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| Status |
Public on Jan 31, 2021 |
| Title |
MYB is an essential regulator of primitive human hematopoiesis in human pluripotent stem cell differentiation cultures. |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
MYB is well recognized to be a key regulator of definitive hematopoiesis that plays an important role in the maintenance and multilineage differentiation of hematopoietic stem cells (HSCs). In the vertebrate developmental context, MYB is widely regarded dispensable for primitive hematopoiesis but critically required for the development of definitive hematopoiesis. To explore the role of MYB in human hematopoietic development we have inactivated the gene by bi-allelic TALEN-supported gene targeting in several lines of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), and subjected these cells to hematopoietic differentiation in well-defined cell culture conditions. Venus gene reporter was inserted into the knock-in allele to monitor MYB expression during the course of the hESC/iPSC differentiation. The gene reporter system showed that MYB is specifically expressed during hematopoietic commitment in the earliest primitive blood cells. Moreover, the level of MYB expression was highest at the commitment stage of differentiation and significantly decreased at the maturations stage. We found that MYB was not required for initial hematopoietic commitment of nascent mesoderm and emergence of primitive, yolk sac-type human hematopoietic progenitors. However, inactivation of MYB severely abrogated proliferation of the primitive erythroid and mixed erythroid-macrophage-megakaryocyte progenitors. In addition, MYB-negative hESC/iPSC lines demonstrated major defects in myeloid cell development and completely failed to generate mature granulocytes. Transposon-mediated rescue of MYB expression in MYB-null cells efficiently restored both the primitive hematopoietic progenitors and immature myeloid cells. Our data indicate that in contrast to its previously attributed exclusive role in definitive hematopoiesis, MYB is indispensable for primitive human hematopoiesis.
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| Overall design |
We inactivated the human MYB gene by biallelic integration of reporter donor cassette through TALENs-supported genetic engineering. The modified lines including MYB+/+, MYB+/- and MYB-/- were differentiated into hematopoietic cells in our new established serum-free, cytokine-free culture system with minimal BMP4 exposure for mesoderm induction. The cells were harvested at different time points for cell-sorting based on the expression of MYB-YFP and CD34 cell surface marker on day 6 and day 12. Subsequently the total mRNA was extracted using trizole reagent and subject to RNA-sequencing. In total 31 samples, 2 or 3 replicate samples per group being biological repeats, were analyzed.
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| Contributor(s) |
Shah Z, Samokhvalov IM |
| Citation missing |
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| Submission date |
Jan 28, 2019 |
| Last update date |
Jan 31, 2021 |
| Contact name |
ZAHIR SHAH |
| E-mail(s) |
zahir@gibh.ac.cn
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| Phone |
13229487422
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| Organization name |
GIBH,CAS, China
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| Department |
Stem Cells and Regenerative Medicines
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| Street address |
190 Kaiyuan avenue, Haungpu district, Guangzhou, China
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| City |
Guangzhou |
| State/province |
Guangdong |
| ZIP/Postal code |
510530 |
| Country |
China |
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| Platforms (2) |
| GPL16791 |
Illumina HiSeq 2500 (Homo sapiens) |
| GPL21290 |
Illumina HiSeq 3000 (Homo sapiens) |
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| Samples (31)
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| Relations |
| BioProject |
PRJNA517462 |
| SRA |
SRP182678 |
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