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| Status |
Public on Dec 09, 2019 |
| Title |
Prolonged generation of erythroid cells from human induced pluripotent stem cells in a three-dimensional culture system with reduced cytokine support |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by array
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| Summary |
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.
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| 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
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| Contributor(s) |
Bernecker C, Ackermann M, Lachmann N, Rohrhofer L, Zaehres H, Araúuzo-Bravo MJ, van den Akker E, Schlenke P, Dorn I |
| Citation(s) |
31595840 |
| Submission date |
Jan 24, 2019 |
| Last update date |
Jan 15, 2022 |
| Contact name |
Marcos J. Araúzo-Bravo |
| E-mail(s) |
mararabra@yahoo.co.uk
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| Phone |
+34 943 00 6108
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| Organization name |
Max Planck Institute for Molecular Biomedicine
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| Department |
Cell and Developmental Biology
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| Lab |
Computational Biology and Bionformatics
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| Street address |
Rogentstrasse
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| City |
Muenster |
| ZIP/Postal code |
48149 |
| Country |
Germany |
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| Platforms (2) |
| GPL6883 |
Illumina HumanRef-8 v3.0 expression beadchip |
| GPL10558 |
Illumina HumanHT-12 V4.0 expression beadchip |
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| Samples (3) |
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| Relations |
| BioProject |
PRJNA516933 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE125624_GSM3579083_non-normalized_all_probes.txt.gz |
281.3 Kb |
(ftp)(http) |
TXT |
| GSE125624_GSM3579083_normalized_all_probes.txt.gz |
275.5 Kb |
(ftp)(http) |
TXT |
| GSE125624_RAW.tar |
30.1 Mb |
(http)(custom) |
TAR |
| GSE125624_non-normalized_probes_in_common.txt.gz |
492.6 Kb |
(ftp)(http) |
TXT |
| GSE125624_normalized_probes_in_common.txt.gz |
360.4 Kb |
(ftp)(http) |
TXT |
| Processed data included within Sample table |
| Processed data are available on Series record |
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