# Gradient Fractionated Separation of Chondrogenically Committed Cells Derived from Human Embryonic Stem Cells

## TL;DR

This paper describes a method to separate chondrogenically committed cells from human embryonic stem cells using a density gradient, improving cell homogeneity for cartilage regeneration.

## Contribution

A new strategy using micromass culture and density gradient separation to obtain more homogeneous chondrogenic cell populations from hESCs.

## Key findings

- Chondrogenic conditions significantly affected gene expression levels of NANOG, OCT4, SOX5, SOX9, ACAN, and COL2A1 in all fractions.
- Fraction F4 showed higher expression of NANOG, OCT4, and SOX9 compared to F3, while COL2A1 and COL2A1:COL1A1 ratio were lower.
- F3 cells were more morphologically homogeneous than F4 cells based on toluidine blue staining.

## 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.