Abstract 5595: Regulated Expression and Function of c-Myb During Cardiovascular-directed Differentiation of Mouse Embryonic Stem Cells
Bibliographic record
Abstract
Background: c-Myb plays a pivotal role in hematopoiesis and in proliferation of vascular smooth muscle cells (VSMC). We showed that c-myb null (KO) mouse embryonic stem cells (ESC) were unable to form contractile VSMC, but formed contracting cardiomyocytes (CM) in embryoid bodies (EB) with seemingly greater efficiency. This suggested lineage-defining effects of c-Myb during CM and VSMC differentiation. Methods & Results: To investigate underlying mechanisms, we first studied c-Myb levels in wild-type (WT) ESC. In serum-conditioned spontaneously differentiating WT-ESC, Western blot revealed high c-Myb levels on days 0 –2, rapid/complete loss of c-Myb on days 2.5 to 3, and subsequent re-expression during days 4 – 6 of differentiation. The loss of c-Myb between days 2.5–3.0 did not occur with any change in mRNA, but was completely blocked by proteosome inhibitor MG132. We next studied ESC-derived cardiovascular progenitor populations by flow cytometry using VEGFR2 and PDGFR α makers. By day 3, VEGFR2+/PDGFR α - cells were significantly fewer, while VEGFR2-/PDGFR α + and VEGFR2+/PDGFR α + cells were more frequent, in KO vs. WT EB. In serum-free cardiac-directed differentiation cultures of WT ESC, Western blot revealed the highest levels of c-Myb expression at day 2, with rapid loss by day 3 in VEGFR2+/PDGFR α + cells. Of interest, VEGFR2+/PDGFR α - cells showed the highest levels of c-Myb, while VEGFR2+/PDGFR α + cells expressed the lowest. Subsequently, c-Myb was not detectable in beating CM populations and differentiating CM that emerged under these conditions. Lentiviral constructs enabling inducible expression of a shRNA against c-Myb were used to temporally knock down c-Myb expression and observe corresponding effects on cardiovascular differentiation. Finally, co-IP and mass spectrometry identified TIF1 β , a known negative regulator of c-Myb, as a putative c-Myb partner proteins affecting c-Myb-modulated cardiovascular lineage specification. Summary: These data show that c-Myb is tightly regulated by proteosomal degradation and protein-protein interactions during ESC differentiation, and that it plays a role in the differentiation of VEGFR2+/PDGFR α - cells, which may represent the source of the contractile VSMC observed in EBs.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".