Macroscopic cartilage formation with embryonic stem-cell-derived mesodermal progenitor cells.

The totipotent embryonic stem cell generates various mesodermal cells when stimulated with BMP4. Among the resulting cells, those expressing flk-1 and/or PDGFRalpha displayed chondrogenic activity in the presence of TGFbeta3 and expressed cartilage-specific genes in 7 to 16 day pellet cultures. Depositions of cartilage matrix and type II collagen were detected by day 14. TGFbeta-stimulated chondrogenesis was synergistically enhanced by PDGF-BB, resulting in a larger cartilage particle filled with a cartilaginous area containing type II collagen, with a surface cell layer expressing type I collagen. In contrast, noggin inhibited both the TGFbeta- and TGFbeta+PDGF-stimulated cartilage formation, suggesting that a BMP-dependent pathway is involved. In fact, replacement of TGFbeta3 with BMP4 on days 10 to 12 markedly elevated the cartilage matrix deposition during the following 7 to 8 days. Moreover, culture with TGFbeta3 and PDGF-BB, followed by the incubation with BMP4 alone, resulted in a cartilage particle lacking type I collagen in the matrix and the surface layer, which suggests hyaline cartilage formation. Furthermore, such hyaline cartilage particles were mineralized. These studies indicate that the PDGFRalpha+ and/or flk-1+ cells derived from embryonic stem cells possess the full developmental potential toward chondrocytes, in common with embryonic mesenchymal cells.

Polyethylene glycolated recombinant TNF receptor I improves insulitis and reduces incidence of spontaneous and cyclophosphamide-accelerated diabetes in nonobese diabetic mice.

We have conducted three studies to examine the role of TNFalpha in islet destruction in female nonobese diabetic mouse (NOD) mice, a model of human autoimmune diabetes, using polyethylene glycolated (PEGylated) soluble TNF receptor type I (PEG sTNF-RI) as TNFalpha antagonist. PEG sTNF-RI (3 mg/kg, sc) was given every other day to NOD mice from age wk 8 for 12 wk (study 1), from age wk 12 for 8 wk (study 2), or from age wk 8 for 3 wk, with cyclophosphamide (6 mg/mouse) injected at wk 9 to accelerate the onset of diabetes (study 3). Diabetic incidence was reduced (control vs. PEG sTNF-RI) from 68.7% (11 of 16) to 18.3% (3 of 16) in study 1, from 84.6% (11 of 13) to 28.5% (4 of 14) in study 2, and from 66.6% (8 of 12) to 23.1% (3 of 13) in study 3, respectively. The incidence of insulitis was also reduced from 91.6% (11 of 12) to 12.5% (2 of 16) in study 1 and from 100% (7 of 7) to 16.6% (2 of 12) in study 2 by PEG sTNF-RI. PEG sTNF-RI also largely preserved islet insulin content, reduced mRNA of inducible nitric oxide synthase and IL-6 in pancreases, and lowered plasma corticosterone, glycerol, and free fatty acid levels. These results confirm a pathogenic role of TNFalpha in mediating insulitis in NOD mice and suggest the prophylactic and therapeutic potential of PEG sTNF-RI for human autoimmune diabetes.