Evaluation of 2D and 3D Erythroid Differentiation Protocols Using Sickle Cell Disease and Healthy Donor Induced Pluripotent Stem Cells
Background: Sickle cell disease (SCD) is a highly prevalent genetic disease caused by a point mutation in the <i>HBB</i> gene, which can lead to chronic hemolytic anemia and vaso-occlusive events. Patient-derived induced pluripotent stem cells (iPSCs) hold promise for the development of...
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-04-01
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| Online Access: | https://www.mdpi.com/2073-4409/12/8/1121 |
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| author | Gabriele Louise Soares Martins Carolina Kymie Vasques Nonaka Erik Aranha Rossi Adne Vitória Rocha de Lima Corynne Stephanie Ahouefa Adanho Moisés Santana Oliveira Setondji Cocou Modeste Alexandre Yahouedehou Clarissa Lima e Moura de Souza Marilda de Souza Gonçalves Bruno Diaz Paredes Bruno Solano de Freitas Souza |
| author_facet | Gabriele Louise Soares Martins Carolina Kymie Vasques Nonaka Erik Aranha Rossi Adne Vitória Rocha de Lima Corynne Stephanie Ahouefa Adanho Moisés Santana Oliveira Setondji Cocou Modeste Alexandre Yahouedehou Clarissa Lima e Moura de Souza Marilda de Souza Gonçalves Bruno Diaz Paredes Bruno Solano de Freitas Souza |
| author_sort | Gabriele Louise Soares Martins |
| collection | DOAJ |
| description | Background: Sickle cell disease (SCD) is a highly prevalent genetic disease caused by a point mutation in the <i>HBB</i> gene, which can lead to chronic hemolytic anemia and vaso-occlusive events. Patient-derived induced pluripotent stem cells (iPSCs) hold promise for the development of novel predictive methods for screening drugs with anti-sickling activity. In this study, we evaluated and compared the efficiency of 2D and 3D erythroid differentiation protocols using a healthy control and SCD-iPSCs. Methods: iPSCs were subjected to hematopoietic progenitor cell (HSPC) induction, erythroid progenitor cell induction, and terminal erythroid maturation. Differentiation efficiency was confirmed by flow cytometry analysis, colony-forming unit (CFU) assay, morphological analyses, and qPCR-based gene expression analyses of <i>HBB</i> and <i>HBG2</i>. Results: Both 2D and 3D differentiation protocols led to the induction of CD34<sup>+</sup>/CD43<sup>+</sup> HSPCs. The 3D protocol showed good efficiency (>50%) and high productivity (45-fold) for HSPC induction and increased the frequency of BFU-E, CFU-E, CFU-GM, and CFU-GEMM colonies. We also produced CD71<sup>+</sup>/CD235a<sup>+</sup> cells (>65%) with a 630-fold cell expansion relative to that at the beginning of the 3D protocol. After erythroid maturation, we observed 95% CD235a<sup>+</sup>/DRAQ5- enucleated cells, orthochromatic erythroblasts, and increased expression of fetal <i>HBG2</i> compared to adult <i>HBB</i>. Conclusion: A robust 3D protocol for erythroid differentiation was identified using SCD-iPSCs and comparative analyses; however, the maturation step remains challenging and requires further development. |
| first_indexed | 2024-03-11T05:09:01Z |
| format | Article |
| id | doaj.art-6556d48984b94c92862d351916038858 |
| institution | Directory Open Access Journal |
| issn | 2073-4409 |
| language | English |
| last_indexed | 2024-03-11T05:09:01Z |
| publishDate | 2023-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Cells |
| spelling | doaj.art-6556d48984b94c92862d3519160388582023-11-17T18:42:49ZengMDPI AGCells2073-44092023-04-01128112110.3390/cells12081121Evaluation of 2D and 3D Erythroid Differentiation Protocols Using Sickle Cell Disease and Healthy Donor Induced Pluripotent Stem CellsGabriele Louise Soares Martins0Carolina Kymie Vasques Nonaka1Erik Aranha Rossi2Adne Vitória Rocha de Lima3Corynne Stephanie Ahouefa Adanho4Moisés Santana Oliveira5Setondji Cocou Modeste Alexandre Yahouedehou6Clarissa Lima e Moura de Souza7Marilda de Souza Gonçalves8Bruno Diaz Paredes9Bruno Solano de Freitas Souza10Gonçalo Moniz Institute, Oswaldo Cruz Foundation (FIOCRUZ), Salvador 40296-710, BrazilCenter for Biotechnology and Cell Therapy (CBTC), São Rafael Hospital (HSR), Salvador 41253-190, BrazilGonçalo Moniz Institute, Oswaldo Cruz Foundation (FIOCRUZ), Salvador 40296-710, BrazilGonçalo Moniz Institute, Oswaldo Cruz Foundation (FIOCRUZ), Salvador 40296-710, BrazilGonçalo Moniz Institute, Oswaldo Cruz Foundation (FIOCRUZ), Salvador 40296-710, BrazilCenter for Biotechnology and Cell Therapy (CBTC), São Rafael Hospital (HSR), Salvador 41253-190, BrazilGonçalo Moniz Institute, Oswaldo Cruz Foundation (FIOCRUZ), Salvador 40296-710, BrazilHospital Universitário Professor Edgard Santos, Federal University of Bahia (UFBA), Salvador 40110-060, BrazilGonçalo Moniz Institute, Oswaldo Cruz Foundation (FIOCRUZ), Salvador 40296-710, BrazilCenter for Biotechnology and Cell Therapy (CBTC), São Rafael Hospital (HSR), Salvador 41253-190, BrazilGonçalo Moniz Institute, Oswaldo Cruz Foundation (FIOCRUZ), Salvador 40296-710, BrazilBackground: Sickle cell disease (SCD) is a highly prevalent genetic disease caused by a point mutation in the <i>HBB</i> gene, which can lead to chronic hemolytic anemia and vaso-occlusive events. Patient-derived induced pluripotent stem cells (iPSCs) hold promise for the development of novel predictive methods for screening drugs with anti-sickling activity. In this study, we evaluated and compared the efficiency of 2D and 3D erythroid differentiation protocols using a healthy control and SCD-iPSCs. Methods: iPSCs were subjected to hematopoietic progenitor cell (HSPC) induction, erythroid progenitor cell induction, and terminal erythroid maturation. Differentiation efficiency was confirmed by flow cytometry analysis, colony-forming unit (CFU) assay, morphological analyses, and qPCR-based gene expression analyses of <i>HBB</i> and <i>HBG2</i>. Results: Both 2D and 3D differentiation protocols led to the induction of CD34<sup>+</sup>/CD43<sup>+</sup> HSPCs. The 3D protocol showed good efficiency (>50%) and high productivity (45-fold) for HSPC induction and increased the frequency of BFU-E, CFU-E, CFU-GM, and CFU-GEMM colonies. We also produced CD71<sup>+</sup>/CD235a<sup>+</sup> cells (>65%) with a 630-fold cell expansion relative to that at the beginning of the 3D protocol. After erythroid maturation, we observed 95% CD235a<sup>+</sup>/DRAQ5- enucleated cells, orthochromatic erythroblasts, and increased expression of fetal <i>HBG2</i> compared to adult <i>HBB</i>. Conclusion: A robust 3D protocol for erythroid differentiation was identified using SCD-iPSCs and comparative analyses; however, the maturation step remains challenging and requires further development.https://www.mdpi.com/2073-4409/12/8/1121sickle cell diseaseerythropoiesisiPSCs |
| spellingShingle | Gabriele Louise Soares Martins Carolina Kymie Vasques Nonaka Erik Aranha Rossi Adne Vitória Rocha de Lima Corynne Stephanie Ahouefa Adanho Moisés Santana Oliveira Setondji Cocou Modeste Alexandre Yahouedehou Clarissa Lima e Moura de Souza Marilda de Souza Gonçalves Bruno Diaz Paredes Bruno Solano de Freitas Souza Evaluation of 2D and 3D Erythroid Differentiation Protocols Using Sickle Cell Disease and Healthy Donor Induced Pluripotent Stem Cells Cells sickle cell disease erythropoiesis iPSCs |
| title | Evaluation of 2D and 3D Erythroid Differentiation Protocols Using Sickle Cell Disease and Healthy Donor Induced Pluripotent Stem Cells |
| title_full | Evaluation of 2D and 3D Erythroid Differentiation Protocols Using Sickle Cell Disease and Healthy Donor Induced Pluripotent Stem Cells |
| title_fullStr | Evaluation of 2D and 3D Erythroid Differentiation Protocols Using Sickle Cell Disease and Healthy Donor Induced Pluripotent Stem Cells |
| title_full_unstemmed | Evaluation of 2D and 3D Erythroid Differentiation Protocols Using Sickle Cell Disease and Healthy Donor Induced Pluripotent Stem Cells |
| title_short | Evaluation of 2D and 3D Erythroid Differentiation Protocols Using Sickle Cell Disease and Healthy Donor Induced Pluripotent Stem Cells |
| title_sort | evaluation of 2d and 3d erythroid differentiation protocols using sickle cell disease and healthy donor induced pluripotent stem cells |
| topic | sickle cell disease erythropoiesis iPSCs |
| url | https://www.mdpi.com/2073-4409/12/8/1121 |
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