利用镰状细胞病和健康供体诱导多能干细胞评估 2D 和 3D 红细胞分化方案。

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

机构信息

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.

出版信息

Cells. 2023 Apr 10;12(8):1121. doi: 10.3390/cells12081121.

DOI:10.3390/cells12081121

PMID:37190030

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10137038/

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.

摘要

背景

镰状细胞病（SCD）是一种由基因突变引起的高度流行的遗传性疾病，可导致慢性溶血性贫血和血管阻塞性事件。患者来源的诱导多能干细胞（iPSC）为开发具有抗镰变活性的新型药物筛选方法提供了希望。在这项研究中，我们评估并比较了使用健康对照和 SCD-iPSC 的 2D 和 3D 红细胞分化方案的效率。

方法

将 iPSC 进行造血祖细胞（HSPC）诱导、红细胞祖细胞诱导和终末红细胞成熟。通过流式细胞术分析、集落形成单位（CFU）测定、形态分析和基于 qPCR 的和基因表达分析来确认分化效率。

结果

两种 2D 和 3D 分化方案均诱导了 CD34/CD43 HSPC。3D 方案对 HSPC 诱导具有良好的效率（>50%）和高产量（45 倍），并增加了 BFU-E、CFU-E、CFU-GM 和 CFU-GEMM 集落的频率。我们还产生了 CD71/CD235a 细胞（>65%），与 3D 方案开始时相比，细胞扩增了 630 倍。在红细胞成熟后，我们观察到 95%的 CD235a/DRAQ5 去核细胞、正染红细胞和胎儿的表达增加，而成年的表达减少。