Molecular Validation of Chondrogenic Differentiation and Hypoxia Responsiveness of Platelet-Lysate Expanded Adipose Tissue–Derived Human Mesenchymal Stromal Cells

Abstract

Objective: To determine the optimal environmental conditions for chondrogenic differentiation of human adipose tissue-derived mesenchymal stromal/stem cells (AMSCs). In this investigation we specifically investigate the role of oxygen tension and 3-dimensional (3D) culture systems. Design: Both AMSCs and primary human chondrocytes were cultured for 21 days in chondrogenic media under normoxic (21% oxygen) or hypoxic (2% oxygen) conditions using 2 distinct 3D culture methods (high-density pellets and poly-ε-caprolactone [PCL] scaffolds). Histologic analysis of chondro-pellets and the expression of chondrocyte-related genes as measured by reverse transcriptase quantitative polymerase chain reaction were used to evaluate the efficiency of differentiation. Results: AMSCs are capable of expressing established cartilage markers including COL2A1, ACAN, and DCN when grown in chondrogenic differentiation media as determined by gene expression and histologic analysis of cartilage markers. Expression of several cartilage-related genes was enhanced by low oxygen tension, including ACAN and HAPLN1. The pellet culture environment also promoted the expression of hypoxia-inducible cartilage markers compared with cells grown on 3D scaffolds. Conclusions: Cell type-specific effects of low oxygen and 3D environments indicate that mesenchymal cell fate and differentiation potential is remarkably sensitive to oxygen. Genetic programming of AMSCs to a chondrocytic phenotype is effective under hypoxic conditions as evidenced by increased expression of cartilage-related biomarkers and biosynthesis of a glycosaminoglycan-positive matrix. Lower local oxygen levels within cartilage pellets may be a significant driver of chondrogenic differentiation.

Adipose-Derived Mesenchymal Stem Cell Chondrospheroids Cultured in Hypoxia and a 3D Porous Chitosan/Chitin Nanocrystal Scaffold as a Platform for Cartilage Tissue Engineering

“…Our data revealed a significantly higher expression of HIF-1α at 7 and 14 days in chondrospheroids cultured under hypoxia than in those cultured under normoxia, which explains the higher COL2A1 and aggrecan expression at the same time points. These findings are in line with those obtained by other authors, in which hypoxic conditions were associated with the increased expression of cartilage-related biomarkers and biosynthesis of a glycosaminoglycan-positive matrix in 3D structures [15,49] and the maintenance of the chondrogenic phenotype in chondrocytes [50].…”

Human Platelet Lysate Improves Bone Forming Potential of Human Progenitor Cells Expanded in Microcarrier-Based Dynamic Culture

“…Schallmoser et al and Capelli et al also reported the feasibility of clinical scale expansion of MSCs from BM aspirate without any prior manipulation using HPL . It has also been reported that use of HPL enhances osteogenic and chondrogenic differentiation capabilities of MSCs in vitro .…”

In vitro study on chondrogenic differentiation of human adipose-derived stem cells on treated bovine pericardium

“…Thus, the stable expression of Col10a1 in our study indicated that hADSCs might approach an end-stage differentiation. The decreasing relative transcription level of COMP from day 7 to day 14 and increasing Col10a1 expression were also observed in human chondrocytes when cultured in a cell culture incubator (Galeano-Garces et al, 2017). Consequently, GAG products from differentiated cells did not aggregate enough to form a large amount of aggrecan.…”