Hematopoietic Stem Cell Expansion, without Exhaustion or Transformation, by Stable Microrna Antagonism in Vivo
Notice bibliographique
Résumé
Abstract Abstract 30 The precise mechanisms controlling hematopoietic stem cell (HSC) quiescence, self renewal and differentiation remain incompletely understood, and HSC expansion remains a long sought after goal for cell- and gene therapy. Genetic manipulation of single transcription factors and signal transduction pathways has so far failed to provide sustained HSC expansion, often resulting in stem cell exhaustion or the emergence of malignancy. MicroRNAs (miRNAs) post-transcriptionally regulate multiple genes in a coordinated fashion, making them attractive targets to manipulate signaling networks and complex cellular functions. We have systematically manipulated miR-126, a miRNA enriched in the primitive hematopoietic compartment, in mouse and human HSC by stably knocking down its activity with a sponge lentiviral vector (126/KD LV) or forcing its overexpression by a 126/OE LV. Hematochimeric mice were generated by transplanting 126/OE LV- or 126/KD LV- transduced progenitors into congenic mice (murine HSC) or NSG mice (human HSC), and the effects of altered miR-126 levels on hematopoiesis were investigated in vivo. Steady state hematopoiesis was comparatively normal, with no alterations in complete blood cell counts except a minor skewing towards or against lymphopoiesis upon 126/OE or 126/KD, respectively. Strikingly, under stress conditions such as 5-fluorouracil exposure, secondary transplantation or the NSG xenograft environment, 126/OE resulted in a progressive loss of HSC, while 126/KD increased HSC numbers as measured by immunophenotype and functional output upon competitive transplantation into secondary recipients. Limiting dilution analysis confirmed a 3–4 fold expansion of mouse and human HSC upon miR-126/KD, and the competitive advantage of 126/KD HSC was maintained long-term including post tertiary transplantation. To study the mechanism by which miR-126 regulates HSC numbers, cell cycle analysis was performed on 126/KD- or 126/OE progenitors isolated from steady state bone marrow. Human CD34+CD38-CD90+ and murine Lineage-Kit+Sca+CD150+ HSC were shifted towards the S/G2/M or the G0 phase upon 126/KD or 126/OE, respectively, indicating that miR-126 antagonism increased cycling while excessive levels of miR-126 favoured HSC quiescence within the niche microenvironment. Interestingly, this effect was highly specific for HSC and the most primitive progenitors. An in vitro culture model of HSC-enriched cord blood cells was set up to study the molecular basis of miR-126 mediated control of HSC cycling. This model reproduced key aspects of the HSC phenotype observed in vivo, i.e. increased or decreased proliferation of primitive progenitors upon 126/KD or 126/OE, respectively. Transcriptome analysis indicated that miR-126 manipulation altered several cellular functions and pathways, most significantly the PI3K/AKT/GSK3β signalling axis. We confirmed PIK3R2 and CRKII as direct miR-126 targets in CB CD34+38- cells and show enhanced PI3K pathway activation as reflected by increased AKT and GSK3β phosphorylation upon 126/KD, while 126/OE caused opposite effects. Pharmacologic inhibition of the beta subunit of PI3K abrogated the 126/KD phenotype, indicating that miR-126 mainly acts through the PI3K pathway. In contrast to published studies showing HSC expansion upon hyperactivation of the PI3K pathway using various genetic approaches, miR-126/KD did not cause HSC exhaustion or leukemogenesis. On the contrary, evidence will be provided that 126/OE is oncogenic in hematopoietic cells. Thus, miR-126 represents a principle target for HSC expansion for cell and gene therapy applications. In summary, we propose that in vivo HSC pool size is influenced by endogenous levels of miR-126 through modulation of the HSC quiescence/proliferation equilibrium. Disclosures: No relevant conflicts of interest to declare.
Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.
Comment cette classification a été obtenuedéplier
Prédiction machine sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Le volet Gemma est une étiquette directe du modèle pour chaque travail de la base, lue sur la notice réduite au titre. Le volet Codex est un classifieur appris des 10 348 étiquettes directes de Codex et calibré sur les taux pondérés de l'échantillon; les champs sans appui suffisant ne portent aucun appel Codex. Le mode candidate est l'union des deux volets; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont pas des étiquettes humaines.
Scores du classifieur distillé par catégorie (deux têtes)
| 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 | 0,001 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,001 | 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 source (Gemma direct ou Codex distillé), pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».