Comparison of Different Strategies of MSC Isolation Revels Advantage To Expand MSC Directly from Purified CD105+ and CD271+ Cells.
Notice bibliographique
Résumé
Abstract The potential for multilineage differentiation together with the ability to expand in cultures are the reasons why Mesenchymal Stem Cells (MSC) are considered to be the population of stem cells for potential treatment for variety of disorders (e. g. Osteogenesis Imperfecta, Myocardium Infarction, GvHD). MSC are isolated from the bone marrow mononuclear cells (MNC) based on their adhesive properties. There have been few attempts to isolate MSC directly based on the expression of selected surface antigens, but these isolation strategies were not compared with “the gold standard” procedure which is still selection by plastic adherence. Nevertheless, it is obvious that a presence of different populations of cells “contaminating” MSC in adherent cell cultures (e.g., endothelial cells, macrophages, dendritic cells) may affect expansion of MSC. In this study we proposed new isolation strategies of bone marrow MSC based on RosetteSep Isolation Kit (Stem Cells Technologies Inc., Vancouver, Canada) and immunomagnetic isolation of CD105+ or CD271+ cell populations (Miltenyi Biotec, Germany). Four fractions of bone marrow mononuclear cells i) non-purified MNC, ii) MNC enriched in MSC by RosetteSep Isolation Kit, iii) sorted CD105+ and iv) sorted CD271+ cells were cultured for three passages. Subsequently, we evaluated i) number of CFU-F colonies, ii) expression of selected surface antigens (CD105, CD166, CD44, CD73, CD45, CD34), iii) in vitro osteogenic differentiation of expanded cells and iv) changes in the expression of genes related to osteogenesis (RQ-PCR). We found that the mean number of CFU-F colonies counted on the 9th day of culture was 26 (range 14,5–41,4), 49 (range 21,2–97,1), 105 (range 36,5–221) and 148 (range 55,3–211) per 107 MNC for non-purified MNC, MNC enriched in MSC by RosetteSep Isolation Kit, purified CD105+cells and purified CD271+cells, respectively. After 3rd passage the phenotype of cells was similar as we observed a comparable percentage of cells positive (over 90%) for CD105, CD166, CD44, CD73 and negative (below 5%) for CD45, CD34 surface antigens in all fractions. The RQ - PCR analysis of mRNA level of osteogenic (osteocalcin, PTHR, α1collagen), adipogenic (lipoprotein lipase, leptin, PPARγ2) and chondrogenic (aggrecan1) genes in all four populations revealed that MSC isolated by means of expression of 105 and CD271 antigens had higher level of mRNA for all assessed genes except for lipoprotein lipase and α1collagen prior to differentiation. After 30 days of osteogenic differentiation RQ - PCR analysis was repeated and compared with that before differentiation. We noticed an increased level of mRNA for osteocalcin and PTHR (markers of osteogenic differentiation) in all four populations, with the highest expression in MSC derived from non-purified MNC. However, this fraction had also the highest mRNA level of PPARγ2, lipoprotein lipase, and aggrecan genes (adipogenic and chondrogenic lineage respectively). Since the highest number of CFU-F was derived from purified CD105+ and CD271+ cells as well as these two populations seem to be the most homogenous based on RQ-PCR data, these cell fractions should be employed to expand most efficiently MSC for potential therapeutic purposes. Our data suggest that, non-MSC cells present in MNC and RosetteSep cultures may negatively affect both the expansion efficiency and differentiation along desired MSC lineage.
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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) | 0,001 | 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 ».