Expansion Culture Of Hematopoietic Stem & Progenitor Cells From Frozen-Thawed, Non-Enriched Human Umbilical Cord Blood In Animal Component– & Serum–Free Medium Enhances Engraftment & Reduces Graft-Versus-Host-Disease
Notice bibliographique
Résumé
Hematopoietic stem cell transplantation in adults using umbilical cord blood (UCB) is limited by low cell dosage & post-thaw viability. In several clinical trials cytokine supplementation & stromal cell support have been shown to enhance total nucleated cells (TNC). However, clinical safety is compromised due to source inconsistency & population heterogeneity of stromal cells along with animal components of the conventional growth media. In this study, we demonstrate effective use of an animal component– & serum–free growth medium to enhance the viability & ex vivo expansion of SCID repopulating cells (SRC) from frozen-thawed, non-enriched UCB–mononucleated cells (UCB-MNC). UCB-MNC were cultured in a commercially available animal component– & serum–free medium, StemSpanTM–ACF (ACF), while StemSpanTM–SFEM (SFEM), a conventional serum–free medium with human and bovine components served as control. Both media (from STEMCELL Technologies INC. Vancouver, Canada) were supplemented with clinical grade SCF, Flt-3 ligand, TPO, & IGFBP2. The expansion effects were characterized based on cell viability, phenotypic stem & progenitor cells & functional in vitro & in vivo assays. After 3-days of culturing, viability of CD45+ UCB-MNC was maintained at a significantly higher level in ACF (90.7±0.2%) compared to SFEM (75.4±0.1%) (p<0.0001; n=3). Culturing for 11-days significantly (p<0.0001; n=6) increased CD45+CD34+CD38– hematopoietic progenitors in ACF (90.6±13.5 fold) compared to control (4.8±0.4 fold). Further phenotypic study of ACF expanded cells showed significant increases of 4.1-fold for CD45+CD34+C38–CD90+ stem cells (p<0.0001), 2.1-fold for CD45+CD34+CD13+CD33+ myeloid progenitors (p<0.01) and 2.3-fold for CD45+CD34+C38–CD7+(p<0.01) lymphoid progenitors compared to SFEM (n=6). Viable TNC expansions were 4.3±0.2 fold and 5.9±0.7 fold in ACF and SFEM respectively (n=6; p<0.05). Colony forming unit (CFU) assay showed that ACF supported significantly higher expansion of GM progenitors than SFEM (60.1±7.9 vs. 14.6±2.1 fold; p<0.00001; n=16). The numbers of multi-potent progenitors, CFU-GEMM, were maintained in ACF but decreased in SFEM (0.83±0.21 vs. 0.09±0.04 fold relative to non-expanded UCB; p<0.01; n=16). UCB-MNC cultured for 11 days reconstituted the bone marrow (BM) of sub-lethally irradiated NOD/SCID gamma (NSG) mice with human CD45+/71+ cells as measured 16 weeks after transplantation at a dosage of 1x108 cells/kg. The frequency of human cells was higher for UCB expanded in ACF (38.1±15.4%; n=5) than for UCB expanded in SFEM (3.4±2.1; n=14; p<0.01). Human CD34+ progenitors were also detected in BM of the engrafted mice at frequencies of 2.4±1.4% and 0.2±0.1% for ACF and SFEM expanded cells respectively (p<0.05). Human hematopoiesis was multi-lineage with significantly higher numbers of CD45+/71+ & CD15+/66b+ granulopoietic cells (71.4-fold; p<0.001) and CD19+/20+ B-lineage cells (23.1-fold; p<0.001) in mice transplanted with cells expanded in ACF (n=5) as compared to SFEM (n=14). At a transplantation dosage of 2.5x107 cells/kg, non-expanded grafts (n=10) had similar engraftment of CD45+/71+cells compared to ACF expanded grafts (n=5; p=0.14), while engraftment was lower for SFEM expanded grafts (n=12; p<0.01). Limiting dilution analysis revealed that SRC frequencies were increased, on average, 7.9– and 1.2–fold in ACF relative to SFEM expanded & non-expanded grafts respectively. NSG mice transplanted with non-expanded grafts had a significantly lower (p<0.001) survival rate (40.4%, n=47) compared to those transplanted with grafts expanded in ACF (90.9%, n=11) or SFEM (92.3% n=26), or injected with saline only (100%, n=7). The high mortality rate in recipients of non-expanded grafts was due to higher incidence of graft-versus-host-disease (GVHD) associated with significantly (p<0.01; n=6) higher CD45+CD7+T cells in comparison to expanded grafts. In conclusion, expansion of freeze-thawed, non-enriched UCB-MNC in animal component– & serum–free medium improves in vivo repopulation and reduces mortality due to GVHD in a xenotransplantation model. These findings could set the platform for developing safer, cheaper & time efficient clinical transplantation, since no animal components, in the form of serum albumin or stromal cells, are required to achieve desired ex vivo expansion of hematopoietic stem & progenitor cells & pre-clinical outcomes. Disclosures: No relevant conflicts of interest to declare.
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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 | 0,000 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,002 | 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 ».