An RNA Interference Screen Identifies the Cell Fate Determinants Msi2 and Prox1 as Novel Regulators of Hematopoietic Stem Cell Self-Renewal.
Bibliographic record
Abstract
Abstract Abstract 394 Hematopoietic stem cells (HSC) can not yet be unambiguously prospectively identified, a fact which has made it difficult to determine whether a segregation of cell fate determinants underlies the asymmetric/symmetric self-renewal of these cells or whether deregulation of such determinants could contribute to the pathogenesis of hematopoietic malignancies by inducing constitutive symmetric self-renewal divisions. We have addressed these questions through a functional genetics approach taking advantage of systematic RNAi to evaluate the function of conserved polarity factors and cell fate determinants in HSCs. From a list of 72 of such factors identified in the literature, 30 murine homologues were chosen based on their differentially higher level of expression in HSC-enriched populations as measured by qRT-PCR. For each candidate we designed 3 unique short hairpin RNA (shRNA) encoding retroviral constructs also carrying EGFP for the purposes of following transduced cells. Primitive hematopoietic cells enriched for HSC were infected at high efficiency with the library in an arrayed 96-well format and their in vivo reconstituting potential was then evaluated through competitive repopulating unit assays. Genes for which shRNA vectors altered late transplant EGFP levels below or above thresholds as defined by a control shRNA to luciferase were considered as hits. Using this approach, we identified and comprehensively validated 4 genes, including the RNA binding protein Msi2, for which shRNA-mediated depletion dramatically impairs repopulation but does not induce cell death or a cell cycle block. Importantly, we show that the loss in the repopulating ability of these shRNA transduced cells is mediated at the stem cell level and is not due to progenitor or downstream cell toxicity or to any defect in the process of bone marrow homing. Subsequent expression profiling indicated that Msi2 is also upregulated in HOXB4-overexpressing symmetrically expanding HSC in line with our findings that it functions as a positive HSC regulator and further suggesting that it represents a potential novel HSC marker. As well as finding HSC agonists, the RNAi screen identified the homeodomain containing transcription factor Prox1 as a negative HSC regulator since its shRNA-mediated transcript loss consistently led to the dramatic in vivo accumulation of EGFP+ transduced cells. Grafts comprised of Prox1 shRNA-transduced cells did not exhibit any lineage skewing however, repeatedly contained an average of 10-fold more primitive Lin-Sca+CD150+48- cells as compared to non-transduced donor cells within the same recipient or to control shRNA-luciferase grafts indicating Prox1 knockdown leads to a significant in vivo expansion of phenotypic HSCs. Moreover, following a 7 day in vitro culture, cells infected with shRNAs to Prox1 were both morphologically and immunophenotypically more primitive than control cells and when transplanted at this time yielded a significantly enhanced engraftment level relative to control shRNAs (51+/-6% GFP vs 8+/-3% GFP). These results further suggest that Prox1 reduction by RNAi expands functional HSCs in vitro. Together these findings have identified conserved cell fate determinants as important and novel regulators of murine hematopoietic stem cells. Disclosures: No relevant conflicts of interest to declare.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.001 | 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) | 0.001 | 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 source (direct Gemma or distilled Codex), 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".