Targeting Unique Epitopes on Highly Similar Proteins GDF-11 and GDF-8 with Modified DNA Aptamers.

The mature forms of the TGF-ß family members GDF-11 and GDF-8 are highly similar 25 kDa homodimers with 90% amino acid sequence identity and 99% similarity. Cross-reactivity of GDF-11 and GDF-8 binding reagents is common, making it difficult to attribute distinct roles of these two proteins in biology. We report the selection of GDF-11 and GDF-8 specific SOMAmer (Slow Off-rate Modified Aptamer) reagents aided by a combination of positive selection for one protein coupled with counter-selection against the other. We identified GDF-11 specific SOMAmer reagents from four modified DNA libraries that showed a high affinity (Kd range 0.05-1.2 nM) for GDF-11 but did not bind to GDF-8 (Kd > 1 µM). Conversely, we identified one SOMAmer reagent for GDF-8 from one of the modified libraries that demonstrated excellent affinity (Kd = 0.23 nM) and specificity. In contrast, standard protocols that utilized only positive selection produced binding reagents with similar affinity for both proteins. High affinity and specificity were efficiently encoded in minimal sequences of 21 nucleotides for GDF-11 and 24 nucleotides for GDF-8. Further characterization in pull-down, competition, sandwich-binding, and kinetic studies revealed robust binding under a wide range of buffer and assay conditions. For highly similar proteins like GDF-11 and GDF-8, our method of selection coupled with counter-selection was essential for identification of high-affinity, specific reagents that have the potential to elucidate the fundamental distinction of these growth factors in biology.

GDF11 expression in the adult rat central nervous system.

Growth differentiation factor 11 (GDF11), also known as bone morphogenetic protein 11 (BMP11), is a member of the transforming growth factor ß (TGF-ß) superfamily. Although GDF11 plays pivotal roles during development, including anterior/posterior patterning, formation of the kidney, stomach, spleen and endocrine pancreas, little information is available for GDF11 expression in the adult central nervous system (CNS). We, thus, investigated GDF11 expression in the adult rat CNS using immunohistochemistry. GDF11 was intensely expressed in most neurons and their axons. Furthermore, we found that astrocytes and ependymal cells also express GDF11 protein. These data indicate that GDF11 is widely expressed throughout the adult CNS, and its abundant expression in the adult brain strongly supports the idea that GDF11 plays important roles in the adult brain.

Development of an ultra-sensitive Simoa assay to enable GDF11 detection: a comparison across bioanalytical platforms.

BACKGROUND: Four bioanalytical platforms were evaluated to optimize sensitivity and enable detection of recombinant human GDF11 in biological matrices; ELISA, Meso Scale Discovery, Gyrolab xP Workstation and Simoa HD-1. Results & methodology: After completion of custom assay development, the single-molecule ELISA (Simoa) achieved the greatest sensitivity with a lower limit of quantitation of 0.1 ng/ml, an improvement of 100-fold over the next sensitive platform (MSD). DISCUSSION & CONCLUSION: This improvement was essential to enable detection of GDF11 in biological samples, and without the technology the sensitivity achieved on the other platforms would not have been sufficient. Other factors such as ease of use, cost, assay time and automation capability can also be considered when developing custom immunoassays, based on the requirements of the bioanalyst.

Characteristic changes of periodontal ligament-derived cells during passage.

BACKGROUND AND OBJECTIVE: Although periodontal ligament-derived cells are expected to be a useful source of cells for periodontal tissue engineering, the characteristic changes of primary cultured cells have not been well studied. Therefore, the aim of this study was to investigate the characteristics of periodontal ligament-derived cells and their changes during passage. MATERIAL AND METHODS: Human periodontal ligament tissue was obtained from extracted third molars. Cells were subcultured until passage 6 and the cell characteristics from early to late passages were evaluated using immunofluorescence microscopy, alkaline phosphatase activity analyses, reverse transcription-polymerase chain reaction and quantitative real-time polymerase chain reaction. To examine the function of periodontal ligament-derived cells further, cells were transplanted into the renal subcapsule of an immunocompromised rat. RESULTS: Immunofluorescence results showed relatively uniform expression of MSX-2 and osteonectin from passage 1 until passage 6. The STRO-1-positive fraction was 33.5% at passage 0, which was reduced to 14.7% at passage 3. Cultured cells at passage 1 expressed mRNA for collagen type I, collagen type XII, Runx2, alkaline phosphatase, osteonectin, osteopontin, scleraxis, tenomodulin, Msx2, GDF5 and GDF7 genes, but not for bone sialoprotein. The level of mRNA expression from tenomodulin and collagen type XII genes decreased after passage 3. Alkaline phosphatase activity decreased in cells at later passages. Osteogenic induction of periodontal ligament-derived cells resulted in a down-regulation of the tenomodulin gene. Transplanted cells from both early and late passages produced dense collagen fiber bundles without calcified tissue. CONCLUSION: Cultured periodontal ligament-derived cells were a morphologically homogeneous population, although expression of STRO-1 was limited in primary culture. Cultured cells showed de-differentiation during passage for both osteogenesis- and tendo/ligamentogenesis-related genes.