Integrity of Induced Pluripotent Stem Cell (iPSC) Derived Megakaryocytes as Assessed by Genetic and Transcriptomic Analysis

Abstract

Previously, we have described our feeder-free, xeno-free approach to generate megakaryocytes (MKs) in culture from human induced pluripotent stem cells (iPSCs). Here, we focus specifically on the integrity of these MKs using: (1) genotype discordance between parent cell DNA to iPSC cell DNA and onward to the differentiated MK DNA; (2) genomic structural integrity using copy number variation (CNV); and (3) transcriptomic signatures of the derived MK lines compared to the iPSC lines. We detected a very low rate of genotype discordance; estimates were 0.0001%-0.01%, well below the genotyping error rate for our assay (0.37%). No CNVs were generated in the iPSCs that were subsequently passed on to the MKs. Finally, we observed highly biologically relevant gene sets as being upregulated in MKs relative to the iPSCs: platelet activation, blood coagulation, megakaryocyte development, platelet formation, platelet degranulation, and platelet aggregation. These data strongly support the integrity of the derived MK lines.

Gene and protein expression in human megakaryocytes derived from induced pluripotent stem cells

“…In addition, we found that the percent of cells expressing the MK surface markers CD41 and CD42a varied among subjects despite a consistent derivation protocol, and that the expression profile of genes and proteins was strongly dependent on MK CD41+CD42a+ expression. We previously showed in 14 subjects that megakaryocyte‐ and platelet‐related genes are highly expressed in iPSC‐derived MKs and that their transcriptome differs markedly from their parent iPSCs 9 . Our prior study verified that there is high genomic structural integrity and genotype concordance between MKs and their parent iPSCs.…”

“…In this work we extend our prior study 9 by describing the complete transcriptome of MKs derived from iPSCs in 194 healthy European American (EA) and African American (AA) subjects. We relate gene expression to the expression of MK markers in each subject.…”

“…The MKs also express the MK markers CD42b and CD61 and display polyploidy. We have shown using RNA sequencing that megakaryocyte‐related genes are highly expressed and that the transcriptome of these iPSC‐derived MKs differs markedly from their parent iPSCs 9 …”

Platelet “-omics” in health and cardiovascular disease

“…Until recently, our understanding of the megakaryocyte transcriptome has relied on bulk analysis of in vitro differentiated CD34 + progenitors, immortalised MEG-01 cells or induced pluripotent stem cells [104][105][106], while insufficient sample numbers of this rare cell type have impeded direct transcriptomic analysis of native megakaryocytes. Only by using scRNA-seq, Davizon-Castillo et al made it recently possible to analyse the whole coding transcriptome of native, single megakaryocytes [15].…”

Induced Pluripotent Stem Cell-Derived Red Blood Cells and Platelet Concentrates: From Bench to Bedside

“…Kammers et al studied genetic and transcript integrity of iPS cells-derived MKs detecting a very low rate of genotype discordance with parent cell (0.0001–0.01%), no CNVs, and highly biologically-relevant gene expression profiles [ 64 ].…”