Stromal Cell Regulation of Murine Hematopoietic Stem Cells.
Bibliographic record
Abstract
Abstract Abstract 1566 Recent advances in purifying murine hematopoietic stem cells (HSCs) to near homogeneity (>20%) have made it possible to analyze their in vivo clonal growth, self-renewal and differentiation properties over prolonged periods and the effects of various manipulations on these key functional parameters. However, conditions that allow genetically unaltered HSCs to maintain their original functional properties over equivalent periods of prolonged proliferation in vitro have not yet been identified. Since initial studies showed that the UG26 stromal cell could support murine HSC maintenance for limited periods, we first asked whether the addition of cytokines that also maintain HSCs for short periods might synergize with UG26 cells to enable HSC expansion to occur. Limiting dilution transplants that used a 6-month read-out of reconstituted blood elements (>1%) showed that the addition of 100 ng/ml Steel Factor (SF) and 20 ng/ml IL-11 to cultures containing UG26 cells and single purified (50%) HSCs (EPCR+CD150+CD48-, ESLAM cells) consistently stimulated a 3–5 fold HSC expansion after 7 days (3 expts). Furthermore, the effect of the UG26 cells could be replaced by UG26 conditioned medium (CM) and, in the presence of the CM+SF/IL-11 cocktail, the HSCs showed sustained longterm in vivo lympho-myeloid reconstituting activity in both primary and secondary recipients. Under these conditions, every ESLAM cell isolated proliferated several times within 7 days, but individual analysis of paired daughter cells showed that most first divisions (13/42) were, nevertheless, asymmetrical in terms of the numbers and types of different lineages produced by each of the 2 daughter cells for at least 4 months, although occasional evidence of symmetry was obtained (2/42 divisions). Interestingly, these first divisions showed a biphasic curve with 75% of the cells dividing before and 25% after 48 hours - the late dividers being more highly enriched for HSCs (95% vs 20%). We next asked whether TGF-β might be an important factor in UG26 CM, since UG26 cells exert a strong cell cycle inhibitory effect, and produce abundant TGF-beta. Accordingly, we next analyzed the effect of adding a neutralizing anti-TGF-β antibody or replacing the CM with TGF-β in the same type of single HSC cultures by tracking the survival and division kinetics of the cells as well as measuring the repopulating activity of their in vitro progeny present after 7 days. Strikingly, the addition of anti-TGF-β to the CM+SF/IL-11 supplemented HSC cultures eliminated the late wave of first cell divisions and caused an accompanying loss of myeloid reconstituting ability in recipients transplanted with the cultured cells. Conversely, replacement of the CM with TGF-β restored a biphasic division kinetics curve to cultures supplemented with SF/IL-11 but no CM. However, this did not protect against the early 50% loss of cells by apoptosis. These findings provide evidence of a new role of TGF-β in preserving the integrity of HSC functionality in vitro, but suggest a requirement for other types of factors released by certain stromal cells to achieve sustained symmetrical HSC self-renewal in vitro. 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 | 0.000 | 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) | 0.002 | 0.001 |
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".