Sujittra Khampang1,2, Rangsun Parnpai2, Wiriya Mahikul3, Charles A Easley1,4,5, In Ki Cho6,7, Anthony W S Chan8,9. 1. Division of Neuropharmacology and Neurologic Diseases, Yerkes National Primate Research Center, Atlanta, GA, USA. 2. Embryo Technology and Stem Cell Research Center, School of Biotechnology, Suranaree University of Technology, Nakhon Ratchasima, Thailand. 3. Faculty of Medicine and Public Health, HRH Princess Chulabhorn College of Medical Science, Chulabhorn Royal Academy, Bangkok, Thailand. 4. Department of Environmental Health Science, College of Public Health, University of Georgia, Athens, GA, USA. 5. Regenerative Bioscience Center, University of Georgia, Athens, GA, USA. 6. Division of Neuropharmacology and Neurologic Diseases, Yerkes National Primate Research Center, Atlanta, GA, USA. inkicho@outlook.com. 7. Department of Human Genetics, Emory University, Atlanta, GA, 30322, USA. inkicho@outlook.com. 8. Division of Neuropharmacology and Neurologic Diseases, Yerkes National Primate Research Center, Atlanta, GA, USA. awschan@hotmail.com. 9. Department of Human Genetics, Emory University, Atlanta, GA, 30322, USA. awschan@hotmail.com.
Abstract
PURPOSE: The expansion of CAG (glutamine; Q) trinucleotide repeats (TNRs) predominantly occurs through male lineage in Huntington's disease (HD). As a result, offspring will have larger CAG repeats compared to their fathers, which causes an earlier onset of the disease called genetic anticipation. This study aims to develop a novel in vitro model to replicate CAG repeat instability in early spermatogenesis and demonstrate the biological process of genetic anticipation by using the HD stem cell model for the first time. METHODS: HD rhesus monkey embryonic stem cells (rESCs) were cultured in vitro for an extended period. Male rESCs were used to derive spermatogenic cells in vitro with a 10-day differentiation. The assessment of CAG repeat instability was performed by GeneScan and curve fit analysis. RESULTS: Spermatogenic cells derived from rESCs exhibit progressive expansion of CAG repeats with high daily expansion rates compared to the extended culture of rESCs. The expansion of CAG repeats is cell type-specific and size-dependent. CONCLUSIONS: Here, we report a novel stem cell model that replicates genome instability and CAG repeat expansion in in vitro derived HD monkey spermatogenic cells. The in vitro spermatogenic cell model opens a new opportunity for studying TNR instability and the underlying mechanism of genetic anticipation, not only in HD but also in other TNR diseases.
PURPOSE: The expansion of CAG (glutamine; Q) trinucleotide repeats (TNRs) predominantly occurs through male lineage in Huntington's disease (HD). As a result, offspring will have larger CAG repeats compared to their fathers, which causes an earlier onset of the disease called genetic anticipation. This study aims to develop a novel in vitro model to replicate CAG repeat instability in early spermatogenesis and demonstrate the biological process of genetic anticipation by using the HD stem cell model for the first time. METHODS: HD rhesus monkey embryonic stem cells (rESCs) were cultured in vitro for an extended period. Male rESCs were used to derive spermatogenic cells in vitro with a 10-day differentiation. The assessment of CAG repeat instability was performed by GeneScan and curve fit analysis. RESULTS: Spermatogenic cells derived from rESCs exhibit progressive expansion of CAG repeats with high daily expansion rates compared to the extended culture of rESCs. The expansion of CAG repeats is cell type-specific and size-dependent. CONCLUSIONS: Here, we report a novel stem cell model that replicates genome instability and CAG repeat expansion in in vitro derived HD monkey spermatogenic cells. The in vitro spermatogenic cell model opens a new opportunity for studying TNR instability and the underlying mechanism of genetic anticipation, not only in HD but also in other TNR diseases.
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