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Publications

Journal Article Research Support, Non-U.S. Gov't Epublish 2 citations

PubMed: 37190030 DOI: 10.3390/cells12081121

Evaluation of 2D and 3D Erythroid Differentiation Protocols Using Sickle Cell Disease and Healthy Donor Induced Pluripotent Stem Cells.

Cells 12(8)

Authors: 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

Affiliations

  • Gonçalo Moniz Institute, Oswaldo Cruz Foundation (FIOCRUZ), Salvador 40296-710, Brazil.
  • Center for Biotechnology and Cell Therapy (CBTC), São Rafael Hospital (HSR), Salvador 41253-190, Brazil.
  • D'Or Institute for Research and Education (IDOR), Salvador 41253-190, Brazil.
  • Gonçalo Moniz Institute, Oswaldo Cruz Foundation (FIOCRUZ), Salvador 40296-710, Brazil. Center for Biotechnology and Cell Therapy (CBTC), São Rafael Hospital (HSR), Salvador 41253-190, Brazil.
  • Hospital Universitário Professor Edgard Santos, Federal University of Bahia (UFBA), Salvador 40110-060, Brazil.

Abstract

BACKGROUND

Sickle cell disease (SCD) is a highly prevalent genetic disease caused by a point mutation in the 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 and .

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 compared to adult .

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.

MeSH

Cells, Genes, Microscopy, Electron, Scanning Transmission, Anemia, Sickle Cell, Pluripotent Stem Cells, Stem Cells, Induced Pluripotent Stem Cells, Erythropoiesis

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