliver defined factors in reprogramming process, availability of the cell types for easy introduction of factors without acquired DNA damages, and optimal culture conditions for deriving iPSCs. Therefore, it is essential to improve the methodologies of iPSC generation in human for therapeutic purposes.From the perspectives of safety, two kinds of novel polycistronic plasmid vectors were constructed in this study. Oct4, Sox2, Klf4, and c-Myc genes were amplified by polymerase chain reaction (PCR) using DNA polymerase. After confirmation by DNA sequencing, such correct DNA sequences were joined with self-cleaving2A sequence as a fusion gene (Oct4-P2A-Sox2-T2A-Klf4-E2A-c-Myc, OSKM) within a single open reading frame (ORF). Then, this ORF was cloned upstream of an internal ribosome entry site (IRES) in the pIRES2-EGFP plasmid and driven by a common CMV promoter (Pcmv IE). The GFP gene was promoted by IRES, because IRES permits both the upstream OSKM gene and downstream GFP gene to be translated from a single mRNA. The vector backbone also contains an SV40origin for replication in mammalian cells expressing the SV40T antigen. A neomycin-resistance cassette (Neor) of plasmid vector allows transfected eukaryotic cells to be selected using G418. Thus, taking pIRES2-EGFP plasmid as a basic backbone, we constructed a polycistronic vector pIRES2-OSKM-EGFP. To construct another plamid vector, using pIRES2-OSKM-EGFP as template, amplified the sequences of Oct4and Sox2linked with T2A peptide sequence as a fusion gene (Oct4-P2A—Sox2, OS) within a single ORF. Then, this ORF was inserted upstream of IRES in the pIRES2-EGFP plasmid. This constructed polycistronic plasmid was designated as pIRES2-OS-EGFP. Thus, taking pIRES2-EGFP plasmid as a basic backbone, we constructed two kinds of polycistronic vector and designated as pIRES2-OSKM-EGFP and pIRES2-OS-EGFP respectively.Human ADSCs were isolated and then enriched by serial plate passages. Human adipose-derived mesenchymal stem cells (ADSCs) of passage4were transfected with pIRES2-OSKM-EGFP using Calcium-phosphate-mediated Transfection Method. A second round of transfection was performed on day4. The transfected cells not only exhibited GFP expression, but also formed more aggregates with upheaval-shaped morphology after transfection. These gathered cells adopted a tightly packed morphology and formed a number of undifferentiated clones at3-4weeks after transfection. The cell clones fulfill the critical criteria for successful reprogramming:They exhibited typical iPSCs morphology, and expressed alkaline phosphatase (AP) positive. They expressed high levels of Oct4, Sox2, Klf4and c-Myc mRNAs, while the corresponding vector genes were lost and negative expressed. Like human ESCs, the cell clones were positive for Oct4, Nanog, TRA-1-60and SSEA4by immunofluorescent staining. These cell clones could differentiate into three embryonic germ layers both in vitro by embryoid body generation and in vivo by teratoma formation after being injected into immunodeficient mice. More importantly, the plasmid DNA does not integrate into the genome of cells as revealed by Southern blotting experiments. Karyotypic analysis also demonstrated that the reprogramming of ADSCs by the defined factors did not induce chromosomal abnormalities. Therefore, ADSCs derived iPSCs were generated without the use of a feeder layer, by ectopic expression of the defined transcription factors Oct4, Sox2, Klf4and c-Myc using a polycistronic plasmid.The iPSCs are the ideal and promising sources since they may provide a personalized source of tissue to replace lost cells for degenerative and age-associated diseases. However, the clinical use of iPSCs is hindered by the issue of safety concerns. In view of the fact that the use of proto-oncogenes, such as Klf4or c-Myc, would increases the risk of tumor formation, ADSCs were transfected with pIRES2-OS-EGFP and cultured in stem cell medium supplemented with4ng/ml basic fibroblast growth factor (bFGF), and as a consequence the clear-cut cell colonies were gradually derived. The cells in such colonies had large translucent nuclei and a high nucleocytoplasmic ratio. All of the cells grew at similar rates, requiring sub-culture every4days. They exhibited normal karyotype, and expressed AP positive. They were immunoreactive for Oct4, Nanog, TRA-1-60and SSEA4and were similar to human ESCs in gene expression. Moreover, they can differentiate into cell types of the three embryonic germ layers in both in vitro and in vivo assays. Remarkably, the plasmid DNA did not insert into the genome of cells as revealed by Southern blotting experiments. This system provides a valuable tool for generation of iPSCs, and our data suggest that ADSCs derived iPSCs can be successful generated using only Oct4and Sox2transcription factors. Somatic cell reprogramming may become a powerful approach to generate specific human cell types for cell-fate determination studies and potential transplantation therapies of neurological diseases. Human ADSCs were first transduced with individual polycistronic plasmid on day0. The transduction was repeated on day4using the same volume of plasmid DNA, and cultured in complete DMEM medium. A week later, the medium was replaced with growth medium adding G418for drug selection, and another week later, changed to neural induced medium for neural differentiation culturing. Reprogrammed hADSCs were expanded and characterized for pluripotency as revealed by immunofluorescence and RT-PCR. Moreover, the generated cells had normal karyotypes and exogenous vector sequences were not inserted into the genomes. Three weeks after being cultured in neural induce medium, the cells expressed neural cells related markers, Nestin, GFAP, TUJ1, RIP and Nurrl. As determined by counting the number of positive cells per vision under a fluorescence microscope, the number of GFAP, RIP, TUJ1and Nurrl-positive cells were70.5±8.0%(n=258),60.4±5.2%(n=212),82.7±7.3%(n=266) and73.6±9.2%(n=244) respectively. Thus, induction of neural differentiation in reprogrammed ADSCs with defined transcription factors methodology bypasses the risk of mutation, gene instability, and provides an alternative strategy for patient-specific neural cells for basic research and therapeutic application.