Inhibition of LSD1 promotes the differentiation of human induced pluripotent stem cells into insulin-producing cells
Abstract Background Human induced pluripotent stem cells (hiPSCs) represent a potentially unlimited source of pancreatic endocrine lineage cells. Although insulin-producing β cells derived from hiPSCs have been successfully induced, much work remains to be done to achieve mature β cells. Lysine-spec...
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| Format: | Article |
| Language: | English |
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BMC
2020-05-01
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| Series: | Stem Cell Research & Therapy |
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| Online Access: | http://link.springer.com/article/10.1186/s13287-020-01694-8 |
| _version_ | 1818852464465543168 |
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| author | Xiao-Fei Yang Shu-Yan Zhou Ce Wang Wei Huang Ning Li Fei He Fu-Rong Li |
| author_facet | Xiao-Fei Yang Shu-Yan Zhou Ce Wang Wei Huang Ning Li Fei He Fu-Rong Li |
| author_sort | Xiao-Fei Yang |
| collection | DOAJ |
| description | Abstract Background Human induced pluripotent stem cells (hiPSCs) represent a potentially unlimited source of pancreatic endocrine lineage cells. Although insulin-producing β cells derived from hiPSCs have been successfully induced, much work remains to be done to achieve mature β cells. Lysine-specific demethylase 1 (LSD1) plays an important role in the regulation of hiPSC self-renewal and differentiation. We propose a new strategy to acquire insulin-producing cells (IPCs) from hiPSCs by knocking down LSD1. Methods Knockdown of LSD1 in hiPSCs with five shRNA. Assessment of the effects of shRNA on hiPSC proliferation, cell cycle, and apoptosis. Using knockdown hiPSCs with 31.33% LSD1 activity, we achieved a four-step differentiation into IPCs and test its differentiation efficiency, morphology, and marker genes and proteins. We implanted the IPCs into the renal subcapsular of SCID-Beige diabetic mice to evaluate the hypoglycemic effect in vivo. We tested LSD1 and HDAC1 whether they are present in the CoREST complex through IP-WB, and analyzed LSD1, CoREST, HDAC1, H3K4me2/me3, and H3K27me3 protein expression before and after knockdown of LSD1. Results Differentiated hiPSCs were 38.32% ± 3.54% insulin-positive cells and released insulin/C-peptide in response to glucose stimulus in a manner comparable to adult human islets. Most of the IPCs co-expressed mature β cell-specific markers. When transplanted under the left renal capsule of SCID-Beige diabetic mice, these IPCs reversed hyperglycemia, leading to a significant increase in the definitive endoderm cells. IP-WB results showed that LSD1, HDAC1, and CoREST formed a complex in hiPSCs. Chip-PCR results showed that LSD1, HDAC1, and CoREST were enriched in the same district during the SOX17 and FOXA2 promoter region. Inhibition of LSD1 would not affect the level of CoREST but decreased the HDAC1 expressions. The H3K4me2/me3 and H3K9act level of SOX17 and FOXA2 promoter region increased after inhibited of LSD1, and promoted transcriptional activation. The H3K4me2/me3 and H3K9act level of OCT4 and SOX2 promoter region decreased with the transcriptional repressed. Conclusions LSD1 regulated histone methylation and acetylation in promoter regions of pluripotent or endodermal genes. Our results suggest a highly efficient approach to producing IPCs from hiPSCs. |
| first_indexed | 2024-12-19T07:21:20Z |
| format | Article |
| id | doaj.art-28ad016137b949608760b78ec988fda4 |
| institution | Directory Open Access Journal |
| issn | 1757-6512 |
| language | English |
| last_indexed | 2024-12-19T07:21:20Z |
| publishDate | 2020-05-01 |
| publisher | BMC |
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| series | Stem Cell Research & Therapy |
| spelling | doaj.art-28ad016137b949608760b78ec988fda42022-12-21T20:30:55ZengBMCStem Cell Research & Therapy1757-65122020-05-0111111310.1186/s13287-020-01694-8Inhibition of LSD1 promotes the differentiation of human induced pluripotent stem cells into insulin-producing cellsXiao-Fei Yang0Shu-Yan Zhou1Ce Wang2Wei Huang3Ning Li4Fei He5Fu-Rong Li6Translational Medicine Collaborative Innovation Center, The Second Clinical Medical College (Shenzhen People’s Hospital), Ji’nan UniversityTranslational Medicine Collaborative Innovation Center, The Second Clinical Medical College (Shenzhen People’s Hospital), Ji’nan UniversityTranslational Medicine Collaborative Innovation Center, The Second Clinical Medical College (Shenzhen People’s Hospital), Ji’nan UniversityDepartment of Biology, Southern University of Science and TechnologyTranslational Medicine Collaborative Innovation Center, The Second Clinical Medical College (Shenzhen People’s Hospital), Ji’nan UniversityGuangdong Engineering Technology Research Center of Stem Cell and Cell therapyTranslational Medicine Collaborative Innovation Center, The Second Clinical Medical College (Shenzhen People’s Hospital), Ji’nan UniversityAbstract Background Human induced pluripotent stem cells (hiPSCs) represent a potentially unlimited source of pancreatic endocrine lineage cells. Although insulin-producing β cells derived from hiPSCs have been successfully induced, much work remains to be done to achieve mature β cells. Lysine-specific demethylase 1 (LSD1) plays an important role in the regulation of hiPSC self-renewal and differentiation. We propose a new strategy to acquire insulin-producing cells (IPCs) from hiPSCs by knocking down LSD1. Methods Knockdown of LSD1 in hiPSCs with five shRNA. Assessment of the effects of shRNA on hiPSC proliferation, cell cycle, and apoptosis. Using knockdown hiPSCs with 31.33% LSD1 activity, we achieved a four-step differentiation into IPCs and test its differentiation efficiency, morphology, and marker genes and proteins. We implanted the IPCs into the renal subcapsular of SCID-Beige diabetic mice to evaluate the hypoglycemic effect in vivo. We tested LSD1 and HDAC1 whether they are present in the CoREST complex through IP-WB, and analyzed LSD1, CoREST, HDAC1, H3K4me2/me3, and H3K27me3 protein expression before and after knockdown of LSD1. Results Differentiated hiPSCs were 38.32% ± 3.54% insulin-positive cells and released insulin/C-peptide in response to glucose stimulus in a manner comparable to adult human islets. Most of the IPCs co-expressed mature β cell-specific markers. When transplanted under the left renal capsule of SCID-Beige diabetic mice, these IPCs reversed hyperglycemia, leading to a significant increase in the definitive endoderm cells. IP-WB results showed that LSD1, HDAC1, and CoREST formed a complex in hiPSCs. Chip-PCR results showed that LSD1, HDAC1, and CoREST were enriched in the same district during the SOX17 and FOXA2 promoter region. Inhibition of LSD1 would not affect the level of CoREST but decreased the HDAC1 expressions. The H3K4me2/me3 and H3K9act level of SOX17 and FOXA2 promoter region increased after inhibited of LSD1, and promoted transcriptional activation. The H3K4me2/me3 and H3K9act level of OCT4 and SOX2 promoter region decreased with the transcriptional repressed. Conclusions LSD1 regulated histone methylation and acetylation in promoter regions of pluripotent or endodermal genes. Our results suggest a highly efficient approach to producing IPCs from hiPSCs.http://link.springer.com/article/10.1186/s13287-020-01694-8LSD1Human induced pluripotent stem cellsInsulin-producing cellsDifferentiationDiabetes |
| spellingShingle | Xiao-Fei Yang Shu-Yan Zhou Ce Wang Wei Huang Ning Li Fei He Fu-Rong Li Inhibition of LSD1 promotes the differentiation of human induced pluripotent stem cells into insulin-producing cells Stem Cell Research & Therapy LSD1 Human induced pluripotent stem cells Insulin-producing cells Differentiation Diabetes |
| title | Inhibition of LSD1 promotes the differentiation of human induced pluripotent stem cells into insulin-producing cells |
| title_full | Inhibition of LSD1 promotes the differentiation of human induced pluripotent stem cells into insulin-producing cells |
| title_fullStr | Inhibition of LSD1 promotes the differentiation of human induced pluripotent stem cells into insulin-producing cells |
| title_full_unstemmed | Inhibition of LSD1 promotes the differentiation of human induced pluripotent stem cells into insulin-producing cells |
| title_short | Inhibition of LSD1 promotes the differentiation of human induced pluripotent stem cells into insulin-producing cells |
| title_sort | inhibition of lsd1 promotes the differentiation of human induced pluripotent stem cells into insulin producing cells |
| topic | LSD1 Human induced pluripotent stem cells Insulin-producing cells Differentiation Diabetes |
| url | http://link.springer.com/article/10.1186/s13287-020-01694-8 |
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