Modelling the Erythroblastic Island Niche of Dyserythropoietic Anaemia Type IV patients using Induced Pluripotent Stem Cells
Modelling the Erythroblastic Island Niche of Dyserythropoietic Anaemia Type IV patients using Induced Pluripotent Stem Cells
复制标题
使用诱导多能干细胞对 IV 型红细胞生成不良性贫血患者的成红细胞岛生态位进行建模
DOI：
10.1101/2023.02.02.526657
复制
发表时间：
2023
期刊：
影响因子：
--
通讯作者：
May A
中科院分区：
文献类型：
--
作者：
May A
引言：先天性红细胞生成障碍性贫血（CDA）IV型与转录因子KLF 1中的氨基酸取代Glu 325 Lys（E325 K）有关。这些患者表现出一系列症状，包括外周血中持续存在有核红细胞（RBC），这反映了KLF 1在红系细胞谱系中的已知作用。红细胞成熟和去核的最后阶段发生在与EBI巨噬细胞密切相关的成红细胞岛（EBI）龛内。目前尚不清楚KLF 1中E325 K突变的有害影响是否仅限于红细胞系，或者与其生态位相关的巨噬细胞中的缺陷是否也有助于疾病pathology.Methods：为了解决这个问题，我们使用来自一名CDA IV型患者的诱导多能干细胞（iPSC）以及两个经遗传修饰的iPSC系来表达可被4 OH-他莫昔芬激活的KLF 1-E325 K-ERT 2蛋白，生成了人EBI生态位的体外模型。将一个患者iPSC系与来自两个健康供体的对照系进行比较，并将KLF 1-E325 K-ERT 2 iPSC系与从相同亲本iPSCS.Results产生的一个诱导型KLF 1-ERT 2系进行比较：CDA患者来源的iPSC和表达活化的KLF 1-E325 K-ERT 2蛋白的iPSC在红系细胞的产生中显示出显著缺陷，并伴有一些已知KLF 1靶基因的相关破坏。巨噬细胞可以从所有iPSC系产生，但是当E325 K-ERT 2融合蛋白被激活时，我们注意到产生了由CD 93标记的稍微不太成熟的巨噬细胞群体。一个微妙的趋势，他们的能力降低，以支持红细胞去核也与巨噬细胞携带的E325 K-ERT 2 transgene.Discussion：综合这些数据支持的概念，即KLF 1-E325 K突变的临床显着影响主要与红细胞系的缺陷，但它是可能的，在小生境的缺陷可能有可能加剧病情。我们所描述的策略提供了一种强有力的方法来评估KLF 1中其他突变以及与EBI生态位相关的其他因素的影响。
Introduction:Congenital dyserythropoietic anaemia (CDA) type IV has been associated with an amino acid substitution, Glu325Lys (E325K), in the transcription factor KLF1. These patients present with a range of symptoms, including the persistence of nucleated red blood cells (RBCs) in the peripheral blood which reflects the known role for KLF1 within the erythroid cell lineage. The final stages of RBCs maturation and enucleation take place within the erythroblastic island (EBI) niche in close association with EBI macrophages. It is not known whether the detrimental effects of the E325K mutation in KLF1 are restricted to the erythroid lineage or whether deficiencies in macrophages associated with their niche also contribute to the disease pathology.Methods:To address this question, we generated anin vitromodel of the human EBI niche using induced pluripotent stem cells (iPSCs) derived from one CDA type IV patient as well as two iPSC lines genetically modified to express an KLF1-E325K-ERT2protein that could be activated with 4OH-tamoxifen. The one patient iPSC line was compared to control lines from two healthy donors and the KLF1-E325K-ERT2iPSC line to one inducible KLF1-ERT2line generated from the same parental iPSCS.Results:The CDA patient-derived iPSCs and iPSCs expressing the activated KLF1-E325K-ERT2protein showed significant deficiencies in the production of erythroid cells with associated disruption of some known KLF1 target genes. Macrophages could be generated from all iPSC lines but when the E325K-ERT2fusion protein was activated, we noted the generation of a slightly less mature macrophage population marked by CD93. A subtle trend in their reduced ability to support RBC enucleation was also associated with macrophages carrying the E325K-ERT2transgene.Discussion:Taken together these data support the notion that the clinically significant effects of the KLF1-E325K mutation are primarily associated with deficiencies in the erythroid lineage but it is possible that deficiencies in the niche might have the potential to exacerbate the condition. The strategy we describe provides a powerful approach to assess the effects of other mutations in KLF1 as well as other factors associated with the EBI niche.