Analysis of the Effects of Five Factors Relevant to In Vitro Chondrogenesis of Human Mesenchymal Stem Cells Using Factorial Design and High Throughput mRNA-Profiling

Abstract

The in vitro process of chondrogenic differentiation of mesenchymal stem cells for tissue engineering has been shown to require three-dimensional culture along with the addition of differentiation factors to the culture medium. In general, this leads to a phenotype lacking some of the cardinal features of native articular chondrocytes and their extracellular matrix. The factors used vary, but regularly include members of the transforming growth factor β superfamily and dexamethasone, sometimes in conjunction with fibroblast growth factor 2 and insulin-like growth factor 1, however the use of soluble factors to induce chondrogenesis has largely been studied on a single factor basis. In the present study we combined a factorial quality-by-design experiment with high-throughput mRNA profiling of a customized chondrogenesis related gene set as a tool to study in vitro chondrogenesis of human bone marrow derived mesenchymal stem cells in alginate. 48 different conditions of transforming growth factor β 1, 2 and 3, bone morphogenetic protein 2, 4 and 6, dexamethasone, insulin-like growth factor 1, fibroblast growth factor 2 and cell seeding density were included in the experiment. The analysis revealed that the best of the tested differentiation cocktails included transforming growth factor β 1 and dexamethasone. Dexamethasone acted in synergy with transforming growth factor β 1 by increasing many chondrogenic markers while directly downregulating expression of the pro-osteogenic gene osteocalcin. However, all factors beneficial to the expression of desirable hyaline cartilage markers also induced undesirable molecules, indicating that perfect chondrogenic differentiation is not achievable with the current differentiation protocols.

Identification of IL‐1β and LPS as optimal activators of monolayer and alginate‐encapsulated mesenchymal stromal cell immunomodulation using design of experiments and statistical methods

“…This design allows for accurate estimation of the effects of all individual factors and all two‐factor interactions, assuming that higher order interactions (≤4th order) are negligible. DoE methodology is extensively used in biotechnological fields for bioprocess development/optimization and is increasingly being utilized for basic and preclinical research applications, such as optimization of stem cell differentiation and antiviral drug studies . We have previously utilized FFD to screen the effects of 13 MSC‐secreted factors on the conversion of macrophages from a proinflammatory to an anti‐inflammatory phenotype .…”

“…DoE methodology is extensively used in biotechnological fields for bioprocess development/optimization and is increasingly being utilized for basic and preclinical research applications, such as optimization of stem cell differentiation and antiviral drug studies. [29][30][31][32] We have previously utilized FFD to screen the effects of 13 MSC-secreted factors on the conversion of macrophages from a proinflammatory to an anti-inflammatory phenotype. 8 This study identified PGE2 as the primary MSC-secreted molecule involved in this macrophage conversion.…”

Transcriptomics of human multipotent mesenchymal stromal cells: Retrospective analysis and future prospects

“…Comparative transcriptome analysis has been employed to investigate the cell response, particularly MSCs, in such 3D culture systems (Gatta et al, 2013;Hu et al, 2014;Kim et al, 2014;Pedersen et al, 2013;Roobrouck et al, 2011;Wu et al, 2012;Han et al, 2013; A C C E P T E D M A N U S C R I P T Jakobsen et al, 2014;Neuss et al, 2011). For example, a study by Han et on the scaffolds made with poly(lactic acid) than those made with poly(ɛ-caprolactone) (Neuss et al, 2011).…”

“…However, this also induced undesirable molecules, thus, indicating a need for improvement in the differentiation protocols (Jakobsen et al, 2014).…”

A promising novel formulation for articular cartilage regeneration: Preclinical evaluation of a treatment that produces SOX9 overexpression in human synovial fluid cells

“…Corticosteroids are one such bioactive substance. When acting alone, corticosteroids may facilitate tissue atrophy and joint destruction; however, when acting in synergy with other factors, they have been demonstrated to increase a number of chondrogenic markers (10), in addition to aiding in the accumulation of extracellular matrix, which is necessary for the formation of cartilage (11). In addition, substances that contain insulin or certain organic acids favor chondrogenesis (12–14).…”