One-step genetic correction of hemoglobin E/beta-thalassemia patient-derived iPSCs by the CRISPR/Cas9 system
Abstract Background Thalassemia is the most common genetic disease worldwide; those with severe disease require lifelong blood transfusion and iron chelation therapy. The definitive cure for thalassemia is allogeneic hematopoietic stem cell transplantation, which is limited due to lack of HLA-matche...
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| Format: | Article |
| Language: | English |
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BMC
2018-02-01
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| Series: | Stem Cell Research & Therapy |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s13287-018-0779-3 |
| _version_ | 1811192786695749632 |
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| author | Methichit Wattanapanitch Nattaya Damkham Ponthip Potirat Kongtana Trakarnsanga Montira Janan Yaowalak U-pratya Pakpoom Kheolamai Nuttha Klincumhom Surapol Issaragrisil |
| author_facet | Methichit Wattanapanitch Nattaya Damkham Ponthip Potirat Kongtana Trakarnsanga Montira Janan Yaowalak U-pratya Pakpoom Kheolamai Nuttha Klincumhom Surapol Issaragrisil |
| author_sort | Methichit Wattanapanitch |
| collection | DOAJ |
| description | Abstract Background Thalassemia is the most common genetic disease worldwide; those with severe disease require lifelong blood transfusion and iron chelation therapy. The definitive cure for thalassemia is allogeneic hematopoietic stem cell transplantation, which is limited due to lack of HLA-matched donors and the risk of post-transplant complications. Induced pluripotent stem cell (iPSC) technology offers prospects for autologous cell-based therapy which could avoid the immunological problems. We now report genetic correction of the beta hemoglobin (HBB) gene in iPSCs derived from a patient with a double heterozygote for hemoglobin E and β-thalassemia (HbE/β-thalassemia), the most common thalassemia syndrome in Thailand and Southeast Asia. Methods We used the CRISPR/Cas9 system to target the hemoglobin E mutation from one allele of the HBB gene by homology-directed repair with a single-stranded DNA oligonucleotide template. DNA sequences of the corrected iPSCs were validated by Sanger sequencing. The corrected clones were differentiated into hematopoietic progenitor and erythroid cells to confirm their multilineage differentiation potential and hemoglobin expression. Results The hemoglobin E mutation of HbE/β-thalassemia iPSCs was seamlessly corrected by the CRISPR/Cas9 system. The corrected clones were differentiated into hematopoietic progenitor cells under feeder-free and OP9 coculture systems. These progenitor cells were further expanded in erythroid liquid culture system and developed into erythroid cells that expressed mature HBB gene and HBB protein. Conclusions Our study provides a strategy to correct hemoglobin E mutation in one step and these corrected iPSCs can be differentiated into hematopoietic stem cells to be used for autologous transplantation in patients with HbE/β-thalassemia in the future. |
| first_indexed | 2024-04-11T23:57:18Z |
| format | Article |
| id | doaj.art-4699d70776364a1992e161843252eaa3 |
| institution | Directory Open Access Journal |
| issn | 1757-6512 |
| language | English |
| last_indexed | 2024-04-11T23:57:18Z |
| publishDate | 2018-02-01 |
| publisher | BMC |
| record_format | Article |
| series | Stem Cell Research & Therapy |
| spelling | doaj.art-4699d70776364a1992e161843252eaa32022-12-22T03:56:19ZengBMCStem Cell Research & Therapy1757-65122018-02-019111110.1186/s13287-018-0779-3One-step genetic correction of hemoglobin E/beta-thalassemia patient-derived iPSCs by the CRISPR/Cas9 systemMethichit Wattanapanitch0Nattaya Damkham1Ponthip Potirat2Kongtana Trakarnsanga3Montira Janan4Yaowalak U-pratya5Pakpoom Kheolamai6Nuttha Klincumhom7Surapol Issaragrisil8Siriraj Center of Excellence for Stem Cell Research, Faculty of Medicine Siriraj Hospital, Mahidol UniversitySiriraj Center of Excellence for Stem Cell Research, Faculty of Medicine Siriraj Hospital, Mahidol UniversitySiriraj Center of Excellence for Stem Cell Research, Faculty of Medicine Siriraj Hospital, Mahidol UniversityDepartment of Biochemistry, Faculty of Medicine Siriraj Hospital, Mahidol UniversitySiriraj Center of Excellence for Stem Cell Research, Faculty of Medicine Siriraj Hospital, Mahidol UniversitySiriraj Center of Excellence for Stem Cell Research, Faculty of Medicine Siriraj Hospital, Mahidol UniversityDivision of Cell Biology, Department of Pre-clinical Sciences, Faculty of Medicine, Thammasat UniversityDepartment of Anatomy, Faculty of Dentistry, Chulalongkorn UniversitySiriraj Center of Excellence for Stem Cell Research, Faculty of Medicine Siriraj Hospital, Mahidol UniversityAbstract Background Thalassemia is the most common genetic disease worldwide; those with severe disease require lifelong blood transfusion and iron chelation therapy. The definitive cure for thalassemia is allogeneic hematopoietic stem cell transplantation, which is limited due to lack of HLA-matched donors and the risk of post-transplant complications. Induced pluripotent stem cell (iPSC) technology offers prospects for autologous cell-based therapy which could avoid the immunological problems. We now report genetic correction of the beta hemoglobin (HBB) gene in iPSCs derived from a patient with a double heterozygote for hemoglobin E and β-thalassemia (HbE/β-thalassemia), the most common thalassemia syndrome in Thailand and Southeast Asia. Methods We used the CRISPR/Cas9 system to target the hemoglobin E mutation from one allele of the HBB gene by homology-directed repair with a single-stranded DNA oligonucleotide template. DNA sequences of the corrected iPSCs were validated by Sanger sequencing. The corrected clones were differentiated into hematopoietic progenitor and erythroid cells to confirm their multilineage differentiation potential and hemoglobin expression. Results The hemoglobin E mutation of HbE/β-thalassemia iPSCs was seamlessly corrected by the CRISPR/Cas9 system. The corrected clones were differentiated into hematopoietic progenitor cells under feeder-free and OP9 coculture systems. These progenitor cells were further expanded in erythroid liquid culture system and developed into erythroid cells that expressed mature HBB gene and HBB protein. Conclusions Our study provides a strategy to correct hemoglobin E mutation in one step and these corrected iPSCs can be differentiated into hematopoietic stem cells to be used for autologous transplantation in patients with HbE/β-thalassemia in the future.http://link.springer.com/article/10.1186/s13287-018-0779-3Induced pluripotent stem cellsThalassemiaHematopoietic differentiationGenetic correctionCRISPR/Cas9 |
| spellingShingle | Methichit Wattanapanitch Nattaya Damkham Ponthip Potirat Kongtana Trakarnsanga Montira Janan Yaowalak U-pratya Pakpoom Kheolamai Nuttha Klincumhom Surapol Issaragrisil One-step genetic correction of hemoglobin E/beta-thalassemia patient-derived iPSCs by the CRISPR/Cas9 system Stem Cell Research & Therapy Induced pluripotent stem cells Thalassemia Hematopoietic differentiation Genetic correction CRISPR/Cas9 |
| title | One-step genetic correction of hemoglobin E/beta-thalassemia patient-derived iPSCs by the CRISPR/Cas9 system |
| title_full | One-step genetic correction of hemoglobin E/beta-thalassemia patient-derived iPSCs by the CRISPR/Cas9 system |
| title_fullStr | One-step genetic correction of hemoglobin E/beta-thalassemia patient-derived iPSCs by the CRISPR/Cas9 system |
| title_full_unstemmed | One-step genetic correction of hemoglobin E/beta-thalassemia patient-derived iPSCs by the CRISPR/Cas9 system |
| title_short | One-step genetic correction of hemoglobin E/beta-thalassemia patient-derived iPSCs by the CRISPR/Cas9 system |
| title_sort | one step genetic correction of hemoglobin e beta thalassemia patient derived ipscs by the crispr cas9 system |
| topic | Induced pluripotent stem cells Thalassemia Hematopoietic differentiation Genetic correction CRISPR/Cas9 |
| url | http://link.springer.com/article/10.1186/s13287-018-0779-3 |
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