Journal: Orphanet Journal of Rare Diseases

Article Title: Impaired osteoblast and osteoclast function characterize the osteoporosis of Snyder - Robinson syndrome

doi: 10.1186/s13023-015-0235-8

Figure Lengend Snippet: Characterization of the bone and osteoblast pathology. A . Photograph of the bone biopsy. B . Steady state SMS mRNA levels relative to GAPDH expression in cultured fibroblasts and osteoblasts. The patient’s cells did not differ significantly from controls. Data were derived by qRT-PCR analysis of 3 independent extractions of total RNA. C . Immunoblot showing steady state SMS protein expression in patient and control osteoblasts. ß-tubulin is shown as a loading control. D . Graph showing steady state SMS protein levels in the patient and control hBMSCs relative to ß-tubulin levels; there was no significant difference. The data are based on 3 independent experiments for each cell line. E - J . Immunofluorescent detection of SMS protein subcellular distribution in unaffected (E-G) and Patient II-1 (H-J) hBMSCs. SMS protein is shown in red and the nucleus is shown in blue. K . Graph quantifying immunoblot detected steady state SMS protein levels in the cytoplasm and nuclei of patient and control hBMSCs. The cytoplasmic expression was normalized to β-tubulin expression and the nuclear expression to p84 expression. L . Polyamine quantification in fibroblasts and osteoblasts. Note that the patient hBMSCs have a more striking imbalance of spermidine and spermine levels than do the patient fibroblasts, * p < 0.05, *** p < 0.005. M . Osteogenic potential of bone marrow stromal cells (hBMSCs) isolated from Patient II-1 sample is markedly lower than that of an unaffected control (cnt). The hBMSCs were seeded in triplicates (6x10 4 /12-well) and either kept untreated (-) or treated (+) with osteogenic differentiation media (see ) for 18 days. After the treatment, cells were fixed and were stained with Alizarin Red S to check for calcium deposition, a marker of osteogenic differentiation.

Article Snippet: We also measured spermine and spermidine levels in cultured human bone marrow stromal cells (hBMSCs) and fibroblasts using the Biochrom 30 polyamine protocol and assessed the osteogenic potential of hBMSCs.

Techniques: Expressing, Cell Culture, Derivative Assay, Quantitative RT-PCR, Western Blot, Control, Isolation, Staining, Marker

Journal: bioRxiv

Article Title: DONOR VARIABILITY IN HUMAN MESENCHYMAL STEM CELL OSTEOGENIC RESPONSE AS A FUNCTION OF PASSAGE CONDITIONS AND DONOR SEX

doi: 10.1101/2023.11.12.566781

Figure Lengend Snippet: (A) Experimental overview for passage scheme study. Human mesenchymal stem cells (hMSCs) from 4 donors (2 male, 2 female) were cultured on mineralized collagen scaffolds after varying passage schemes to compare the influence of passage number and culture length on cell proliferation and response. hMSCs were thawed at passage 3 and seeded at passage 6 (p3-6) undergoing 2 passages in culture prior to being seeded on mineralized collagen scaffolds. hMSCs were thawed at passage 3 and seeded at passage 5 (p3-5) undergoing 1 passage in culture prior to being seeded on mineralized collagen scaffolds. hMSCs were thawed at passage 4 and seeded at passage 5 (p4-5) undergoing 1 passage in culture prior to being seeded on mineralized collagen scaffolds. These cells were subsequently cultured for 21 days and their metabolic activity, cell proliferation and secretome were quantified. (B) Experimental overview for donor variability study. hMSCs from 8 donors (4 male, 4 female) were cultured as p3-5 or p4-5 groups and cultured on mineralized collagen scaffolds for 56 days. Cell response on these scaffolds was quantified to identify donor and sex-based differences.

Article Snippet: Human mesenchymal stem cells (hMSCs) were purchased from Essent Biologics (Centennial, Colorado USA) and RoosterBio (Frederick, Maryland, USA).

Techniques: Cell Culture, Activity Assay