BioProject

Ex vivo red blood cell (RBC) differentiation from human induced pluripotent stem cells (hiPSCs) offers great potential for developmental studies and innovative therapies. More...

Ex vivo red blood cell (RBC) differentiation from human induced pluripotent stem cells (hiPSCs) offers great potential for developmental studies and innovative therapies. However, ex vivo erythropoiesis from hiPSCs is currently limited by its complexity and the absence of a physiological niche, which may impair cell growth and lineage-specific differentiation. We describe a simplified culture system for RBC generation over 6 weeks from hiPSCs using 1) low cytokine support (SCF, EPO, and IL-3) and 2) maintenance of a three-dimensional organization during hematopoietic specification. CD43+ hematopoietic cells containing high percentages of CD34+/CD45+ and CD36+/CD45+ progenitors were continuously released into the supernatant. During further erythroid differentiation into >90% GPA+ cells, up to 60% of cells extruded their nuclei depending upon the hiPSC origin. These advantages may be explained by the generation of an artificial niche within adherent spheroids that sufficiently mimics the spatiotemporal signaling of the physiological microenvironment in which erythropoiesis occurs RNA samples for microarray analysis were prepared using RNeasy columns (Qiagen, Germany) with on-column DNA digestion. 300 ng of total RNA per sample were used as the input in the linear amplification protocol (Ambion), which involved the synthesis of T7-linked double-stranded cDNAs and 12 hrs of in vitro transcription incorporating biotin-labeled nucleotides. Purified and labeled cRNA was then hybridized for 18 hrs onto HumanHT-12 v4 expression BeadChips (Illumina, USA) following the manufacturer’s instructions. After the recommended washing, the chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and the accompanying software. The samples were exclusively hybridized as biological replicates. The bead intensities were mapped to the corresponding gene information using BeadStudio 3.2 (Illumina) and background correction was performed using the Affymetrix Robust Multi-array Analysis (RMA) background correction model (Irizarry et al., 2003). Variance stabilization was performed using the log2 scaling, and gene expression normalization was calculated with the quantile method implemented in the lumi package of R-Bioconductor. Data post-processing and graphics were performed with in-house developed functions in MATLAB. Overall design: 3 samples were analyzed H1-ESC, H1 humam Embryonics Stem Cells (ESCs), 2 replicates PEB#1 iPS, Human Basophilic Erythroblasts (PEB) induced Pluripotent Stem Cells (iPSCs), 1 replicate Less...