High-Throughput Screening for Modulators of Mesenchymal Stem Cell Chondrogenesis

Abstract

Mesenchymal stem cells (MSCs) are an attractive cell source for regenerative medicine and the study of skeletal development. Despite considerable interest in MSC chondrogenesis, the signal transduction and molecular mechanisms underlying this process remain largely undefined. To explore the signaling topology regulating chondrogenic differentiation, as well as to discover novel modulators, we developed and validated a high-throughput screening (HTS) assay for MSC chondrogenesis. Adapting standard assay procedures to enable HTS, we successfully minimized cell number, handling, and culture duration. Using our optimized methodology with automation, we evaluated a comprehensive screen using four growth factors, TGF-beta3, BMP-2, IGF-1, and FGF-2, to demonstrate the feasibility of large combinatorial screens. We examined the chondrogenic effects of these growth factors in different combinations and doses (81 combinations total with 16 replicates per group) and found variable effects on GAG content with different combinations. In general, TGF-beta3 had a pro-chondrogenic effect while FGF-2 had a proliferative effect. BMP-2 was both proliferative and pro-chondrogenic while the effect of IGF-1 in our system was variable. We also carried out an HTS campaign of the National Institute of Neurological Disorders and Stroke (NINDS) chemical library of small molecules (1040 compounds) and identified 5 potential inducers and 24 potential inhibitors of chondrogenesis. Of these compounds, several were identified from the hypnotic, anti-neoplastic, or anti-protein synthesis classes of molecules. These studies demonstrate our ability to carry out high-throughput screening assays for modulators of chondrogenesis.

Compound screening platform using human induced pluripotent stem cells to identify small molecules that promote chondrogenesis

“…Recent reports have used hMSCs to screen for compounds that promote chondrogenesis (Huang et al, 2008;Johnson et al, 2012). However, due to variations between donors, limitations in expanding the hMSC populations, and cost (Siddappa et al, 2007), these cells were not ideal.…”

Deconstructing the third dimension – how 3D culture microenvironments alter cellular cues

“…Highthroughput screening approaches can be used to expose cells to libraries of soluble factors and examine the resulting biological response. Huang and co-workers have used a high-throughput approach to optimize the soluble factors that promote mesenchymal stem cell chondrogenesis (Huang et al, 2008). Microfluidic devices (G,H in Figure) that are based on soft lithography can be used to control the finer features of the soluble microenvironment, including the timing and spatial presentation of biological factors (Quake and Scherer, 2000), or can simply reduce the effective culture volume and, thus, amplify autocrine signals (Yu et al, 2007).…”

Micromass co-culture of human articular chondrocytes and human bone marrow mesenchymal stem cells to investigate stable neocartilage tissue formation in vitro

“…In contrast to our study, the number of cells for pellet preparation in co-cultures differed between the individual cell pellets. It has been shown that the correlation in the increase of cell numbers and GAG production in pellets in not linear (Huang et al, 2008), suggesting that direct comparison of pellets containing different cell numbers could be misleading. Furthermore, the physiological properties of cells in the two studies were dissimilar; while we employed HAC with retained intrinsic chondrogenic potential (Giovannini et al, 2010), Fischer et al (2010) used HAC which required the presence of TGFβ1 for chondrogenesis.…”