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Enhanced Ex Vivo Generation of Erythroid Cells from Human Induced Pluripotent Stem Cells in a Simplified Cell Culture System with Low Cytokine Support.

ABSTRACT: 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 low efficiency and unphysiological conditions of common culture systems. Especially, the absence of a physiological niche may impair cell growth and lineage-specific differentiation. We here describe a simplified, xeno- and feeder-free culture system for prolonged RBC generation that uses low numbers of supporting cytokines [stem cell factor (SCF), erythropoietin (EPO), and interleukin 3 (IL-3)] and is based on the intermediate development of a "hematopoietic cell forming complex (HCFC)." From this HCFC, CD43+ hematopoietic cells (purity >95%) were continuously released into the supernatant and could be collected repeatedly over a period of 6 weeks for further erythroid differentiation. The released cells were mainly CD34+/CD45+ progenitors with high erythroid colony-forming potential and CD36+ erythroid precursors. A total of 1.5?×?107 cells could be harvested from the supernatant of one six-well plate, showing 100- to 1000-fold amplification during subsequent homogeneous differentiation into GPA+ erythroid cells. Mean enucleation rates near 40% (up to 60%) further confirmed the potency of the system. These benefits may be explained by the generation of a niche within the HCFC that mimics the spatiotemporal signaling of the physiological microenvironment in which erythropoiesis occurs. Compared to other protocols, this method provides lower complexity, less cytokine and medium consumption, higher cellular output, and better enucleation. In addition, slight modifications in cytokine addition shift the system toward continuous generation of granulocytes and macrophages.

SUBMITTER: Bernecker C

PROVIDER: S-EPMC6882453 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Enhanced Ex Vivo Generation of Erythroid Cells from Human Induced Pluripotent Stem Cells in a Simplified Cell Culture System with Low Cytokine Support.

Bernecker Claudia C Ackermann Mania M Lachmann Nico N Rohrhofer Lisa L Zaehres Holm H Araúzo-Bravo Marcos J MJ van den Akker Emile E Schlenke Peter P Dorn Isabel I

Stem cells and development 20191119 23

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 low efficiency and unphysiological conditions of common culture systems. Especially, the absence of a physiological niche may impair cell growth and lineage-specific differentiation. We here describe a simplified, xeno- and feeder-free culture system for prolonged RBC ...[more]

PMID: 31595840

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Project description: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.

Project description:BackgroundPigs have emerged as one of the most popular large animal models in biomedical research, which in many cases is considered as a superior choice over rodent models. In addition, transplantation studies using pig pluripotent stem (PS) cell derivatives may serve as a testbed for safety and efficacy prior to human trials. Recently, it has been shown that mouse and human PS cells cultured in LCDM (recombinant human LIF, CHIR 99021, (S)-(+)-dimethindene maleate, minocycline hydrochloride) medium exhibited extended developmental potential (designated as extended pluripotent stem cells, or EPS cells), which could generate both embryonic and extraembryonic tissues in chimeric mouse conceptus. Whether stable pig induced pluripotent stem (iPS) cells can be generated in LCDM medium and their chimeric competency remains unknown.MethodsiPS cells were generated by infecting pig pericytes (PC) and embryonic fibroblasts (PEFs) with a retroviral vector encoding Oct4, Sox2, Klf4, and cMyc reprogramming factors and subsequently cultured in a modified LCDM medium. The pluripotency of PC-iPS and PEF-iPS cells was characterized by examining the expression of pluripotency-related transcription factors and surface markers, transcriptome analysis, and in vitro and in vivo differentiation capabilities. Chimeric contribution of PC-iPS cells to mouse and pig conceptus was also evaluated with fluorescence microscopy, flow cytometry, and PCR analysis.ResultsIn this study, using a modified version of the LCDM medium, we successfully generated iPS cells from both PCs and PEFs. Both PC-iPS and PEF-iPS cells maintained the stable "dome-shaped" morphology and genome stability after long-term culture. The immunocytochemistry analyses revealed that both PC-iPS and PEF-iPS cells expressed OCT4, SOX2, and SALL4, but only PC-iPS cells expressed NANOG and TRA-1-81 (faint). PC-iPS and PEF-iPS cells could be differentiated into cell derivatives of all three primary germ layers in vitro. The transcriptome analysis showed that PEF-iPS and PC-iPS cells clustered with pig ICM, Heatmap and volcano plot showed that there were 1475 differentially expressed genes (DEGs) between PC-iPS and PEF-iPS cells (adjusted p value < 0.1), and the numbers of upregulated genes and downregulated genes in PC-iPS cells were 755 and 720, respectively. Upregulated genes were enriched with GO terms including regulation of stem cell differentiation, proliferation, development, and maintenance. And KEGG pathway enrichment in upregulated genes revealed Wnt, Jak-STAT, TGF-β, P53, and MAPK stem cell signaling pathways. Fluorescence microscopy and genomic PCR analyses using pig mtDNA-specific and GFP primers showed that the PC-iPS cell derivatives could be detected in both mouse and pig pre-implantation blastocysts and post-implantation conceptuses. Quantitative analysis via flow cytometry revealed that the chimeric contribution of pig PC-iPS cells in mouse conceptus was up to 0.04%.ConclusionsOur findings demonstrate that stable iPS cells could be generated in LCDM medium, which could give rise to both embryonic and extraembryonic cells in vivo. However, the efficiency and level of chimeric contribution of pig LCDM-iPS cells were found low.

Dataset Information

Enhanced Ex Vivo Generation of Erythroid Cells from Human Induced Pluripotent Stem Cells in a Simplified Cell Culture System with Low Cytokine Support.

Publications

Enhanced Ex Vivo Generation of Erythroid Cells from Human Induced Pluripotent Stem Cells in a Simplified Cell Culture System with Low Cytokine Support.

Similar Datasets

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