Modelling the Erythroblastic Island Niche of Dyserythropoietic Anaemia Type IV patients using Induced Pluripotent Stem Cells

Abstract

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. To address this question, we generated an in vitro model of the human EBI niche using induced pluripotent stem cells (iPSCs) derived from a CDA type IV patient as well as iPSCs genetically modified to express an KLF1-E325K-ERT2 protein that could be activated with 4OH-tamoxifen. CDA patient-derived iPSCs and iPSCs expressing the activated KLF1-E325K-ERT2 protein 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-ERT2 fusion protein was activated, we noted the generation of a slightly less mature macrophage population marked by CD93. A subtle reduction in their ability to support RBC maturation was also associated with macrophages carrying the E325K-ERT2 transgene. 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 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.

Proteomic analysis reveals a potential role for extracellular vesicles within the erythroblastic island niche

“…It was recently shown that proteins encoded by gene networks associated with mitochondrial biogenesis were upregulated in erythroid cells carrying the E325K mutation that is associated with Congenital Dyserythropoietic Anemia Type IV (Ferrer-Vicens et al, 2023). Proteomic analysis of macrophages carrying this pathogenic mutation will reveal whether comparable changes are associated with the EI niche using our recently developed model of this disease (May et al, 2023).…”

“…RNA extraction was performed using the RNAeasy Mini Kit (74106, QIAGEN) following the manufacturer's instructions and cDNA was generated as previously described (May et al, 2023). Quantitative real time PCR reactions were performed on the Roche LightCycler ® 480 Instrument.…”

“…The production and maturation of red blood cells (RBCs) takes place within the erythroblastic island (EBI) niche where central macrophages provide support for proliferating and maturing erythroid cells (Lopez-Yrigoyen et al, 2019;Li et al, 2020;May and Forrester, 2020;May et al, 2023). The inaccessible location of this important niche within the bone marrow makes it difficult to study so the cellular and molecular mechanisms associated with its function remain poorly understood.…”

“…Several model systems have been used to study the EBI niche including relatively simple coculture models in vitro and sophisticated genetic depletion strategies in vivo (Chow et al, 2011;Heideveld et al, 2015;May and Forrester, 2020;Romano et al, 2022). The ex-vivo proliferation and differentiation of human erythroid progenitor cells were significantly enhanced when co-cultured with macrophages derived from various sources including from peripheral blood mononuclear cells and from human induced pluripotent stem cells (hiPSCs) (Heideveld et al, 2015;Lopez-Yrigoyen et al, 2019;May et al, 2023). This provided a strategy to study the molecular processes that are associated with the EBI niche.…”

A novel human pluripotent stem cell-based gene activation system identifies IGFBP2 as a mediator in the production of hematopoietic progenitors in vitro

“…iPSC clones that had specifically integrated the iSAM cassette into the AAVS1 locus were validated by genomic PCR and sequencing (Figure S2A-B). The AAVS1 locus has been reported to be a "safe harbour" site that is resistant to epigenetic silencing and indeed, we had previously demonstrated that transgenes inserted into the AAVS1 locus under the control of the constitutively active CAG promoter were efficiently expressed both in undifferentiated and in differentiated iPSCs 20,[22][23][24] . However, after the iSAM line had been established and cultured under self-renewal conditions, we noted a dramatic reduction in the number of mCherry+ cells in undifferentiated iSAM iPSCs upon DOX induction, that we predicted to be due to transgene-silencing of the rTTA DOX-inducible cassette (Figure S3C).…”