Culture Conditions for Generating Human Bone Marrow Stromal Cells Influence Cell Immunophenotype and In Vivo Biodistribution in Immune Deficient Mice.
Notice bibliographique
Résumé
Abstract Bone marrow stromal cells (MSCs) show promise for cell and gene therapies but their utility is currently limited by low levels of engraftment. A better understanding of factors underlying cell trafficking in vivo is likely to lead to enhanced MSC engraftment and tissue targeting. To address this issue, we studied the immunophenotype and transplantation potential of MSCs derived from two different culture systems, serially passaged adherent layers from standard long-term bone marrow cultures (LTBMC) and a novel stirred suspension bioreactor culture (SSBC) supplemented with IL-3 and SCF. MSCs were characterized as CD45 (−), VCAM-1 (+), CD44 (+), SH-3 (+), and CD49e (+) by flow cytometry. At earlier time points, the SSBC system generated 1.8x more MSCs compared with the LTBMC system when analyzing the CD45-negative fraction by flow cytometry. By the end of culture, however, the LTBMC system generated 7.3x more cells. Interestingly, 66% of the CD45 (−) cells from the bioreactor system were negative for both HLA Class I and II antigens. Furthermore, flow cytometry revealed that the bioreactor-derived cells expressed very low levels of the cell adhesion markers, VCAM-1 and CD44. These data suggest that the SSBC system generates cells that may have greater migratory freedom in vivo. Biodistribution patterns of the human MSCs derived from the two sources were examined in 11 completely unconditioned six-week old SCID mice. Six weeks after intravenous infusion of MSCs, femoral and tibial bone marrow, lung, liver, bone, spleen, brain, heart, and blood were analyzed for donor human cell engraftment by PCR and fluorescence in situ hybridization (FISH) against the murine background. Engraftment was determined by PCR with primers from the human alpha-satellite region (chromosome 17) that amplify a specific 850 bp fragment. One tibial bone marrow sample, two femoral bone marrow samples, one bone sample, and four lung samples from nine mice receiving LTBMC MSC were positive by PCR. In contrast, all of the same tissues from the mouse receiving SSBC MSC were negative for human donor cells, including the lung, with the intriguing exception of a positive PCR signal in heart tissue. The presence of donor MSC was confirmed in PCR+ specimens by FISH using a human Cy3- and a murine FITC-pan-centromeric probe. The frequencies of donor LTBMC MSC in the tibial bone marrow, femoral bone marrow, bone, and lung were 0.79%, 1.60%, 0.58%, and 1.99%, respectively. Donor bioreactor-derived MSCs accounted for 0.41% of the cardiomyocytes from one recipient, despite the absence of cardiac injury. Compared with LTBMC MSCs, SSBC-derived cells appear to display an unusual biodistribution pattern which may be attributable to the altered immunophenotype. The present study underscores the importance of culture conditions in influencing the immunophenotype of MSCs and holds promise for developing targeted cell therapy.
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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 | 0,001 | 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 ».