Maintaining undifferentiated mouse embryonic stem cell (mESC) culture has been a major challenge as mESCs cultured in Leukemia Inhibitory Factor (LIF) conditions exhibit spontaneous differentiation, fluctuating expression of pluripotency genes, and genes of specialized cells. Here we show that, in sharp contrast to the mESCs seeded on the conventional rigid substrates, the mESCs cultured on the soft substrates that match the intrinsic stiffness of the mESCs and in the absence of exogenous LIF for 5 days, surprisingly still generated homogeneous undifferentiated colonies, maintained high levels of Oct3/4, Nanog, and Alkaline Phosphatase (AP) activities, and formed embryoid bodies and teratomas efficiently. A different line of mESCs, cultured on the soft substrates without exogenous LIF, maintained the capacity of generating homogeneous undifferentiated colonies with relatively high levels of Oct3/4 and AP activities, up to at least 15 passages, suggesting that this soft substrate approach applies to long term culture of different mESC lines. mESC colonies on these soft substrates without LIF generated low cell-matrix tractions and low stiffness. Both tractions and stiffness of the colonies increased with substrate stiffness, accompanied by downregulation of Oct3/4 expression. Our findings demonstrate that mESC self-renewal and pluripotency can be maintained homogeneously on soft substrates via the biophysical mechanism of facilitating generation of low cell-matrix tractions.
We previously reported that CORS26 gene, isolated from C3H10T1/2 cells treated with transforming growth factor-beta1, was predominantly expressed in cartilage. Because the gene product is a kind of secretory protein produced by cartilage tissue, we named it "cartducin". Cartducin shares a similar modular organization to adipocyte-derived hormone, adiponectin. In this study, we investigated cartducin function during chondrogenesis and cartilage development. In situ hybridization analysis showed that cartducin transcripts were restricted to the proliferating chondrocytes in the growth plate cartilage. Whole-mount in situ hybridization revealed that the first significant induction of cartducin expression occurred in the sclerotome, which contains a chondrogenic cell lineage between days 9.5 and 10.5 postcoitus (p.c.) during mouse embryogenesis. Chondrogenic differentiation by combined treatment with bone morphogenetic protein-2 and insulin induced cartducin expression along with type II and IX collagen expression in chondrogenic progenitor N1511 cells. To elucidate the direct action of cartducin on the cells, recombinant cartducin protein was expressed in and purified from Escherichia coli. The recombinant cartducin potentially forms homo-oligomers and promoted the proliferation of chondrogenic progenitor N1511 cells, and chondrocytic HCS-2/8 cells in a dose-dependent manner. On the other hand, cartducin did not affect the production of sulfated glycosaminoglycan (sGAG) in these cells. These findings indicate that cartducin is a novel growth factor and plays important roles in regulating both chondrogenesis and cartilage development by its direct stimulatory action on the proliferation of chondrogenic precursors and chondrocytes.
Mouse, rat and human molars begin to form root after the completion of crown formation. In these teeth, fibroblast growth factor (Fgf) 10 disappears in the transitional stage from crown formation to root. By contrast, rodent incisors and vole molars demonstrate continuous growth, owing to the formation and maintenance of a stem cell compartment by the constant expression of Fgf10. To clarify the relationship between root formation and disappearance of Fgf10, we carried out two experiments for the loss and gain of Fgf10 function. First, we examined postnatal growth in the incisors of Fgf10-deficient mice, which have the defect of a dental epithelial stem cell compartment referred to as 'apical bud', after implantation under the kidney capsule. The growth at the labial side in the mutant mice mimics the development of limited-growth teeth. 5Ј-Bromo-2Ј-deoxyuridine (BrdU) labeling and cytokeratin (CK) 14 and Notch2 immunostaining suggested that the inhibition of inner enamel epithelium growth and the moreactive proliferation of the outer enamel epithelium and/or stellate reticulum result in Hertwig's epithelial root sheath formation. Second, we examined the effects of Fgf10 overexpression in the transitional stage of molar germs, which led to the formation of apical bud involving in the inhibition of HERS formation. Taken together, these results suggest that the disappearance of Fgf10 signaling leads to the transition from crown to root formation, owing to the loss of a dental epithelial stem cell compartment.KEY WORDS: Fibroblast growth factor 10, Apical bud, Inner enamel epithelium, Outer enamel epithelium, Hertwig's epithelial root sheath (HERS)
An in situ hybridization study of Runx2 , Osterix , and Sox9 in the anlagen of mouse mandibular condylar cartilage in the early stages of embryogenesis AbstractMandibular condylar cartilage is the best-studied mammalian secondary cartilage, differing from primary cartilage in that it originates from alkaline phosphatase-positive progenitor cells. We previously demonstrated that three transcription factors related to bone and cartilage formation, namely Runx2 , Osterix and Sox9 , are simultaneously expressed in the anlage of mandibular condylar cartilage (condylar anlage) at embryonic day (E)14. In this study, expression of these transcription factors was investigated in the anlagen of mandibular bone (mandibular anlagen) from E11.0 to 14.0. Runx2 mRNA was first expressed in the mandibular anlage at E11.5. Osterix mRNA was first expressed at E12.0, and showed a different expression pattern from that of Runx2 from E12.5 to E14.0, confirming that Osterix acts downstream of Runx2. Sox9 mRNA was expressed in Meckel's cartilage and its anlagen throughout the experimental period, but not clearly in the mandibular anlagen until E13.0. At E13.5, the condylar anlage was morphologically identified at the posterior end of the mandibular anlage, and enhanced Sox9 mRNA expression was detected here. At this stage, Runx2 and Osterix mRNA were simultaneously detected in the condylar anlage. These results indicate that the Sox9 mRNA-expressing condylar anlage is derived from Runx2/Osterix mRNAexpressing mandibular anlage, and that upregulation of Sox9 in this region acts as a trigger for subsequent condylar cartilage formation.
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