Dataset Information

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

ABSTRACT:

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.

SUBMITTER: Martins GLS

PROVIDER: S-EPMC10137038 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature

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Publications

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

Martins Gabriele Louise Soares GLS Nonaka Carolina Kymie Vasques CKV Rossi Erik Aranha EA de Lima Adne Vitória Rocha AVR Adanho Corynne Stephanie Ahouefa CSA Oliveira Moisés Santana MS Yahouedehou Setondji Cocou Modeste Alexandre SCMA de Souza Clarissa Lima E Moura CLEM Gonçalves Marilda de Souza MS Paredes Bruno Diaz BD Souza Bruno Solano de Freitas BSF

Cells 20230410 8

<h4>Background</h4>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 an ...[more]

PMID: 37190030

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Project description:BackgroundHuman pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), can undergo erythroid differentiation, offering a potentially invaluable resource for generating large quantities of erythroid cells. However, the majority of erythrocytes derived from hPSCs fail to enucleate compared with those derived from cord blood progenitors, with an unknown molecular basis for this difference. The expression of vimentin (VIM) is retained in erythroid cells differentiated from hPSCs but is absent in mature erythrocytes. Further exploration is required to ascertain whether VIM plays a critical role in enucleation and to elucidate the underlying mechanisms.MethodsIn this study, we established a hESC line with reversible vimentin degradation (dTAG-VIM-H9) using the proteolysis-targeting chimera (PROTAC) platform. Various time-course studies, including erythropoiesis from CD34+ human umbilical cord blood and three-dimensional (3D) organoid culture from hESCs, morphological analysis, quantitative real-time PCR (qRT-PCR), western blotting, flow cytometry, karyotyping, cytospin, Benzidine-Giemsa staining, immunofluorescence assay, and high-speed cell imaging analysis, were conducted to examine and compare the characteristics of hESCs and those with vimentin degradation, as well as their differentiated erythroid cells.ResultsVimentin expression diminished during normal erythropoiesis in CD34+ cord blood cells, whereas it persisted in erythroid cells differentiated from hESC. Depletion of vimentin using the degradation tag (dTAG) system promotes erythroid enucleation in dTAG-VIM-H9 cells. Nuclear polarization of erythroblasts is elevated by elimination of vimentin.ConclusionsVIM disappear during the normal maturation of erythroid cells, whereas they are retained in erythroid cells differentiated from hPSCs. We found that retention of vimentin during erythropoiesis impairs erythroid enucleation from hPSCs. Using the PROTAC platform, we validated that vimentin degradation by dTAG accelerates the enucleation rate in dTAG-VIM-H9 cells by enhancing nuclear polarization.

Dataset Information

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

Background

Methods

Results

Conclusion

Publications

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

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