Abstract
Cartilage regeneration is a fast growing field that combines biotechnology and molecular techniques in creating new tissue mimicking the native microenvironment. Human embryonic stem cells (hESCs) are a highly potent cell source for cartilage regeneration owing to their infinite proliferation capacity and pluripotency. Thus, lineage-specific differentiation of hESCs often results in populations with cellular heterogeneity. Chondrogenesis was induced through high-density micromass culture of hESCs and by addition of chondrogenic medium; 1:100 ITS+, 100 nM dexamethasone, 40 μg/ml l-proline, 50 μg/mL ascorbic acid-2-phosphate, 1:100 Knockout serum, and 10 ng/mL TGFβ3. At day 14 micromasses were dissociated and chondrogenically committed cell separated in a fraction-based discontinuous density gradient. After fractionation the chondrogenically committed cells were analyzed with regard to embryonic- and chondrogenic gene expression and fraction F3 and F4 with histology. In general, we found that the chondrogenic condition compared with the control condition had a significant effect on the following gene expression levels: NANOG, OCT4, SOX5, SOX9, ACAN, and COL2A1 in all fractions. Furthermore, we found in the chondrogenic condition that NANOG, OCT4, and SOX9 were significantly higher in F4 compared with F3, whereas COL2A1 and the ratio COL2A1:COL1A1 were significantly lower. Additionally, toluidine blue pH 4 stains of pellet cultures of F3 and F4 revealed that cells from F3 were more homogenous in morphology than F4. In conclusion, we propose a simple strategy to obtain more homogenous population of chondrogenically committed cells from hESCs using micromass culture and discontinuous density gradient separation.
Highlights
Articular cartilage regeneration represents one of the major challenges in orthopedic surgery
Even though autologous articular chondrocytes have the potential to produce various degrees of hyaline cartilage in vivo the cells are very limited in availability.[1]. This facilitates the interest of using human embryonic stem cells for cartilage regeneration owing to their infinite proliferation capacity and pluripotency
Compared with the control condition, chondrogenic medium significantly reduced the expression of NANOG and OCT4 and increased the expression of SOX5, sex determining region Y box 9 (SOX9), ACAN, and collagen type 2 alpha 1 (COL2A1) in all fractions (Fig. 2A)
Summary
Articular cartilage regeneration represents one of the major challenges in orthopedic surgery. Even though autologous articular chondrocytes have the potential to produce various degrees of hyaline cartilage in vivo the cells are very limited in availability.[1] This facilitates the interest of using human embryonic stem cells (hESCs) for cartilage regeneration owing to their infinite proliferation capacity and pluripotency. Several studies report that lineage commitment of hESCs in a certain direction often results in low differentiation efficiency and cellular heterogeneity in the differentiated cell population, which poses the risk of tumorigenicity in vivo.[2] Previously, the fate decision of hESCs has been induced by the use a high-density micromass system for chondrogenic commitment.[3,4,5].
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