Supplementation of vitamin C promotes early germ cell specification from human embryonic stem cells
Abstract Background As the precursors of sperm and eggs, human primordial germ cells (hPGCs) emerge as early as weeks 2 to 3 of post-implantation development. Recently, robust hPGC induction models have been established in vitro with different protocols, but global 5mC/5hmC epigenetic reprogramming...
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| Format: | Article |
| Language: | English |
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BMC
2019-11-01
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| Series: | Stem Cell Research & Therapy |
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| Online Access: | http://link.springer.com/article/10.1186/s13287-019-1427-2 |
| _version_ | 1818532522892460032 |
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| author | Zili Li Fang Fang Qian Zhao Honggang Li Chengliang Xiong |
| author_facet | Zili Li Fang Fang Qian Zhao Honggang Li Chengliang Xiong |
| author_sort | Zili Li |
| collection | DOAJ |
| description | Abstract Background As the precursors of sperm and eggs, human primordial germ cells (hPGCs) emerge as early as weeks 2 to 3 of post-implantation development. Recently, robust hPGC induction models have been established in vitro with different protocols, but global 5mC/5hmC epigenetic reprogramming is not initiated in vitro. Previous studies found that vitamin C can enhance Tet (ten-eleven translocation) enzyme expression and improve 5hmC level in cells. But the effect of vitamin C supplementation on hPGC in vitro induction is still unknown. Methods We generated a gene-edited human embryonic stem cell (hESC) line carrying a BLIMP1-mkate2 reporter by CRISPR/Cas9 technology and used flow cytometry to optimize the PGC differentiation protocol; meanwhile, the expression of PGC genes (BLIMP1, TFAP2C, SOX17, OCT4) was evaluated by qRT-PCR. When different concentrations of vitamin C were added to the induction medium, the percentage of hPGCLCs (hPGC-like cells) was analyzed by flow cytometry; dot blot and ELISA were used to detect the levels of 5hmC and 5mC. The expression of TET enzymes was also evaluated by qRT-PCR. Results We optimized the PGC differentiation protocol with the BLIMP1-mkate reporter hESCs, and the efficiency of PGC induction in vitro can be improved to 30~40%. When 50 μg/mL vitamin C was added, the derived hPGCLCs not only upregulated the expression of key genes involved in human early germ cell development such as NANOS3, TFAP2C, BLIMP1, and SOX17, but also increased the levels of 5hmC and TET enzymes. Conclusions Taken together, supplementation of vitamin C can promote the in vitro induction of hPGCLCs from hESCs, which might be related to vitamin C-mediated epigenetic regulations during the differentiation process. |
| first_indexed | 2024-12-11T17:46:38Z |
| format | Article |
| id | doaj.art-a0fad603937f4e438b0aa05293d34e94 |
| institution | Directory Open Access Journal |
| issn | 1757-6512 |
| language | English |
| last_indexed | 2024-12-11T17:46:38Z |
| publishDate | 2019-11-01 |
| publisher | BMC |
| record_format | Article |
| series | Stem Cell Research & Therapy |
| spelling | doaj.art-a0fad603937f4e438b0aa05293d34e942022-12-22T00:56:21ZengBMCStem Cell Research & Therapy1757-65122019-11-011011910.1186/s13287-019-1427-2Supplementation of vitamin C promotes early germ cell specification from human embryonic stem cellsZili Li0Fang Fang1Qian Zhao2Honggang Li3Chengliang Xiong4Institute of Reproductive Health, Tongji Medical College, Huazhong University of Science and TechnologyDepartment of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and TechnologyInstitute of Reproductive Health, Tongji Medical College, Huazhong University of Science and TechnologyInstitute of Reproductive Health, Tongji Medical College, Huazhong University of Science and TechnologyInstitute of Reproductive Health, Tongji Medical College, Huazhong University of Science and TechnologyAbstract Background As the precursors of sperm and eggs, human primordial germ cells (hPGCs) emerge as early as weeks 2 to 3 of post-implantation development. Recently, robust hPGC induction models have been established in vitro with different protocols, but global 5mC/5hmC epigenetic reprogramming is not initiated in vitro. Previous studies found that vitamin C can enhance Tet (ten-eleven translocation) enzyme expression and improve 5hmC level in cells. But the effect of vitamin C supplementation on hPGC in vitro induction is still unknown. Methods We generated a gene-edited human embryonic stem cell (hESC) line carrying a BLIMP1-mkate2 reporter by CRISPR/Cas9 technology and used flow cytometry to optimize the PGC differentiation protocol; meanwhile, the expression of PGC genes (BLIMP1, TFAP2C, SOX17, OCT4) was evaluated by qRT-PCR. When different concentrations of vitamin C were added to the induction medium, the percentage of hPGCLCs (hPGC-like cells) was analyzed by flow cytometry; dot blot and ELISA were used to detect the levels of 5hmC and 5mC. The expression of TET enzymes was also evaluated by qRT-PCR. Results We optimized the PGC differentiation protocol with the BLIMP1-mkate reporter hESCs, and the efficiency of PGC induction in vitro can be improved to 30~40%. When 50 μg/mL vitamin C was added, the derived hPGCLCs not only upregulated the expression of key genes involved in human early germ cell development such as NANOS3, TFAP2C, BLIMP1, and SOX17, but also increased the levels of 5hmC and TET enzymes. Conclusions Taken together, supplementation of vitamin C can promote the in vitro induction of hPGCLCs from hESCs, which might be related to vitamin C-mediated epigenetic regulations during the differentiation process.http://link.springer.com/article/10.1186/s13287-019-1427-2Embryonic stem cellsPrimordial germ cellsVitamin CEpigenetic |
| spellingShingle | Zili Li Fang Fang Qian Zhao Honggang Li Chengliang Xiong Supplementation of vitamin C promotes early germ cell specification from human embryonic stem cells Stem Cell Research & Therapy Embryonic stem cells Primordial germ cells Vitamin C Epigenetic |
| title | Supplementation of vitamin C promotes early germ cell specification from human embryonic stem cells |
| title_full | Supplementation of vitamin C promotes early germ cell specification from human embryonic stem cells |
| title_fullStr | Supplementation of vitamin C promotes early germ cell specification from human embryonic stem cells |
| title_full_unstemmed | Supplementation of vitamin C promotes early germ cell specification from human embryonic stem cells |
| title_short | Supplementation of vitamin C promotes early germ cell specification from human embryonic stem cells |
| title_sort | supplementation of vitamin c promotes early germ cell specification from human embryonic stem cells |
| topic | Embryonic stem cells Primordial germ cells Vitamin C Epigenetic |
| url | http://link.springer.com/article/10.1186/s13287-019-1427-2 |
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