Abstract
Objective: Stem cell replacement therapy through the reprogramming of somatic cells is a significant prospective therapy for neurodegenerative diseases and personalized medicine. As virus-carrying reprogramming genes impart a considerable risk for tumor formation, current approaches tend to replace the viral pattern with a non-viral system. However, there is still great concern regarding the oncogenic properties of these reprogramming genes. At present no direct evidence can verify that non-viral systems do not influence genomic DNA integration and mutagenesis events in the final products after reprogramming. Methods: We evaluated the potential cancer risk of reprogrammed stem cell-derived dopaminergic neurons using a unique non-viral vector containing four reprogramming genes. Reprogrammed stem cell-derived dopaminergic neurons were the final products after 50 days of cell culture from adult mice fibroblasts. After 6 months of culture, these cells were assessed for cancer risk. Results: Overall cancer risk assessments were first examined using multiple stem cell biomarkers. We did not detect any overexpression of these biomarkers. Moreover, we assessed whether or not the most important neurotransmission factors were expressed after 6 months in mass cell culture. Our results determined that only robust gene expression of tyrosine hydroxylase (TH), as the rate-limiting enzyme in dopamine synthesis, was detected. Furthermore, the final product of TH-positive dopaminergic neurons was confirmed by sequencing TH genomic DNA. Our data suggested that none of the four reprogramming genes integrated into the host cell’s genomic DNA and that no other mutagenesis events occurred after gene sequencing TH genomic DNA related complete 13 coding exons with two-ended partial untranslated regions. Conclusion: Re-programmed adult mouse fibroblasts-derived dopaminergic neurons were proven to be a safe technology and approach as a potential therapy for neurodegenerative diseases.
Highlights
Stem cell replacement therapy has potential clinical applications by inducing neuroplasticity in neurodegenerative disorders, such as Parkinson’s disease (PD), Alzheimer’s disease (AD) and spinal cord injury, and possibly neuropsychiatric illnesses [1,2]
Our results determined that only robust gene expression of tyrosine hydroxylase (TH), as the rate-limiting enzyme in dopamine synthesis, was detected
Our data suggested that none of the four reprogramming genes integrated into the host cell’s genomic DNA and that no other mutagenesis events occurred after gene sequencing TH genomic DNA related complete 13 coding exons with two-ended partial untranslated regions
Summary
Stem cell replacement therapy has potential clinical applications by inducing neuroplasticity in neurodegenerative disorders, such as Parkinson’s disease (PD), Alzheimer’s disease (AD) and spinal cord injury, and possibly neuropsychiatric illnesses [1,2]. One critical limitation of the use of induced pluripotent stem cells (iPSCs) derived from re-programmed somatic cells for longterm cell culturing is the fact that the viral-based (usually retroviral) transfection methods applied in the host cells pose a considerable risk of tumor formation. This is attributable to the random and permanent integration of viral genomic DNA into the host cells’ genome. There is no straight proof that a non-viral reprogramming system does not have a proficient to inspire genomic DNA integration and mutagenesis events for the final product such as re-programmed dopaminergic neurons, in particular, in the long-term in vitro cell culture surroundings
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