Expansion, differentiation, and culture of hematopoietic stem & progenitor cells using an ancillary GMP-compliant medium
Notice bibliographique
Résumé
Abstract Hematopoietic stem and/or progenitor cells (HSPCs) from human primary or pluripotent stem cells (hPSCs) and their derivatives, natural killer (NK), B, and T cells, hold promise as effective cellular therapies to treat various diseases, including malignancies. A significant challenge in manufacturing HSPC-derived cellular therapies is culturing sufficient numbers of HSPCs while preserving their potency and self-renewal. Concurrently, ex vivo differentiation of HSPCs into immune cells offers a cost-effective, allogeneic source for immune cell therapy. However, this entire process relies on reliable cell culture reagents that can support the expansion of functional HSPCs, as well as their subsequent differentiation into various blood cells, including T and NK cells. For clinical-grade cell and gene therapy products, high-quality ancillary materials compliant with current good manufacturing practice (cGMP) regulations are a requirement. We developed a cGMP-compliant culture medium designed to support the maintenance, expansion, and differentiation of HSPCs from both human primary and hPSC-derived progenitors. This novel HSPC medium is serum- and phenol red-free, and specifically designed to support further cell therapy manufacturing of HSPCs and HSPC-derived T and NK cells. To evaluate the novel HSPC medium, CD34+ HSPCs were isolated immunomagnetically from cord blood (CB) and cultured in HSPC medium supplemented with StemSpan™ CD34+ Expansion Supplement containing Flt3L, SCF, TPO, IL-3, and IL-6 plus 1 μM UM729 for 7 days. Expanded CB-derived CD34+ cells showed an average fold expansion of 117 ± 21 cells per input HSPC, with 53 ± 5% retaining CD34 expression (n = 6; mean ± SEM) after 7 days of culture. Notably, 6 ± 1% of the cells were also CD34+CD45RA-CD90+EPCR+, a phenotype indicative of a hematopoietic stem cell-enriched population. The yield of CD34+CD45RA-CD90+EPCR+ cells was 8 ± 2 per input cell. T and NK Differentiation potential was assessed by using HSPC medium supplemented with commercially available reagents. For differentiation to CD4+CD8+ double-positive (DP) T cells, CB- and hPSC-derived CD34+ HSPCs were differentiated for 42 and 28 days, respectively. CB-derived cultures displayed an average frequency of 20 ± 8% CD4+CD8+ cells with a yield of 21,300 ± 16,800 cells per input CD34+ HSPC (n = 6), while hPSC-derived cultures generated 34 ± 14% to CD4+CD8+ cells with a yield of 31 ± 7 cells per input CD34+ cell (n = 3; mean ± SEM). Notably, 10 ± 2% of the CB-derived and 23 ± 8% of the hPSC-derived CD4+CD8+ DP T cells also expressed CD3 and TCRαß, indicating a more differentiated phenotype. DP T cells from both cell sources were able to differentiate to more mature CD8 single-positive T cells. For differentiation to CD56+ NK cells, CB- and hPSC-derived CD34+ HSPCs were differentiated for 28 and 21 days, respectively. CB-derived cultures displayed an average frequency of 89 ± 1% CD56+ cells with a yield of 20,300 ± 3,700 cells per input CD34+ HSPC (n = 6; mean ± SEM ), while hPSC-derived cultures generated 84 ± 3% to CD56+ cells with a yield of 464 ± 132 cells per input CD34+ cell (n = 13; mean ± SEM). These results highlight the utility of this HSPC medium to generate high yields of T and NK cells required for large-scale cell manufacturing. Taken together, our data show that this novel cGMP-compliant HSPC medium enables efficient cord blood- and hPSC-derived HSPC expansion and differentiation to T and NK cells—both of which are crucial to meet the demands of immunotherapy research. These results demonstrate its potential to facilitate the development of cell and gene therapies, where their successful clinical manufacturing relies on robust and scalable cell expansion and differentiation protocols.
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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 | 0,001 |
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 ».