Article
Evaluation of 2D and 3D erythroid differentiation protocols using sickle cell disease and healthy donor induced pluripotent stem cells
Registro en:
MARTINS, Gabriele Louise Soares et al. Evaluation of 2D and 3D erythroid differentiation protocols using sickle cell disease and healthy donor induced pluripotent stem cells. Cells, v. 12, p. 1-17, 2023.
1097-4172
10.3390/cells12081121
Autor
Martins, Gabriele Louise Soares
Nonaka, Carolina Kymie Vasques
Rossi, Erik Aranha
Lima, Adne Vitória Rocha de
Adanho, Corynne Stephanie Ahouefa
Oliveira, Moisés Santana
Yahouedehou, Setondji Cocou Modeste Alexandre
Souza, Clarissa Lima E Moura de
Gonçalves, Marilda de Souza
Paredes, Bruno Diaz
Souza, Bruno Solano de Freitas
Resumen
Fiocruz - INOVA Novos Talentos.
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). Background: Sickle cell disease (SCD) is a highly prevalent genetic disease caused by a point mutation in the HBB 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 HBB and HBG2. Results: Both 2D and 3D differentiation protocols led to the induction of CD34+/CD43+ 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+/CD235a+ 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+/DRAQ5- enucleated cells, orthochromatic erythroblasts, and increased expression of fetal HBG2 compared to adult HBB. 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.