Global MicroRNA Profiling Uncovers miR-206 as a Negative Regulator of Hematopoietic Commitment in Human Pluripotent Stem Cells
Abstract
:1. Introduction
2. Results
2.1. Overview of the Protocol
2.2. Characterization of the Hematopoietic Potential of hPSC Lines
2.3. Gene Expression Analysis of the NODAL/ACTIVIN Signaling Pathway
2.4. Hematopoiesis-Related miRNA Expression during Hematopoietic Differentiation
2.5. Global microRNA Expression Profiling in Human PSCs
2.6. Human PSCs Hematopoietic Potential and miRNA Expression
2.7. Integrative miRNome and Transcriptome Analysis
2.8. Overexpression of miR206 in the H1 ES Cell Line
3. Discussion
4. Materials and Methods
4.1. Cell Lines and Culture
4.2. Differentiation of hPSCs in EB Cells
4.3. Clonogenic Progenitor Assays
4.4. Flow Cytometry
4.5. Total RNA, miRNA Extraction, and cDNA Synthesis
4.6. Whole Transcriptome and qRT-PCR Analyses
4.7. Hematopoiesis-Related miRNA qRT-PCR Analysis
4.8. TLDA Analysis
4.9. Integrative miRNome and Transcriptome Analysis
4.10. Overexpression of miR-206 in a Blood-Competent ES Cell Line
4.11. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
ath | Arabidopsis thaliana |
BFU-E | Burst-forming unit-erythroid |
CFC | Colony-forming cell |
CFU-E | Colony-forming unit-erythroid |
CFU-GEMM | Colony-forming unit-granulocytes erythrocytes macrophages megakaryocytes |
CFU-GM | Colony-forming unit-granulocytes macrophages |
CGH | Comparative genomic hybridization |
EB | Embryoid bodies |
ESCs | Embryonic stem cells |
hiPSCs | Human induced pluripotent stem cells |
hPSCs | Human pluripotent stem cells |
LiMMa | Linear models for microarrays |
miRNAs | Micrornas |
PSCs | Pluripotent stem cells |
qRT-PCR | Quantitative real-time RT-PCR |
TLDA | TaqMan low-density array |
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Cell Line | Passage | Karyotype/CGH | Origin |
---|---|---|---|
iPSC lines | |||
PB3 | 43 | 46, XX | Normal amniocyte |
PB4 | 26 | 46, XY | Amniocyte, sickle cell anemia |
PB6 | 23 | 47, XY, +21 | Amniocyte, Down syndrome |
PB6.1 | 14 | 47, XY, +21 | Amniocyte, Down syndrome |
PB7 | 63 | 46, XY | Normal amniocyte |
PB9 | 17 | 46, XY | Normal amniocyte |
PB10 | 16 | 46, XY | Normal amniocyte |
PB12.1 | 33 | 47, XY, +21 | Amniocyte, Down syndrome |
PB13 | 28 | 46, XY | Endothelial progenitor cell from peripheral blood |
PB14.3 | 41 | 47, XX, +21 | Amniocyte, Down syndrome |
PB17 | 38 | 46, XY | Endothelial progenitor cell from peripheral blood |
ESC lines | |||
SA01 | 22+4+7 | 46, XY | Cellartis AB, Sweden |
H1 | 67 | 46, XY | WiCell, Wisconsin, USA |
H9 | 31 | 46, XX | WiCell, Wisconsin, USA |
CL01 | 16 | 46, XY, der21, t(1;21) | Paris Sud University, France |
Cell line | CFC/105 Cells | Distribution of Hematopoietic Colony Subtypes (/105 cells) | %CD34+ Cells | Hematopoietic Potential | ||||
---|---|---|---|---|---|---|---|---|
CFU-E | BFU-E | CFU-GM | CFU-GEMM | |||||
iPSCs | PB3 | 140 | 0 | 1 | 139 | 0 | 35 ± 10 | +++ |
PB4 | 80 | 0 | 1 | 79 | 0 | 13 ± 3 | ++ | |
PB6 | 2 | 1 | 0 | 1 | 0 | 2 ± 1 | 0 | |
PB6.1 | 120 | 41 | 46 | 30 | 3 | 17 ± 7 | +++ | |
PB7 | 144 | 52 | 33 | 58 | 1 | 13 ± 6 | +++ | |
PB9 | 0 | 0 | 0 | 0 | 0 | 0.3 | 0 | |
PB10 | 24 | 0 | 4 | 20 | 0 | 3 ± 0.3 | + | |
PB12.1 | 0 | 0 | 0 | 0 | 0 | 0.4 | 0 | |
PB13 | 249 | 115 | 134 | 0 | 0 | 16 ± 10 | +++ | |
PB14.3 | 5 | 0 | 1 | 4 | 0 | 3 ± 0.5 | 0 | |
PB17 | 235 | 47 | 0 | 188 | 0 | 17 ± 4 | +++ | |
ESCs | SA01 | 402 | 145 | 84 | 161 | 12 | 23 ± 9 | +++ |
H1 | 318 | 216 | 29 | 70 | 3 | 33 ± 16 | +++ | |
H9 | 38 | 2 | 1 | 35 | 0 | 9 ± 2 | ++ | |
CL01 | 41 | 2 | 1 | 37 | 1 | 8 ± 2 | ++ |
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Flamant, S.; Chomel, J.-C.; Desterke, C.; Féraud, O.; Gobbo, E.; Mitjavila-Garcia, M.-T.; Foudi, A.; Griscelli, F.; Turhan, A.G.; Bennaceur-Griscelli, A. Global MicroRNA Profiling Uncovers miR-206 as a Negative Regulator of Hematopoietic Commitment in Human Pluripotent Stem Cells. Int. J. Mol. Sci. 2019, 20, 1737. https://doi.org/10.3390/ijms20071737
Flamant S, Chomel J-C, Desterke C, Féraud O, Gobbo E, Mitjavila-Garcia M-T, Foudi A, Griscelli F, Turhan AG, Bennaceur-Griscelli A. Global MicroRNA Profiling Uncovers miR-206 as a Negative Regulator of Hematopoietic Commitment in Human Pluripotent Stem Cells. International Journal of Molecular Sciences. 2019; 20(7):1737. https://doi.org/10.3390/ijms20071737
Chicago/Turabian StyleFlamant, Stéphane, Jean-Claude Chomel, Christophe Desterke, Olivier Féraud, Emilie Gobbo, Maria-Teresa Mitjavila-Garcia, Adlen Foudi, Frank Griscelli, Ali G. Turhan, and Annelise Bennaceur-Griscelli. 2019. "Global MicroRNA Profiling Uncovers miR-206 as a Negative Regulator of Hematopoietic Commitment in Human Pluripotent Stem Cells" International Journal of Molecular Sciences 20, no. 7: 1737. https://doi.org/10.3390/ijms20071737