Enhancement of Soluble Transcription Factor (TAT-HOXB4 and TAT-NUP98HOXA10HD) - Mediated Human Hematopoietic Stem Cell Self-Renewal by Minimizing Inhibitory Endogenous Signalling.
Bibliographic record
Abstract
Abstract Abstract 1493 Poster Board I-516 Predictive control of human hematopoietic stem cell (HSC) self-renewal would enable more effective strategies to treat hematologic disease. Although evidence suggests that both cell autonomous (stem cell-associated transcription factors) and cell non-autonomous (the stem cell niche) mechanisms regulate stem cell fate, the dynamic interplay between these regulatory axis are poorly understood. Using a defined synthetic stem cell niche, we have been investigating the role of the transcription factors HOXB4 and the engineered fusion gene between NUP98 and the homeodomain of HOXA10 (NUP98A10HD), provided as soluble membrane-permeable proteins, as clinically relevant reagents to enhance in vitro HSC self-renewal. To aid our understanding of interactions between these complex processes, we have developed systems biology-based approaches to describe and predict cell supportive and non-supportive cell-cell interaction networks. Using a controlled and automated system to achieve semi-continuous protein delivery, and an accompanying model to predict dynamic intracellular protein concentrations, we have optimized strategies for the addition of the TAT-HOXB4 and TAT-NUP98A10HD fusion proteins to umbilical cord blood cultures. Our results demonstrate that an optimized delivery scheme of 1.5nM (from day 0-4) and 6nM (from day 4-8) every 30min, produces stable intracellular levels of TAT-HOXB4, and results in a increase of primitive progenitor cells, as measured by colony counts from bulk long term culture-initiating cell (LTC-IC) assays, of 1.9x greater than the classic, non-optimized TAT-HOXB4 delivery scheme (40nM every 4h) and 3.1x greater than untreated control cells. Ongoing studies are extending these significantly enhanced primitive progenitor outputs to HSC self-renewal using the NOD/SCID repopulating cell assay. Our results thus far demonstrate that the nuclear concentrations of HSC-associated transcription factors can significantly impact stem cell self-renewal. In these studies we also observed, for the first time, that endogenously produced secreted factors limit HSC output, and that TAT-HOXB4 acts to desensitize the primitive blood progenitor cells to negative feedback regulation by secreted factors. As a means of prospectively regulating the levels of endogenously produced factors in culture, we have implemented a media delivery approach, in which cell culture media volume is adjusted throughout the culture period, to counteract increasing negative inhibitors by endogenously produced secreted factors. Using this “fed-batch” delivery approach, we have achieved significant (p<0.05) improvement in the total cell number (TNC), colony forming cells (CFCs), and LTC-ICs, of 4.6x, 4.9x, and 4.1x respectively, above the blood stem and progenitor numbers obtained from untreated control cells. Furthermore, data suggests that this non-autonomous regulation promotes HSC self-renewal for a more prolonged period in vitro, with total expansions after 12 days of culture reaching 80x for CFCs and 22x for LTC-ICs. Media dilution strategies have been optimized to further limit negative feedback from mature cell types by monitoring and counteracting rising concentrations of specific critical factors, such as TGF-β1. Collectively, these studies shed new insight into the complexity of strict HSC regulation to predictively enhance in vitro HSC self-renewal, and provide evidence that overcoming cell non-autonomous control of HSC self-renewal should enable novel strategies to enhance endogenous stem cell growth. Disclosures: No relevant conflicts of interest to declare.
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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".