Characterisation of novel culturing systems for the in vitro expansion of haematopoietic stem cells
Bibliographic record
Abstract
Haematopoietic stem cell (HSC) self-renewal is essential for the development and homeostasis of the blood system.Self-renewal expansion divisions where one HSC makes two equally potent daughter HSCs can be harnessed to create large numbers of HSCs for cell and gene therapies.Although mouse and human HSCs can be expanded in vitro to unprecedented levels using recently developed expansion cultures, not all cells in culture maintain stem cell properties.Chapter 4 of this thesis focused on refining the identification of functional expanded HSCs by using ESAM as a novel marker and replacing the need for the Fgd5 ZsGreen•ZsGreen/+ reporter, which in turn averts the need to cross genetically modified mice onto the Fgd5 background in order to permit HSC identification.Successive chapters (5 and 6) focused on the newly described expansion system for human HSCs -focusing initially on establishing this system for cord blood samples and testing further refinements to this expansion protocol via the supplementation of additional molecules to the culture media to increase HSC frequency.Chapter 5 of this thesis successfully applied this culture system to expansion of previously difficult to culture peripheral blood samples from healthy individuals and patients with early-stage blood cancers.This thesis then aimed to unpick the specific roles of cytokine agonists used in the human HSC culture media, specifically the thrombopoietin-mimetic butyzamide -a strong candidate for culture refinement which could alter downstream signalling and therefore culture success.To elucidate the effects of butyzamide versus alterative thrombopoietin (TPO) mimetics, in Chapter 6, receptor internalisation assays, fluorescent cell barcoding with phospho-flow cytometry, and phosphoprotomics were used on cell lines expressing either wild-type TPOreceptor (MPL), or JAK2 V617F mutated MPL, and have identified key differences which may be exploitable for further improving and modulating cultures for therapeutic purposes
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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 | 0.001 | 0.001 |
| 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 | 0.001 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| 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".