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- Bigdeli, Narmin, 1974, et al.
(författare)
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Coculture of human embryonic stem cells and human articular chondrocytes results in significantly altered phenotype and improved chondrogenic differentiation.
- 2009
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Ingår i: Stem cells (Dayton, Ohio). - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 27:8, s. 1812-21
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Tidskriftsartikel (refereegranskat)abstract
- Human embryonic stem (hES) cells have been suggested as a cell source for the repair of cartilage lesions. Here we studied how coculture with human articular chondrocytes affects the expansion potential, morphology, expression of surface markers, and differentiation abilities of hES cells, with special regard to chondrogenic differentiation. Undifferentiated hES cells were cocultured with irradiated neonatal or adult articular chondrocytes in high-density pellet mass cultures for 14 days. Cocultured hES cells were then expanded on plastic and their differentiation potential toward the adipogenic, osteogenic, and chondrogenic lineages was compared with that of undifferentiated hES cells. The expression of different surface markers was investigated using flow cytometry and teratoma formation was studied using injection of the cells under the kidney capsule. Our results demonstrate that although hES cells have to be grown on Matrigel, the cocultured hES cells could be massively expanded on plastic with a morphology and expression of surface markers similar to mesenchymal stem cells. Coculture further resulted in a more homogenous pellet and significantly increased cartilage matrix production, both in high-density pellet mass cultures and hyaluronan-based scaffolds. Moreover, cocultured cells formed colonies in agarose suspension culture, also demonstrating differentiation toward chondroprogenitor cells, whereas no colonies were detected in the hES cell cultures. Coculture further resulted in a significantly decreased osteogenic potential. No teratoma formation was detected. Our results confirm the potential of the culture microenvironment to influence hES cell morphology, expansion potential, and differentiation abilities over several population doublings.
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| 3. |
- Ellerström, Catharina, et al.
(författare)
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Facilitated expansion of human embryonic stem cells by single-cell enzymatic dissociation
- 2007
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Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 25:7, s. 1690-1696
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Tidskriftsartikel (refereegranskat)abstract
- Traditionally, human embryonic stem cells (hESCs) are propagated by mechanical dissection or enzymatic dissociation into clusters of cells. To facilitate up-scaling and the use of hESC in various experimental manipulations, such as fluorescence-activated cell sorting, electroporation, and clonal selection, it is important to develop new, stable culture systems based on single-cell enzymatic propagation. Here, we show that hESCs, which were derived and passaged by mechanical dissection, can be rapidly adjusted to propagation by enzymatic dissociation to single cells. As an indication of the stability of this culture system, we demonstrate that hESCs can be maintained in an undifferentiated, pluripotent, and genetically normal state for up to 40 enzymatic passages. We also demonstrate that a recombinant trypsin preparation increases clonal survival compared with porcine trypsin. Finally, we show that human foreskin fibroblast feeders are superior to the commonly used mouse embryonic fibroblast feeders in terms of their ability to prevent spontaneous differentiation after single-cell passaging. Importantly, the culture system is widely applicable and should therefore be of general use to facilitate reliable large-scale cultivation of hESCs, as well as their use in various experimental manipulations.
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