Journal: The Journal of Experimental Medicine

Article Title: Immune regulation by glucocorticoids can be linked to cell type–dependent transcriptional responses

doi: 10.1084/jem.20180595

Figure Lengend Snippet: The direction and magnitude of transcriptional regulation by glucocorticoids are cell type dependent. Four primary human hematopoietic cell types and five primary human nonhematopoietic cell types were studied. For each cell type, cells from four unrelated healthy donors were independently cultured and treated with methylprednisolone (22.7 µM) or vehicle (0.08% ethanol). Total RNA was purified 2 and 6 h after in vitro treatment and RNA-seq was performed. Differential expression was assessed by comparing data from methylprednisolone-treated versus vehicle-treated cells in the four biological replicates. The statistical significance of differential expression was calculated with a Wald test, after accounting for dispersion, library size, and read count. The resulting P values for differential expression were adjusted for multiple testing by the method of . (a) The left panel displays the transcriptional response to glucocorticoids in hematopoietic cells versus nonhematopoietic cells for each of 56,870 genes. The log2 fold change compares methylprednisolone-treated versus vehicle-treated cells after 6 h of in vitro treatment. Each dot represents one gene. The x-axis variable is the mean log2 fold change in the five nonhematopoietic cells (endothelial cells, fibroblasts, myoblasts, osteoblasts, and preadipocytes), and the y-axis variable is the mean log2 fold change (FC) in the four hematopoietic cells (B cells, CD4 + T cells, monocytes, and neutrophils). The four tails of the distribution are color-coded and represent genes with evidence of transcriptional response to glucocorticoid (defined here as a mean log2 fold change ≥ 0.5 or ≤ −0.5) in one group of cells but not in the other. The right panel displays the baseline expression levels in hematopoietic versus nonhematopoietic cells for the genes with strongest evidence of a transcriptional response to glucocorticoid in one group of cells but not in the other (genes at the four tails of the distribution, as defined above). The values displayed are the mean log2 normalized read count at baseline in nonhematopoietic cells (x axis) versus hematopoietic cells (y axis). (b) Transcriptional response of TRIM22 to in vitro glucocorticoid treatment in nine primary human cell types. (c) Transcriptional response of ITGA5 to in vitro glucocorticoid treatment in nine primary human cell types. In b and c, the values displayed are the normalized read counts in vehicle-treated cells (VH; average of 2 and 6 h) and in glucocorticoid-treated cells (GC; 2 or 6 h). Each dot represents one biological replicate (one donor). Multiple-testing-adjusted P values ( q ) are from comparisons of glucocorticoid-treated versus vehicle-treated cells at each of the two time points. ns, not significant ( q > 0.05).

Article Snippet: Human primary osteoblasts from adult (Lonza; cat. no. CC-2538, lot no. 0000435102) or child (Lonza; cat. no. CC-2538, lot nos.

Techniques: Cell Culture, Purification, In Vitro, RNA Sequencing, Quantitative Proteomics, Dispersion, Expressing

Journal: Bone

Article Title: Ex Vivo Construction of Human Primary 3D-Networked Osteocytes

doi: 10.1016/j.bone.2017.09.012

Figure Lengend Snippet: [32] (a) microfluidic perfusion device with 6 culture chambers, (b) cross-sectional view of a 3D culture chamber with the red arrows indicating the overall direction of culture medium flow through the device, (c) schematic illustration of microbeads-guided assembly, and (d) histologic image of 3D-networked osteocytes with the red arrows indicating medium flow direction with respect to the tissue sample. Scale bar: 20 µm.

Article Snippet: A human 3D bone tissue model was developed by constructing ex vivo the 3D network of osteocytes via: (1) the biomimetic assembly of primary human osteoblastic cells with 20–25 μm and BCP microbeads and (2) subsequent microfluidic perfusion culture.

Techniques:

Journal: Bone

Article Title: Ex Vivo Construction of Human Primary 3D-Networked Osteocytes

doi: 10.1016/j.bone.2017.09.012

Figure Lengend Snippet: (a) hip fragment shown as an example; (b) as-isolated cells after 4 collagenase digestion cycles; (c) proliferated osteoblastic cells after 10 days of 2D culture; (d) 3D tissue sample constructed using 20–25 µm microbeads and proliferated cells and 14 days of perfusion culture; (e) H&E histologic images showing the formation of 3D cellular network as indicated by black arrows in (f) and white arrows in (g); and (h) immunostaining for sclerostin (red). (d) –(f) from patient sample #6 and (g)–(h) from patient sample #4. Scale bar: 25 µm.

Article Snippet: A human 3D bone tissue model was developed by constructing ex vivo the 3D network of osteocytes via: (1) the biomimetic assembly of primary human osteoblastic cells with 20–25 μm and BCP microbeads and (2) subsequent microfluidic perfusion culture.

Techniques: Isolation, Construct, Immunostaining