Enhancement of Soluble Transcription Factor (TAT-HOXB4 and TAT-NUP98HOXA10HD) - Mediated Human Hematopoietic Stem Cell Self-Renewal by Minimizing Inhibitory Endogenous Signalling.
Notice bibliographique
Résumé
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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Prédiction distillée sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Apprise à partir de 10 348 étiquettes directes de Codex et de 10 348 étiquettes directes de Gemma. Le mode candidate est l'union des têtes enseignantes seuillées; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont ni des étiquettes humaines ni des étiquettes directes de modèles de pointe.
Scores Codex et Gemma par catégorie
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,000 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,000 | 0,000 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule tête enseignante, pas un consensus.
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