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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.

SUMMARY:Experimental allergic encephalomyelitis (EAE) is a frequently employed animal model of the human disease multiple sclerosis. EAE can be induced by adoptive transfer of CD4 ϩ T cells that are specific for central nervous system (CNS) antigens, typically myelin proteins. Although the pathogenic mechanism or mechanisms responsible for the clinical signs and histological changes in EAE and multiple sclerosis are not fully defined, the entry of T lymphocytes and antigen recognition within the CNS are required. The present study describes the participation of a novel cell surface molecule with properties suggesting a role in cell-cell adhesion or co-stimulation, or both, in the development of EAE in the rat. The molecule is defined by the unique monoclonal antibody (mAb) TLD-4A2. The TLD-4A2 antigen is present on resting and activated T lymphocytes, activated CNS endothelial cells, and microglia. The antigen is normally distributed in many tissues including lymph node, thymus, and spleen, as well as in the inflamed CNS. Both its pattern of tissue distribution and immunoprecipitation and immunoblotting studies suggest that the TLD-4A2 antigen is a novel molecule. Treatment of rats with the purified 4A2 mAb resulted in the inhibition of the clinical signs of EAE and also decreased the number T cells and macrophages accumulating in the CNS parenchyma. TLD-4A2 antibody did not seem to directly interfere with T cell viability in vivo, as demonstrated by the ability to recover and stimulate CD4 ϩ encephalitogenic T cells from cervical lymph nodes of 4A2-treated animals. In vitro, the antibody partially blocked T cell proliferation assays. These data suggest that the TLD-4A2 mAb recognizes a novel molecule expressed on lymphocytes, endothelial cells, and macrophages that may play a role in hematogenous cell traffic and the initiation of CNS inflammation. (Lab Invest 2000, 80:313-326).

Directed differentiation is defined as the ability to program a stem cell at the most primitive level while it still has its reproductive and full proliferative potential. This is in contrast to ex-vivo expansion where the stem cells are forced into specific lineage commitments, limiting the overall therapeutic utility. We have reproducibly induced directed stem cell differentiation towards megakaryopoiesis by capitalizing on inherent changes in sensitivities to inductive cytokine signals in the context of cell cycle position. Murine experiments have been performed on highly purified quiescent G0–1 lineagenegative rhodaminelowHoeschtlow (LRH) marrow stem cells. When exposed to thrombopoietin, FLT3-ligand and steel factor (TFS), they synchronously pass through cell cycle. Megakaryopoiesis is focused at early to mid S-phase, returning to baseline before initial cell division. Population based differentiation cultures after 14-days produced up to 49% megakaryocytes with stem cells sub-cultured during early-mid S-phase with little to no production with colonies cultured from stem cells in G0–1 or G2 phase at time directed differentiation signaling. Gene expression showed over 2 fold increases in FOG, Nfe2 and Fli1. Clonal studies confirm the results. In one experiment, 33% of clonally derived colonies that grew from early-S phase cells and 10% of colonies that grew from mid-S phase cells had megakaryocytes present compared with 0% for G0–1 and G2 cells. We have now worked with human lineagenegative double-effluxed-rhodaminelow double-effluxed-Hoeschtlow G0–1 stem cells. When expose to TFS cytokines, there initial cell cycle lasts more than 80 hours opposed to CD34+ cells and murine LRH cells which have divided by 44–48 hours. This human population of stem cells comprises approximately 0.01% of CD34+ cells and has tremendous promise in replicating our murine work, elucidating opportunities for human translational work targeting patients that have a block of differentiation toward megakaryopoiesis i.e. sub-sets of autologous transplant or myelodysplastic syndrome patients.