|Send to printer »|
GEN News Highlights : Oct 30, 2012
Purified Chondrocytes from iPSCs Repair Cartilage
Scientists have developed an approach to generating uniformly differentiated populations of collagen-producing chondrocytes from induced pluripotent stem cells (iPSCs) that can be used to repair cartilage defects, or harnessed as an in vitro system for testing drugs or studying disease. The method, developed by a Duke University-led team, involves engineering the cells to express GFP only if they also produce collagen, and then using a cell sorting technique to collect the fluorescent cells specifically. Essentially, expression of GFP under the control of a type II collagen promoter indicates that the cell has differentiated into a chondrocyte, rather than remained in a pluirpotent state or differentiated into a cell of any other lineage.
The potential to use iPSC technologies to generate chondrocytes for cartilage repair is particularly attractive because bone-marrow derived stem cells and adipose-derived stem cells have only a limited chondrogenic potential, especially when taken from older patients or those with diseases such as osteoarthritis. However, one of main challenges with using iPSCs is the difficulty in achieving uniform differentiation to the cell type of interest.
The cell identification and sorting approach has been developed by Farshid Guilak, M.D., Brian O. Diekman, M.D., and colleagues to get around this problem. They first prompted mouse tail fibroblasts to reprogram into iPSCs using a previously described approach of forcing the overexpression of four transcription factors, Oct4, Sox3, Klf5, and Myc. They then added growth factors including BMP-4 and dexamethasone to the cultures to prompt cell differentiation into chondrocytes. GFP marker expression indicated that about 10% of the iPSCs had differentiated into chondrocyte cells, and these were sorted by flow cytometry. Quantitative RT-PCR analysis confirmed that the GFP+ cells demonstrated increased expression of chondrogenic genes when compared with the GFP- cells.
The GFP+ cells could also be expanded in vitro, and passaging in pellet culture prompted the synthesis of cartilage-specific matrix that contained characteristic high levels of glycosaminoglycans and type II collagen, and low levels of type I and type X collagen. The pellets also demonstrated increased elasticity indicative of cartilage formation. And encouragingly, when an agarose gel matrix seeded with the GFP+ cells was applied to an in vitro cartilage defect model, the cells integrated into the damaged tissue and produced a cartilaginous matrix.
“We used techniques to purify a defined cell population with enhanced chondrogenic potential from undifferentiated and off-target cell types, providing a pathway for maximizing therapeutic effectiveness while minimizing the risk of teratoma formation,” the authors state in their published paper in PNAS. “This study demonstrates the potential use of iPSCs for cartilage defect repair and for creating tissue models of cartilage that can be matched to specific genetic backgrounds...The ability to derive large numbers of cells with chondrogenic potential from a noninvasively isolated somatic cell starting population is an important advance for the development of cellular therapy strategies to treat osteoarthritis.”
The Duke team’s work is described in a paper titled “Cartilage tissue engineering using differentiated and purified induced pluripotent stem cells.”
© 2013 Genetic Engineering & Biotechnology News, All Rights Reserved