Pharmacological Regulation of Adult Stem Cells: Chondrogenesis Can Be Induced Using a Synthetic Inhibitor of the Retinoic Acid Receptor

Abstract

Conventional methods for regulating the differentiation of stem cells are largely based on the use of biological agents such as growth factors. We hypothesize that stem cell differentiation could be driven by specific synthetic molecules. If true, this would offer the possibility of screening chemical libraries to develop pharmacological agents with improved efficacy. To test our hypothesis, we have determined which, if any, of the nuclear receptor superfamily might be involved in chondrogenesis. We used fluorescence-activated cell sorting, as well as quantitative polymerase chain reaction, to study expression of a range of nuclear receptors in the undifferentiated mesenchymal population and after growth factor-driven differentiation of these cells to chondrocytes. In this way, we identified retinoic acid receptor ␤ (RAR␤) as a potential pharmacological target. A low molecular weight synthetic inhibitor of the RAR␣ and RAR␤ receptors was able to induce chondrogenic differentiation of mesenchymal stem cells derived from osteoarthritis patients, in the absence of serum and growth factors. Furthermore, the pathway is independent of SOX9 upregulation and does not lead to hypertrophy. When mesenchymal cells were seeded on to polyglycolic acid scaffolds and cultured with LE135, there was a dose-dependent formation of cartilage, demonstrated both histologically and by biochemical analysis of the collagen component of the extracellular matrix. These results demonstrate the feasibility of a pharmacological approach to the regulation of stem cell function. STEM CELLS 2007;25: 2460 -2468 Disclosure of potential conflicts of interest is found at the end of this article.

Regulating Chondrogenesis of Human Mesenchymal Stromal Cells with a Retinoic Acid Receptor-Beta Inhibitor: Differential Sensitivity of Chondral Versus Osteochondral Development

“…Together with our study, this demonstrates that RARβ inhibition by LE135 seems to exert similar inhibitory effects on early differentiating cells and on cells at a more mature stage, leading overall to a complete block of MSC chondrogenesis. Stimulation of COL2A1 and AGC in human BMSC monolayer cultures at unaffected COL10A1 levels and LE135-induced cell differentiation in 3D scaffolds with only marginal deposition of cartilaginous matrix as reported by Kafienah et al [22] may be explained by the different culture conditions.…”

“…Opposite to previous studies [22,23] we here used standard long term chondrogenic pellet culture to investigate the effect of the synthetic RARβ antagonist LE135 on chondrogenesis, cartilaginous matrix deposition and cell phenotype of human BMSCs and ASCs in vitro . Our data show that LE135 is no promising inducer or stimulator of MSC chondrogenesis as reported by Kafienah et al [22] and can neither replace nor promote TGF-β action which is believed to unfortunately contribute to cell hypertrophy. Strikingly, LE135 even blocked chondrogenic TGF-β effects and completely inhibited TGF-β-induced deposition of cartilage-specific matrix molecules including collagen type II and glycosaminoglycans.…”

“…Data on retinoic acid signaling in human MSC cultures are, however, sparse and contradicting. In one report, the RARβ antagonist LE135 promoted human BMSC chondrogenesis in monolayer culture [22], while another study found LE135 to inhibit the TGF-β-mediated chondrogenic induction of BMSCs in 3D micromass pellet culture [23]. While cartilaginous matrix deposition remained unconvincing in the first study [22], the second report was restricted to short-term mRNA data only (day 8) and did neither assess extracellular collagen type II deposition nor the chondrogenic phenotype of the cells [23].…”

High-Throughput Screening for Modulators of Mesenchymal Stem Cell Chondrogenesis

“…For example, expression analysis suggests a decrease in retinoic acid receptor b is associated with chondrogenesis; subsequent treatment with a synthetic inhibitor (LE135) of this pathway induced chondrogenesis via a Sox 9-independent pathway. 17 These studies demonstrate that a clearer understanding of the molecular mechanisms governing MSC chondrogenesis may provide insight into methods for optimizing functional differentiation, and that small molecule modulators will be critical in these efforts.…”

Cell- and tissue-engineered organ replacements

“…Atala's group seeded cultured adult autologous urothelial and smooth muscle cells on to collagen membranes 3 , while Macchiarini's group used decellularised human tissue, repopulated in part with cells derived from autologous mesenchymal stem cells (MSCs) 4 . Although the protocol for differentiating stem cells into chondrocytes in the latter situation was well-established and a high purity of culture obtained 5 , it remains likely that residual MSCs were present in the final graft, and these may have contributed to its success.…”