Journal: Journal of Translational Medicine

Article Title: Cryopreservation of human vascular umbilical cord cells under good manufacturing practice conditions for future cell banks

doi: 10.1186/1479-5876-10-98

Figure Lengend Snippet: Expression of cellular marker molecules and extracellular matrix (ECM) proteins by human umbilical cord artery derived cells (HUCAC). Using indirect immunofluorescence staining, highly positive signals (green) were detected for A ) collagen type I of fresh cultivated cells and B ) collagen type I of cryopreserved cells, E ) collagen type III of fresh cultivated cells and F ) collagen type III of cryopreserved cells. The presence of C ) collagen type I (green) and G ) collagen type III (green) was shown in native human umbilical cord artery walls, serving as a control. Immunohistochemical staining verified the presence of D ) collagen type I (red) and H ) collagen type III (red) in native human umbilical cord artery walls. Using flow cytometry analysis, cellular marker expression of short-term (group A, n = 4) and long-term (group B, n = 4) cryopreserved cells from primary cultures (passage 0) was studied directly after I ) thawing and J ) in passage 3 of recultivation. By comparison, non-cryopreserved fresh cells (n = 3) from I ) primary cultures and J ) passage 3 were analyzed in parallel as a control group Using indirect immunofluorescence staining, highly positive signals (green) were detected for all cellular markers tested such as K ) CD90 (green)/ alpha smooth muscle actin (ASMA) (red) of fresh cultivated cells and L ) CD90 (green)/ ASMA (red) of cryopreserved cells, N ) CD29 of fresh cultivated cells and O ) CD29 of cryopreserved cells, P ) CD105 of fresh cultivated cells and Q ) CD105 of cryopreserved cells. Cell nuclei staining is pictured in blue, present in A-H and K-Q. All studies of marker expression are exemplarily shown for cells of passage 3.

Article Snippet: Fixed cross sectioned umbilical cord arteries were used to localize ECM proteins and served as controls by staining with monoclonal mouse anti-human collagen type I (0.5 μg/ml, Acris Antibodies, Herford, Germany), anti-human collagen type III (30 μg/ml, Innovative Diagnostics, Vienna, Austria) and anti-human elastin (1:100, Sigma, St.Louis, USA) for 30 min at RT after blocking with goat serum (10 %, Leica) for 10 min. Cross sections of umbilical cord vein, serving as controls, were incubated with monoclonal mouse anti-human CD31 (1:80, Innovative Diagnostics) and anti-human vWF (1:100, Dako, Hamburg, Germany) for 30 min to detect HUVEC after blocking with goat serum (10 %, Leica) for 10 min.

Techniques: Expressing, Marker, Derivative Assay, Immunofluorescence, Staining, Immunohistochemical staining, Flow Cytometry

Journal: Stem Cell Research & Therapy

Article Title: A new platelet cryoprecipitate glue promoting bone formation after ectopic mesenchymal stromal cell-loaded biomaterial implantation in nude mice

doi: 10.1186/scrt149

Figure Lengend Snippet: Polymerase chain reaction primers used to investigate osteoblastic cell differentiation

Article Snippet: Sections were incubated overnight at 4°C with monoclonal mouse anti-human type I collagen (clone I-8H5, 1 μg/mL; Acris Antibodies, San Diego, CA, USA).

Techniques: Polymerase Chain Reaction

Journal: Stem Cell Research & Therapy

Article Title: A new platelet cryoprecipitate glue promoting bone formation after ectopic mesenchymal stromal cell-loaded biomaterial implantation in nude mice

doi: 10.1186/scrt149

Figure Lengend Snippet: PGCAP improves osteogenic differentiation of mesenchymal stromal cells (MSCs) loaded on biomaterials . Total RNAs were extracted from ni-MSCs and i-MSCs loaded on BM A and BM F at high cell seeding density (200 × 10 3 cells per biomaterial) embedded or not in PGCAP and grown for 21 days. Quantifications of osteopontin, type I collagen, Runx2, osteonectin, osteocalcin, and alkaline phosphatase mRNA expression were analyzed by real-time quantitative-polymerase chain reaction. Transcript levels in arbitrary units are expressed as mean ± standard error of the mean. A statistically significant difference between individual conditions or groups of condition was revealed by analysis-of-variance test followed by Student Newman-Keuls test. Osteopontin: group of ni-MSCs and i-MSCs loaded on BM A embedded in PGCAP was compared with other conditions ( $ * P <0.05). Type I collagen: individual comparisons, * P <0.05, ** P <0.01, *** P <0.001. Runx2, Osteonectin: group with ni-MSCs embedded in PGCAP (loaded in both biomaterials) was compared with other conditions with i-MSCs ( # * P <0.05). Osteocalcin: comparison of group osteogenic-induced or not ( £ * P <0.05). Alkaline phosphatase: group with i-MSCs without PGCAP (loaded in both biomaterials) was compared with other conditions with ni-MSCs ( § ** P <0.01). i-MSC, not osteogenic induced-mesenchymal stromal cell; ni-MSC, not induced-mesenchymal stromal cell; PGCAP, platelet glue obtained from cryoprecipitation of apheresis platelet products.

Article Snippet: Sections were incubated overnight at 4°C with monoclonal mouse anti-human type I collagen (clone I-8H5, 1 μg/mL; Acris Antibodies, San Diego, CA, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Stem Cell Research & Therapy

Article Title: A new platelet cryoprecipitate glue promoting bone formation after ectopic mesenchymal stromal cell-loaded biomaterial implantation in nude mice

doi: 10.1186/scrt149

Figure Lengend Snippet: Bone formation areas are increased after ectopic implantation of MSC-loaded BM A embedded in PGCAP . (a) The average ratio of bone formation area in BM A 28 days after grafting was determined in each condition. The data are expressed as mean ± standard error of the mean. A statistically significant difference between individual conditions was revealed by analysis-of-variance test followed by Student Newman-Keuls test. * P <0.05, ** P <0.01, *** P <0.00, **** P <0.0001. (b) Type I collagen immuno-staining (brownish color) was performed on BM A and BM F sections before implantation and 28 days after implantation. Original magnifications: ×4 and ×10. i-MSC, not osteogenic induced-mesenchymal stromal cell; MSC, mesenchymal stromal cell; ni-MSC, not induced-mesenchymal stromal cell; PGCAP, platelet glue obtained from cryoprecipitation of apheresis platelet products.

Article Snippet: Sections were incubated overnight at 4°C with monoclonal mouse anti-human type I collagen (clone I-8H5, 1 μg/mL; Acris Antibodies, San Diego, CA, USA).

Techniques: Immunostaining